HEALTH MONITORING: MAMMALS
BATS
Turner G. G., Meteyer C. U., Barton H., Gumbs J. F., Reeder D. M., Overton B., Blehert D. S. (2014): Nonlethal screening of bat-wing skin with the use of ultraviolet fluorescence to detect lesions indicative of white-nose syndrome. Journal of Wildlife Diseases 50: 566-573.
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Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366–385 nm) elicits a distinct orange–yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange–yellow wing fluorescence (n = 80) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n = 88) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n = 22) and 100% of nonfluorescent (n = 40) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of long-wave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.
Lima F. E. D. S., Cibulski S. P., Dos Santos H. F., Teixeira T. F., Varela A. P. M., Roehe P. M., Delwart E., Franco A. C. (2015): Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil. Plos One 10: e0118070.
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Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.
McMichael L., Edson D., Smith C., Mayer D., Smith I., Kopp S., Meers J., Field H. (2017): Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia. Plos One 12: e0182171.
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Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.
Afonso E., Goydadin A. C. (2018): Molecular detection of Anaplasma phagocytophilum DNA in the lesser horseshoe bat (Rhinolophus hipposideros) guano. Epidemiology and Infection 146: 1253-1258.
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Although bats are increasingly recognised as potential reservoir hosts of human zoonotic pathogens, bacteria in bats are still poorly studied. To investigate the DNA faecal prevalence of the bacterium Anaplasma phagocytophilum, we sampled 23 lesser horseshoe bat (Rhinolophus hipposideros) maternity colonies located in buildings (churches, barns) in rural villages of eastern France. A total of 552 faecal samples were collected from 278 individuals. Anaplasma phagocytophilum DNA was detected in the faeces of 63 individuals (22.7%). Such high prevalence might suggest persistent infection in bats and/or a frequent consumption of insect preys carrying bacteria. Faecal DNA prevalence varied highly among colonies but was not related to the colony size. Faecal DNA prevalence was the highest in the Jura Department, where the density of ticks is known to be the highest across the study area. Because the sampled bats live in close proximity to humans, we discuss how concerning the presence of A. phagocytophilum DNA in bat guano is for humans frequenting places of worship that shelter bats. We also advocate future research to understand what a high faecal DNA prevalence in bat guano really implicates in terms of bacteria transmission.
Ingala M. R., Simmons N. B., Wultsch C., Krampis K., Speer K. A., Perkins S. L. (2018): Comparing microbiome sampling methods in a wild mammal: fecal and intestinal samples record different signals of host ecology, evolution. Frontiers in Microbiology 9: 803.
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The gut microbiome is a community of host-associated symbiotic microbes that fulfills multiple key roles in host metabolism, immune function, and tissue development. Given the ability of the microbiome to impact host fitness, there is increasing interest in studying the microbiome of wild animals to better understand these communities in the context of host ecology and evolution. Human microbiome research protocols are well established, but wildlife microbiome research is still a developing field. Currently, there is no standardized set of best practices guiding the collection of microbiome samples from wildlife. Gut microflora are typically sampled either by fecal collection, rectal swabbing, or by destructively sampling the intestinal contents of the host animal. Studies rarely include more than one sampling technique and no comparison of these methods currently exists for a wild mammal. Although some studies have hypothesized that the fecal microbiome is a nested subset of the intestinal microbiome, this hypothesis has not been formally tested. To address these issues, we examined guano (feces) and distal intestinal mucosa from 19 species of free-ranging bats from Lamanai, Belize, using 16S rRNA amplicon sequencing to compare microbial communities across sample types. We found that the diversity and composition of intestine and guano samples differed substantially. In addition, we conclude that signatures of host evolution are retained by studying gut microbiomes based on mucosal tissue samples, but not fecal samples. Conversely, fecal samples retained more signal of host diet than intestinal samples. These results suggest that fecal and intestinal sampling methods are not interchangeable, and that these two microbiotas record different information about the host from which they are isolated.
Wolkers-Rooijackers J., Rebmann K., Bosch T., Hazeleger W. C. (2018): Fecal bacterial communities in insectivorous bats from the Netherlands and their role as a possible vector for foodborne diseases. Acta Chiropterologica 20: 475-483.
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Bats are commonly regarded as vectors for viruses, but little is known about bacterial communities in bats and the possible role of bats in the transmission cycle of foodborne diseases. To gain more insight, microbial communities in fecal samples from 37 insectivorous bats of different species from the Netherlands were investigated by polymerase chain reaction and denaturant gradient gel electrophoresis (PCR-DGGE). Subsequently, 10 samples from the following bat species: common pipistrelle (Pipistrellus pipistrellus; n = 3), Daubenton’s bat (Myotis daubentonii; n = 3), serotine bat (Eptesicus serotinus; n = 1), whiskered bat (Myotis mystacinus; n = 1), Geoffroy’s bat (Myotis emarginatus; n = 1) and Natterer’s bat (Myotis nattereri; n = 1) were selected and used in bacterial 16S rDNA cloning and sequencing. The fecal microbiota in bats was found to be diverse with predominant bacterial genera Carnobacterium, Serratia, Pseudomonas, Enterococcus and Yersinia. The presence of opportunistic pathogens Citrobacter freundii, Escherichia coli, Enterococcus faecalis, Serratia fonticola and Rahnella aquatilis was also recorded. Based on cloning results, we found no proof that bats in the Netherlands are a major vector for the transmission of bacterial zoonotic diseases, although previous findings in literature reported isolation of foodborne pathogens from bats.
Bergner L. M., Orton R. J., da Silva Filipe A., Shaw A. E., Becker D. J., Tello C., Biek R., Streicker D.G. (2019): Using noninvasive metagenomics to characterize viral communities from wildlife. Molecular Ecology Resources 19: 128-143.
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Microbial communities play an important role in organismal and ecosystem health. While high-throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low-input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time.
Adhikari R. B., Maharjan M., Ghimire T. R. (2020): Prevalence of gastrointestinal parasites in the frugivorous and the insectivorous bats in Southcentral Nepal. Journal of Parasitology Research 2020: 8880033.
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Bats are the only active flying placental mammals and are traditionally classified into mega- and microbats, which are, respectively, herbivorous and insectivorous in feeding habit. Though deforestation, habitat destruction, natural calamities, illegal hunting, and climate changes are the challenging threats for bats, the role of existing gastrointestinal (GI) parasites have not been evaluated yet in Nepal. Thus, the current study aims to determine the prevalence of GI parasites in bats from the Shaktikhor area at the Chitwan district of Southcentral Nepal. From July 2018 to February 2019, a total of 60 fecal samples of bats (30 from frugivorous bats and 30 from the insectivorous bats) were collected. These samples were preserved at 2.5% potassium dichromate solution. The fecal examination was carried out by the direct wet mount, concentrations, acid-fast staining, and sporulation techniques. Overall results showed the prevalence rate of 80% GI parasites. The parasites detected in the insectivorous bats were Ascarid spp., Capillarid sp., Cryptosporidium sp., Eimeria spp., Entamoeba sp., Giardia sp., Hymenolepis spp., Isospora sp., Oxyurid sp., Strongyle, and Strongyloides sp. In contrast, Eimeria sp., Entamoeba sp., and Hymenolepis sp. were detected in the frugivorous bats. Based on a wide diversity of parasite richness and parasitic concurrency measured by the prevalence rates, we suggest that GI parasitism might be a threatening factor in the insectivorous bats in the current study area.
Gerbáčová K., Maliničová L., Kisková J., Maslišová V., Uhrin M., Pristaš P. (2020): The faecal microbiome of building-dwelling insectivorous bats (Myotis myotis and Rhinolophus hipposideros) also contains antibiotic-resistant bacterial representatives. Current Microbiology 77: 2333-2344.
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The bacteriome of bat faeces and bat guano is still not well known, despite the increasing knowledge of it as a potential source of pathogenic and/or antibiotic-resistant species. In this study, the faecal microbiome composition of two building-dwelling insectivorous bat species (Myotis myotis and Rhinolophus hipposideros) was analysed using cultivation-based and non-cultivation-based approaches. The cultivation analyses indicate the dominance of Gram-positive bacteria, represented by the genera Enterococcus, Lactococcus and Lactobacillus. Non-cultivation analysis based on 16S rRNA DGGE assays provided a different pattern, with the genus Rahnella found to predominate in the faecal microbiome. The analyses showed bat species to be the most important factor influencing the structure of the guano-associated microbial population. The presence of several possible human pathogens (Hafnia alvei, Serratia fonticola, S. liquefaciens) and antibiotic-resistant bacteria (e.g. vancomycin resistant Staphylococcus nepalensis) was detected in faeces samples, indicating possible human health risks associated with bat guano. Although the bat-human transmission of infection caused by pathogenic bacteria has not been reliably confirmed to date, this possibility should not be underestimated. Given the very important ecosystem services of bats, particular those studied herein, it is time to think about appropriate management of bat populations inhabiting man-made buildings and potential conflicts with humans.
Hicks L. L., Schwab N. A., Homyack J. A., Jones J. E., Maxell B. A., Burkholder B. O. (2020): A statistical approach to white-nose syndrome surveillance monitoring using acoustic data. Plos One 15: e0241052.
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Traditional pathogen surveillance methods for white-nose syndrome (WNS), the most serious threat to hibernating North American bats, focus on fungal presence where large congregations of hibernating bats occur. However, in the western USA, WNS-susceptible bat species rarely assemble in large numbers and known winter roosts are uncommon features. WNS increases arousal frequency and activity of infected bats during hibernation. Our objective was to explore the effectiveness of acoustic monitoring as a surveillance tool for WNS. We propose a non-invasive approach to model pre-WNS baseline activity rates for comparison with future acoustic data after WNS is suspected to occur. We investigated relationships among bat activity, ambient temperatures, and season prior to presence of WNS across forested sites of Montana, USA where WNS was not known to occur. We used acoustic monitors to collect bat activity and ambient temperature data year-round on 41 sites, 2011–2019. We detected a diverse bat community across managed (n = 4) and unmanaged (n = 37) forest sites and recorded over 5.37 million passes from bats, including 13 identified species. Bats were active year-round, but positive associations between average of the nightly temperatures by month and bat activity were strongest in spring and fall. From these data, we developed site-specific prediction models for bat activity to account for seasonal and annual temperature variation prior to known occurrence of WNS. These prediction models can be used to monitor changes in bat activity that may signal potential presence of WNS, such as greater than expected activity in winter, or less than expected activity during summer. We propose this model-based method for future monitoring efforts that could be used to trigger targeted sampling of individual bats or hibernacula for WNS, in areas where traditional disease surveillance approaches are logistically difficult to implement or because of human-wildlife transmission concerns from COVID-19.
Ogórek R., Kurczaba K., Cal M., Apoznański G., Kokurewicz T. (2020): A culture-based ID of Micromycetes on the wing membranes of greater mouse-eared bats (Myotis myotis) from the “Nietoperek” site (Poland). Animals 10: 1337.
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Bats play important functions in ecosystems and many of them are threatened with extinction. Thus, the monitoring of the health status and prevention of diseases seem to be important aspects of welfare and conservation of these mammals. The main goal of the study was the identification of culturable fungal species colonizing the wing membranes of female greater mouse-eared bat (Myotis myotis) during spring emergence from the “Nietoperek” underground hibernation site by the use of genetic and phenotypic analyses. The study site is situated in Western Poland (52°25′ N, 15°32′ E) and is ranked within the top 10 largest hibernation sites in the European Union. The number of hibernating bats in the winter exceeds 39,000 individuals of 12 species, with M. myotis being the most common one. The wing membranes of M. myotis were sampled using sterile swabs wetted in physiological saline (0.85% NaCl). Potato dextrose agar (PDA) plates were incubated in the dark at 8, 24 and 36 ± 1 °C for 3 up to 42 days. All fungi isolated from the surface of wing membranes were assigned to 17 distinct fungal isolates belonging to 17 fungal species. Penicillium chrysogenum was the most frequently isolated species. Some of these fungal species might have a pathogenic potential for bats and other mammals. However, taking into account habitat preferences and the life cycle of bats, it can be assumed that some fungi were accidentally obtained from the surface of vegetation during early spring activity. Moreover, Pseudogymnoascus destructans (Pd)—the causative agent of the White Nose Syndrome (WNS)—was not found during testing, despite it was found very often in M. myotis during previous studies in this same location.
Fritze M., Puechmaille S. J., Fickel J., Czirják G. Á., Voigt C. C. (2021): A rapid, in-situ minimally-invasive technique to assess infections with Pseudogymnoascus destructans in bats. Acta Chiropterologica 23: 259-270.
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Emerging infectious diseases may become serious threats to wildlife, a prominent example being the white-nose disease (WND). In case of WND, the cold-loving fungus Pseudogymnoascus destructans colonizes bats during hibernation, invades the skin and has already lead to the death of millions of bats in North America. P. destructans most likely originated from Europe, where it also causes lesions but without associated mortalities. However, it is still unclear how European bats cope with the fungus. Hence, it is important to have tools that precisely characterise disease progression. Because hibernation is a physiological state during which bats are vulnerable to disturbance, in-situ assessments of the clinical status should be carried out minimal-invasively to avoid detrimental impacts on bats. However, currently available disease assessment methods require handling/touching bats and are therefore invasive: i) UV-light trans-illumination of wing membranes to detect lesions and ii) a qPCR-based quantification of fungal material from wing membrane swabs. Since P. destructans (‘Pd’) becomes visible on all furless skin with distinct distribution patterns, we investigated the use of visible symptoms to assess levels of infections without handling/touching bats. We introduce a technique which we termed ‘Visual Pd-score’ (a visual classification scheme), which can be applied without disturbing the animals. To assess its reliability, we used P. destructans infected greater mouse-eared bats (Myotis myotis) to compare the novel method with the two existing golden-standard techniques. Our results show that infection levels obtained from all three techniques are correlated. Importantly, the information carried by the Visual Pd-score is most similar to a composite index combining the information from the qPCR-based and UV-light quantification methods. We conclude that the Visual Pd-score represents a promising index to better characterise disease severity as it is simultaneously representative for fungal colonization and wing damage. We discuss advantages and disadvantages of the applied techniques and conclude that the Visual Pd-score is particularly useful for routine hibernacula counts or large-scale P. destructans-surveillance. In combination with the lesion detection technique the new method is also applicable to immunological studies where both fungal colonization and associated damage have to be investigated, while qPCRs from swabs of all body parts are especially useful if it is necessary to detect cryptic infections, e.g. during the early hibernation period.
Giles J. R., Peel A. J., Wells K., Plowright R. K., McCallum H., Restif O. (2021): Optimizing noninvasive sampling of a zoonotic bat virus. Ecology and Evolution 11: 12307-12321.
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Outbreaks of infectious viruses resulting from spillover events from bats have brought much attention to bat-borne zoonoses, which has motivated increased ecological and epidemiological studies on bat populations. Field sampling methods often collect pooled samples of bat excreta from plastic sheets placed under-roosts. However, positive bias is introduced because multiple individuals may contribute to pooled samples, making studies of viral dynamics difficult. Here, we explore the general issue of bias in spatial sample pooling using Hendra virus in Australian bats as a case study. We assessed the accuracy of different under-roost sampling designs using generalized additive models and field data from individually captured bats and pooled urine samples. We then used theoretical simulation models of bat density and under-roost sampling to understand the mechanistic drivers of bias. The most commonly used sampling design estimated viral prevalence 3.2 times higher than individual-level data, with positive bias 5–7 times higher than other designs due to spatial autocorrelation among sampling sheets and clustering of bats in roosts. Simulation results indicate using a stratified random design to collect 30–40 pooled urine samples from 80 to 100 sheets, each with an area of 0.75–1 m2, and would allow estimation of true prevalence with minimum sampling bias and false negatives. These results show that widely used under-roost sampling techniques are highly sensitive to viral presence, but lack specificity, providing limited information regarding viral dynamics. Improved estimation of true prevalence can be attained with minor changes to existing designs such as reducing sheet size, increasing sheet number, and spreading sheets out within the roost area. Our findings provide insight into how spatial sample pooling is vulnerable to bias for a wide range of systems in disease ecology, where optimal sampling design is influenced by pathogen prevalence, host population density, and patterns of aggregation.
Hardmeier I., Aeberhard N., Qi W., Schoenbaechler K., Kraettli H., Hatt J. M., Fraefel C., Kubacki J. (2021): Metagenomic analysis of fecal and tissue samples from 18 endemic bat species in Switzerland revealed a diverse virus composition including potentially zoonotic viruses. Plos One 16: e0252534.
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Many recent disease outbreaks in humans had a zoonotic virus etiology. Bats in particular have been recognized as reservoirs to a large variety of viruses with the potential to cross-species transmission. In order to assess the risk of bats in Switzerland for such transmissions, we determined the virome of tissue and fecal samples of 14 native and 4 migrating bat species. In total, sequences belonging to 39 different virus families, 16 of which are known to infect vertebrates, were detected. Contigs of coronaviruses, adenoviruses, hepeviruses, rotaviruses A and H, and parvoviruses with potential zoonotic risk were characterized in more detail. Most interestingly, in a ground stool sample of a Vespertilio murinus colony an almost complete genome of a Middle East respiratory syndrome-related coronavirus (MERS-CoV) was detected by Next generation sequencing and confirmed by PCR. In conclusion, bats in Switzerland naturally harbour many different viruses. Metagenomic analyses of non-invasive samples like ground stool may support effective surveillance and early detection of viral zoonoses.
Seidlova V., Nemcova M., Pikula J., Bartonička T., Ghazaryan A., Heger T., Kokurewicz T., Orlov O. L., Patra S., Piacek V., Treml F. (2021): Urinary shedding of leptospires in palearctic bats. Transboundary and Emerging Diseases 68: 3089-3095.
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Leptospirosis is a bacterial zoonotic infection of worldwide occurrence. Bats, like other mammalian reservoirs, may be long-term carriers that maintain endemicity of infection and shed viable leptospires in urine. Direct and/or indirect contact with these Leptospira shedders is the main risk factor as regards public health concern. However, knowledge about bat leptospirosis in the Palearctic Region, and in Europe in particular, is poor. We collected urine from 176 specimens of 11 bat species in the Czech Republic, Poland, Republic of Armenia and the Altai Region of Russia between 2014 and 2019. We extracted DNA from the urine samples to detect Leptospira spp. shedders using PCR amplification of the 16S rRNA and LipL32 genes. Four bat species (Barbastella barbastellus n = 1, Myotis bechsteinii n = 1, Myotis myotis n = 24 and Myotis nattereri n = 1) tested positive for Leptospira spp., with detected amplicons showing 100% genetic identity with pathogenic Leptospira interrogans. The site- and species-specific prevalence range was 0%–24.1% and 0%–20%, respectively. All bats sampled in the Republic of Armenia and Russia were negative. Given the circulation of pathogenic leptospires in strictly protected Palearctic bat species and their populations, non-invasive and non-lethal sampling of urine for molecular Leptospira spp. detection is recommended as a suitable surveillance and monitoring strategy. Moreover, our results should raise awareness of this potential disease risk among health professionals, veterinarians, chiropterologists and wildlife rescue workers handling bats, as well as speleologists and persons cleaning premises following bat infestation.
Li A., Li Z., Dai W., Parise K. L., Leng H., Jin L., Liu S., Sun K., Hoyt J. R., Feng J. (2022): Bacterial community dynamics on bats and the implications for pathogen resistance. Environmental Microbiology 24: 1484-1498.
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The bats skin microbiota plays an important role in reducing pathogen infection, including the deadly fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome. However, the dynamic of skin bacterial communities response to environmental perturbations remains poorly described. We characterized skin bacterial community over time and space in Rhinolophus ferrumequinum, a species with high resistance to the infection with P. destructans. We collected environmental covariate data to determine what factors influenced changes in community structure. We observed significant temporal and spatial shifts in the skin bacterial community, which was mainly associated with variation in operational taxonomic units. The skin bacterial community differed by the environmental microbial reservoirs and was most influenced by host body condition, bat roosting temperature and geographic distance between sites, but was not influenced by pathogen infection. Furthermore, the skin microbiota was enriched in particular taxa with antifungal abilities, such as Enterococcus, Burkholderia, Flavobacterium, Pseudomonas, Corynebacterium and Rhodococcus. And specific strains of Pseudomonas, Corynebacterium and Rhodococcus even inhibited P. destructans growth. Our findings provide new insights in characterizing the variation in bacterial communities can inform us about the processes of driving community assembly and predict the host’s ability to resist or survive pathogen infection.
CARNIVORES
Zhang L., Yang X., Wu H., Gu X., Hu Y., Wei F. (2011): The parasites of giant pandas: individual-based measurement in wild animals. Journal of Wildlife Diseases 47: 164-171.
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There is a growing recognition of parasites as a significant factor in the successful conservation of endangered species. Determining parasite infection and load in free-ranging populations traditionally is done via necropsy or coproscopy. For studies of wild animals, fecal sample collection can result in bias because the individual identity of animals is unknown and multiple samples may be collected from the same individual, yet treated as unrelated samples. We studied parasite load in wild giant pandas (Ailuropoda melanoleuca) across six mountain ranges in China. Genetic identification was used to determine the exact number of individuals sampled. The parasite fauna consisted of five species, dominated by Baylisascaris shroederi. The pattern of statistical difference between mountains was artificially inflated when animal identity was not included in the model. Our results suggest that caution should be exercised in inferring patterns from comparative parasitologic studies when samples cannot be attributed to specific individuals. Using noninvasive genetic sampling to avoid such bias should form a standard tool in the management of endangered species and their parasites.
Stuart P., Golden O., Zintl A., de Waal T., Mulcahy G., McCarthy E., Lawton C. (2013): A coprological survey of parasites of wild carnivores in Ireland. Parasitology Research 112: 3587-3593.
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The increasing movement of people to wilderness areas, shrinking of wildlife habitats and the resulting urbanisation of wildlife has led to growing concerns about the transfer of parasitic diseases, particularly from contaminated faeces. Faecal samples from wild carnivores in Ireland were examined for the presence of protozoan and nematode parasites. Red fox (Vulpes vulpes) samples (n = 91) were positive for Uncinaria stenocephala (38 %), Eucoleus aerophilus (26 %), Toxocara canis (20 %), Trichuris vulpis (4 %) and Isospora-like oocysts (9 %). Badger (Meles meles) samples (n = 50) were positive for Uncinaria criniformis (40 %), E. aerophilus (6 %) and Isospora-like oocysts (16 %). No parasites were observed in pine marten (n = 48; Martes martes) faeces. Approximately 5 % of American mink (Mustela vison) samples were positive for Cryptosporidium by polymerase chain reaction (identified as Cryptosporidium andersoni (n = 3) and ‘mink’ genotype (n = 1)). The results suggest that wild carnivores in Ireland have a range of parasites, although it is unclear from the present study to what extent these infections are associated with morbidity. While it can be expected that, via their faeces, wild carnivores contribute to the spread of these parasites, they are unlikely the primary source of environmental contamination. Therefore, they should not always be the principal target of control measures.
Liccioli S., Rogers S., Greco C., Kutz S. J., Chan F., Ruckstuhl K. E., Massolo A. (2015): Assessing individual patterns of Echinococcus multilocularis infection in urban coyotes: non‐invasive genetic sampling as an epidemiological tool. Journal of Applied Ecology 52: 434-442.
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In epidemiological studies of wildlife parasites, faecal genotyping has been introduced to prevent bias in estimates of parasite prevalence from faecal samples collected in the field. Such an approach could be particularly relevant in the study of Echinococcus multilocularis transmission in urban settings, where estimates of prevalence and patterns of infection in wild canid hosts are key parameters used in zoonotic risk assessment and management. However, no previous study has evaluated the reliability of E. multilocularis faecal prevalence, and individual patterns of infection in definitive hosts remain poorly understood. We evaluated faecal genotyping as an epidemiological tool, using E. multilocularis in urban coyotes Canis latrans as our study system. Combining parasitological analysis and multilocus individual genotyping of coyote faeces, we compared faecal parasite prevalence with the prevalence obtained from genotyped faecal samples. Furthermore, we assessed patterns of individual infection, such as re-infection rates and phenology of parasite egg excretion. Of 425 faeces collected in five urban sites, we genotyped 142 samples (33·4%) corresponding to 60 unique individual coyotes. Number of genotyped samples per coyote ranged between 1 and 10 (mean = 2·3). Genotypes were obtained at 4–6 microsatellite loci and had a mean reliability of 0·9975. Faecal prevalence of E. multilocularis in genotyped coyotes was 25·0%, and similar to results previously obtained from non-genotyped faeces. Faecal genotyping allowed estimating a re-infection rate of individual coyotes of 57·1% and to observe temporal patterns of parasite infection that were not detected using non-genotyped faeces. If compared to independent data obtained through coyote post-mortem examination, our results suggest that reliable estimates of overall parasite prevalence in definitive host populations can be efficiently obtained through well-designed field collection and traditional faecal parasitological analysis. However, faecal genotyping allows assessing the dynamics of individual infections, which could otherwise only be estimated by using invasive techniques. Combining faecal genotyping with parasitology has a great potential in assessing zoonotic risk transmission in urban areas, as well as advancing the field of wildlife ecology, disease ecology and conservation.
Napoli E., Anile S., Arrabito C., Scornavacca D., Mazzamuto M. V., Gaglio G., Otranto D., Giannetto S., Brianti E. (2016): Survey on parasitic infections in wildcat (Felis silvestris silvestris Schreber, 1777) by scat collection. Parasitology Research 115: 255-261.
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Wildcats are endangered felid species living in Europe, Asia, and Africa. Regrettably, scientific information on parasites of wildcats is particularly meager and they often rely on data gained by necropsies of a small number of animals. In the present study, scat collection was used to assess the parasite spectrum of European wildcats living in the Etna Park (Sicily, Italy). Scat collection was performed from May to September 2010 by weekly walking four transects for a total of 391 km. Samples were then analyzed by flotation and sedimentation techniques to investigate wildcat parasitic fauna. A total of 121 scats of wildcats were collected, and parasitic forms (i.e., oocysts, eggs, and larvae) were retrieved in 110 (90.9 %) of the samples. Parasites found were Physaloptera sp. (52.1 %), tapeworms (45.5 %), Toxocara cati (43.8 %), Eucoleus aerophilus (27.3 %), Ancylostoma sp. (22.3 %), Troglostrongylus brevior (15.7 %), trematodes (9.9 %), Isospora felis (4.1 %), Cylicospirura sp. (1.7 %), and Acanthocephala (0.8 %). The prevalence of endoparasitic infections herein recorded is similar to that described in other studies conducted using necropsy technique. The species richness of parasites found in the present survey, with a total of nine helminths and one protozoon, is the highest ever reported for wildcat in Europe. Scat collection and examination are reliable and rapid non-invasive tools which can be used in a systematic survey design to study the parasite spectrum of wildcat as well as that of other endangered wild species.
Borka-Vitális L., Domokos C., Földvári G., Majoros G. (2017): Endoparasites of brown bears in Eastern Transylvania, Romania. Ursus 28: 20-30.
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Limited information is currently available on the parasite fauna of brown bears (Ursus arctos) in Transylvania, Romania. During 2011–2015 we identified endoparasites from 211 scat samples collected from free-ranging bears and 37 samples from organ tissue of adult individuals harvested by hunters in Eastern Transylvania. We found endoparasites in 40.7% of samples overall; 37.9% of scats and 56.8% of harvested bears. We identified 6 nematodes including undetermined species of the family Ancylostomatidae, Baylisascaris transfuga, Crenosoma sp., undetermined species of the order Spirurida, Trichinella sp., and Trichuris sp. We also found one undetermined protozoan species of the subclass Coccidia, the cestode Taenia sp., and the trematode Dicrocoelium dendriticum. We did not detect seasonal variation in B. transfuga incidence, nor in overall parasite prevalences. In Transylvania, brown bears’ space use overlaps with that of domestic animals, creating a potential risk for the transmission of parasite infections. Supplemental feeding of bears and other wildlife species as part of current management practices may increase the potential for parasitic infections by artificially concentrating wildlife.
Espinosa‐de Aquino W., Olvera‐Ramírez A., Arellano‐Carbajal F., Lanz‐Mendoza H., Villagrán‐Herrera E., Acevedo‐Whitehouse K. (2017): Protein and RNA extraction from mucosal swabs: a minimally invasive source of ecological data for studies of natural populations. Methods in Ecology and Evolution 8: 370-378.
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Mucosal swabs have long been used to study various physiological processes in humans. In contrast, systematic sampling of mucosae is rare for wild animals, and except for its use as a source of DNA for population genetics, its potential as a tool to study physiologically relevant processes of natural populations has not been explored fully. We collected swabs from the rectal, genital, nasal and oral mucosae of California sea lion pups and investigated differences in RNA and protein yield, quality and integrity between storage times and mucosal types. Downstream applications were tested on the extracted products to determine the potential value of mucosal sampling in free-ranging mammals. For most samples, RNA yield was stable regardless of storage time, and RNA quality and integrity were equal for all mucosal types. Ribosomal fragments of sizes expected for mammals, yeast and bacteria were observable in genital and rectal samples, but in nasal and oral samples only bacterial ribosomal fragments were observable. Amplification of selected transcripts was successful for all samples. The protein profile was distinct between mucosae, and samples with high protein yields were useful for antibody detection. Our study demonstrates that mucosal swabbing is a minimally invasive tool that yields useful physiological data for free-ranging wildlife. Minimum experience is needed to collect samples, processing is inexpensive, and downstream applications for ecological studies are realistic.
Štrkolcová G., Goldová M., Šnábel V., Špakulová M., Orosová T., Halán M., Mojžišová J. (2018): A frequent roundworm Baylisascaris transfuga in overpopulated brown bears (Ursus arctos) in Slovakia: a problem worthy of attention. Acta Parasitologica 63: 167-174.
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The genus Baylisascaris (order Ascaridida) includes numerous relatively host-specific nematodes, which are common in intestines of wild mammals. Some of them may have impact on veterinary and public health, as their larvae have the potential to cause visceral, ocular, and/or neural larva migrans in a wide range of mammals, birds, and humans. Baylisascaris transfuga is a parasite occurring in a range of bear species throughout the world. We present the current data on B. transfuga occurrence in brown bears from a relatively restricted territory of the Poľana Protected Landscape Area in Central Slovakia, obtained by traditional methods (faecal examination, morphology). Species affiliation was confirmed by employing molecular markers generating nuclear 28S and mitochondrial cox1 sequences in adult worms. Based on 17 examined samples (15 excrements and two intestines of young bear females), the occurrence of B. transfuga in the surveyed area was assessed as 52.9%. Both bear females were infected with adult and juvenile worms. Due to the high density of bears in the locality, the high infection rate with ascarids, and the huge number of eggs produced by the parasites, it is apparent that the respective environment, including the inhabited areas, might be markedly contaminated by Baylisascaris eggs. The ability of B. transfuga to serve as a zoonotic agent has not been unambiguously proved; however, this attribute should be considered and subjected to further research.
Ebmer D., Navarrete M. J., Muñoz P., Flores L. M., Gärtner U., Taubert A., Hermosilla C. (2019): Antarctophthirus microchir infestation in synanthropic South American sea lion (Otaria flavescens) males diagnosed by a novel non-invasive method. Parasitology Research 118: 1353-1361.
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Antarctophthirus microchir is a sucking louse species belonging to the family Echinophthiriidae and has been reported to parasitize all species of the subfamily Otariinae, the sea lions. Former studies on this ectoparasite mainly required fixation, immobilization, or death of host species and especially examinations of adult male sea lions are still very rare. Between March and May 2018, adult individuals of a unique “urban” bachelor group of South American sea lions (Otaria flavescens) living directly in the city of Valdivia, Chile, were studied regarding their ectoparasite infestation status. For first time, a non-invasive method in the form of a lice comb screwed on a telescopic rod and grounded with adhesive tape was used for sample taking process. Overall, during combing different stages of A. microchir were detected in 4/5 O. flavescens individuals, especially at the junction between the back and hind flippers. Our findings represent the first report of A. microchir infesting individuals of this synanthropic colony and fulfilling complete life cycle in a sea lion group despite inhabiting freshwater and in absence of females/pups. Our “telescopic lice comb apparatus” offers a new strategy to collect different stages of ectoparasites and a range of epidermal material, such as fur coat hair and superficial skin tissue for a broad spectrum of research fields in wildlife sciences in an unmolested and stress reduced manner.
Figueiredo A. M., Barros T., Valente A. M., Fonseca C., de Carvalho L. M., Torres R. T. (2019): Prevalence of zoonotic parasites in an endangered Iberian wolf (Canis lupus signatus) population in Portugal. Mammalian Biology 98: 154-162.
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As a top predator, the Iberian wolf (Canis lupus signatus) plays a major role shaping interactions within food webs. Due to its conservation status in Portugal, i.e. endangered, it is important to understand the role of parasites in this population, since they can be a limiting factor for the population fitness and trophic interactions and, ultimately, their survival. From November 2017 to August 2018, 33 fresh faecal samples were collected in several transects distributed throughout Montesinho Natural Park. Samples were analysed by means of four coprological techniques. A total of three helminth parasites (Ancylostoma spp., Uncinaria spp. and Eucoleus aerophilus) were identified based on size and morphology. The overall prevalence was low (15.5%), being Ancylostoma spp. the most prevalent parasite. The three parasites found are of major concern, once they are pathogenic to humans and other wild and domestic animals. We suggest surveillance programs that include both parasite and wildlife monitoring. To our knowledge, this is the first coprological study performed with this Iberian wolf population.
Ebmer D., Navarrete M. J., Muñoz P., Flores L. M., Gärtner U., Brabec J., Hermosilla C. (2020): Anthropozoonotic parasites circulating in synanthropic and Pacific colonies of South American Sea Lions (Otaria flavescens): Non-invasive techniques data and a review of the literature. Frontiers in Marine Science 7: 847.
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Since late 1970s, the southern Chilean city Valdivia constitutes home for a unique bachelor group of South American sea lions (Otaria flavescens), initially descendant from colonies at the Pacific coast, but now directly living in a freshwater habitat in close proximity to human population and a vast amount of wild and domestic animal species. In the framework of a parasitological monitoring program, 115 individual fecal samples were collected from synanthropic South American sea lions between March and May 2018. For comparative reasons, 79 individual fecal samples from two free-living O. flavescens colonies at the Pacific coast were also sampled. Coproscopical analyses revealed the presence of nine different parasite taxa in individual fecal samples, including two protozoan (Cryptosporidium spp. and Giardia spp.) and seven metazoan parasites (Anisakidae gen. spp., Diphyllobothriidae gen. spp., Ogmogaster heptalineatus, Trematoda indet. type 1, Trematoda indet. type 2, Otostrongylus circumlitus, and Parafilaroides spp.), and morphological and molecular characterizations of adult helminths confirmed identification of following species: Anisakis simplex/A. pegreffi, Pseudoterranova cattani, Contracaecum ogmorhini, and Adenocephalus pacificus. For the first time, the results of the current study show the presence of zoonotic relevant Giardia- and Cryptosporidium-infections in two free-ranging colonies of South American sea lions apart from human settlement. Furthermore, a detailed literature search of previous publications on the endoparasite fauna of South American sea lions was conducted, revealing reports of at least 50 protozoan and metazoan parasite taxa including findings of the current study. Thereby, at least 25 of reported taxa (50%) have been recorded to bear zoonotic potential. The present study illustrates a successful application of non-invasive screening methods and their applicability in the field of marine mammal parasitology, bringing new insights into the endogenous parasite fauna of South American sea lions in Southern Chile, including anthropozoonotic protozoan and metazoan taxa.
Hou Z., Peng Z., Ning Y., Liu D., Chai H., Jiang G. (2020): An initial coprological survey of parasitic fauna in the wild Amur leopard (Panthera pardus orientalis). Integrative Zoology 15: 375-384.
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The Amur leopard, one of nine recently recognized subspecies of leopard, is still the most threatened by a stochastic procession of extinction. Evaluation of the potential danger to the conservation of the Amur leopard originating from disease urgently needs to be studied. Unfortunately, research on the potential risk to Amur leopards caused by disease is rare. In terms of parasitic diseases that affect this species, even basic data for parasitic fauna are absent. The aim of this study is to acquire this knowledge to improve the general understanding of Amur leopard parasites. Seven parasite species, including 3 nematodes (Toxocara cati, a capillarid-type parasite, and a Metastrongyloidea-type parasite), 2 cestodes (Spirometra sp. and Taenia sp.), 1 trematode (Paragonimus sp.), and 1 protozoan (Cystoisospora felis), were found in this research. Toxocara cati occurred most frequently, followed by Spirometra sp.
Bandoo R. A., Bautista J., Lund M., Newkirk E., Squires J., Varsani A., Kraberger S. (2021): Identification of novel circovirus and anelloviruses from wolverines using a non-invasive faecal sampling approach. Infection, Genetics and Evolution 93: 104914.
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Viruses in the families Circoviridae and Anelloviridae have circular single-stranded DNA genomes and have been identified in various animal species. Some members of the Circoviridae family such as beak and feather disease and porcine circovirus have been found to cause disease in their host animals. Anelloviruses on the other hand have not been identified to cause disease in their hosts but are highly prevalent in mammalian species. Using a non-invasive sampling approach, we identified novel circovirus and anelloviruses from faecal samples of wolverines dwelling in Montana, USA. Wolverines are forest carnivores that feed on a wide variety of carrion and other prey species, and they occupy diverse habitats across northern Europe to North America. Little is known about viruses associated with wild wolverines. Our investigation of the faecal samples resulted in the identification of a novel circovirus from three out of four wolverine samples, two collected in 2018 and one in 2019. Comparison with other circoviruses shows it is most closely related to a porcine circovirus 3, sharing ~69% identity. Additionally, three anellovirus genomes were recovered from two wolverine faecal samples which share 68–69% ORF1 nucleotide similarity with an anellovirus from another mustelid species, pine martens. Here we identify novel single-stranded DNA viruses associated with wolverine and open up new avenues for research.
Brealey J. C., Leitão H. G., Hofstede T., Kalthoff D. C., Guschanski K. (2021): The oral microbiota of wild bears in Sweden reflects the history of antibiotic use by humans. Current Biology 31: 4650-4658.
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Following the advent of industrial-scale antibiotic production in the 1940s, antimicrobial resistance (AMR) has been on the rise and now poses a major global health threat in terms of mortality, morbidity, and economic burden. Because AMR can be exchanged between humans, livestock, and wildlife, wild animals can be used as indicators of human-associated AMR contamination of the environment. However, AMR is a normal function of natural environments and is present in host-associated microbiomes, which makes it challenging to distinguish between anthropogenic and natural sources. One way to overcome this difficulty is to use historical samples that span the period from before the mass production of antibiotics to today. We used shotgun metagenomic sequencing of dental calculus, the calcified form of the oral microbial biofilm, to determine the abundance and repertoire of AMR genes in the oral microbiome of Swedish brown bears collected over the last 180 years. Our temporal metagenomics approach allowed us to establish a baseline of natural AMR in the pre-antibiotics era and to quantify a significant increase in total AMR load and diversity of AMR genes that is consistent with patterns of national human antibiotic use. We also demonstrated a significant decrease in total AMR load in bears in the last two decades, which coincides with Swedish strategies to mitigate AMR. Our study suggests that public health policies can be effective in limiting human-associated AMR contamination of the environment and wildlife.
Ferreira S. C., Veiga M. M., Hofer H., East M. L., Czirják G. Á. (2021): Noninvasively measured immune responses reflect current parasite infections in a wild carnivore and are linked to longevity. Ecology and Evolution 11: 7685-7699.
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Host immune defenses are important components of host–parasite interactions that affect the outcome of infection and may have fitness consequences for hosts when increased allocation of resources to immune responses undermines other essential life processes. Research on host–parasite interactions in large free-ranging wild mammals is currently hampered by a lack of verified noninvasive assays. We successfully adapted existing assays to measure innate and adaptive immune responses produced by the gastrointestinal mucosa in spotted hyena (Crocuta crocuta) feces, including enzyme-linked immunosorbent assays (ELISAs), to quantify fecal immunoglobulins (total IgA, total IgG) and total fecal O-linked oligosaccharides (mucin). We investigated the effect of infection load by an energetically costly hookworm (Ancylostoma), parasite richness, host age, sex, year of sampling, and clan membership on immune responses and asked whether high investment in immune responses during early life affects longevity in individually known spotted hyenas in the Serengeti National Park, Tanzania. Fecal concentrations of IgA, IgG, and mucin increased with Ancylostoma egg load and were higher in juveniles than in adults. Females had higher mucin concentrations than males. Juvenile females had higher IgG concentrations than juvenile males, whereas adult females had lower IgG concentrations than adult males. High IgA concentrations during the first year of life were linked to reduced longevity after controlling for age at sampling and Ancylostoma egg load. Our study demonstrates that the use of noninvasive methods can increase knowledge on the complex relationship between gastrointestinal parasites and host local immune responses in wild large mammals and reveal fitness-relevant effects of these responses.
Murray M. H., Fidino M., Lehrer E. W., Simonis J. L., Magle S. B. (2021): A multi‐state occupancy model to non‐invasively monitor visible signs of wildlife health with camera traps that accounts for image quality. Journal of Animal Ecology 90: 1973-1984.
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Camera traps are an increasingly popular tool to monitor wildlife distributions. However, traditional analytical approaches to camera trap data are difficult to apply to visible wildlife characteristics in single images, such as infection status. Several parasites produce visible signs of infection that could be sampled via camera traps. Sarcoptic mange (Sarcoptes scabiei) is an ideal disease to study using cameras because it results in visible hair loss and affects a broad host range. Here, we developed a multi‐state occupancy model to estimate the occurrence of mange in coyotes (Canis latrans) across an urban gradient. This model incorporates a secondary detection function for apparent by‐image infection status to provide detection corrected estimates of mange occurrence. We analyzed a multi‐year camera trap dataset in Chicago, Illinois, USA to test whether the apparent occurrence of sarcoptic mange in coyotes (Canis latrans) increases with urbanization or varies through time. We documented visible signs consistent with current or recovering mange infection and variables we hypothesized would improve mange detection: the proportion of the coyote in frame, image blur, and whether the image was in color. We were more likely to detect coyotes with mange in images that were less blurry, in color, and if a greater proportion of the coyote was visible. Mangy coyote occupancy was significantly higher in urban developed areas with low housing density and higher canopy cover whereas coyote occupancy, mangy or otherwise, decreased with urbanization. By incorporating image quality into our by‐image detection function, we provide a robust method to non‐invasively survey visible aspects of wildlife health with camera traps. Apparently mangy coyotes were associated with low‐density forested neighborhoods, which may offer vegetated areas while containing sources of anthropogenic resources. This association may contribute to human‐wildlife conflict and reinforces posited relationships between infection risk and habitat use. More generally, our model could provide detection‐corrected occupancy estimates of visible characteristics that vary by image such as body condition or injuries.
Ning S., Lu X., Zhao M., Wang X., Yang S., Shen Q., Wang H., Zhang W. (2021): Virome in fecal samples from wild giant pandas (Ailuropoda melanoleuca). Frontiers in Veterinary Science 8: 767494.
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The giant panda (Ailuropoda melanoleuca) is one of the most endangered mammals in the world; anthropogenic habitat loss and poaching still threaten the survival of wild pandas. Viral infection has become one of the potential threats to the health of these animals, but the available information related to these infections is still limited. In order to detect possible vertebrate viruses, the virome in the fecal samples of seven wild giant pandas from Qinling Mountains was investigated by using the method of viral metagenomics. From the fecal virome of wild giant pandas, we determined six nearly complete genomes belonging to the order Picornavirales, two of which may be qualified as a novel virus family or genus. In addition, four complete genomes belonging to the Genomoviridae family were also fully characterized. This virological investigation has increased our understanding of the gut viral community in giant pandas. Whether these viruses detected in fecal samples can really infect giant panda needs further research.
Rooney T. A., Eshar D., Lee C., Weese J. S. (2021): Characterization of faecal and caecal microbiota of free-ranging black-tailed prairie dogs (Cynomys ludovicianus) using high-throughput sequencing of the V4 region of the 16S rRNA gene. Conservation Physiology 9: coab042.
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Black-tailed prairie dogs (Cynomys ludovicianus) are keystone species within their grassland ecosystems; their population stability affects a multitude of other species. The goals of this study were to explore, describe and compare the bacterial communities in caecal and hard faecal samples from free-ranging black-tailed prairie dogs (n = 36) from KS, USA, using high-throughput sequencing of the V4 region of the 16S rRNA gene and to compare sex and geographic locations. A total of 22 paired faecal and caecal samples were collected post-mortem from free-ranging black-tailed prairie dogs from 5 different geographical locations. The results revealed that the microbiota of both faecal and caecal samples were dominated by the phylum Firmicutes (genera belonging to the Clostridiales order). There was significantly greater richness in faecal compared with caecal samples. There were significant differences between the 5 different geographic regions (P < 0.001), specifically in the relative abundances of genera. There were differences in rare members of the microbiome between faecal samples from male and female prairie dogs but with no significant impact on overall community structure. This study provides novel data and expands our knowledge about the gastrointestinal microbiome composition of free-ranging black-tailed prairie dogs, which has potential to inform conservation efforts and improve their captive management.
Rousseau J., Nakamura M., Rio-Maior H., Álvares F., Choquet R., Madeira de Carvalho L., Godinho R., Santos N. (2021): Non-invasive molecular survey of sarcoptic mange in wildlife: diagnostic performance in wolf faecal samples evaluated by multi-event capture–recapture models. Pathogens 10: 243.
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Sarcoptic mange is globally enzootic, and non-invasive methods with high diagnostic specificity for its surveillance in wildlife are lacking. We describe the molecular detection of Sarcoptes scabiei in non-invasively collected faecal samples, targeting the 16S rDNA gene. We applied this method to 843 Iberian wolf Canis lupus signatus faecal samples collected in north-western Portugal (2006–2018). We further integrated this with serological data (61 samples from wolf and 20 from red fox Vulpes vulpes, 1997–2019) in multi-event capture–recapture models. The mean predicted prevalence by the molecular analysis of wolf faecal samples from 2006–2018 was 7.2% (CI95 5.0–9.4%; range: 2.6–11.7%), highest in 2009. The mean predicted seroprevalence in wolves was 24.5% (CI95 18.5–30.6%; range: 13.0–55.0%), peaking in 2006–2009. Multi-event capture–recapture models estimated 100% diagnostic specificity and moderate diagnostic sensitivity (30.0%, CI95 14.0–53.0%) for the molecular method. Mange-infected individually identified wolves showed a tendency for higher mortality versus uninfected wolves (ΔMortality 0.150, CI95 −0.165–0.458). Long-term serology data highlights the endemicity of sarcoptic mange in wild canids but uncovers multi-year epidemics. This study developed and evaluated a novel method for surveying sarcoptic mange in wildlife populations by the molecular detection of S. scabiei in faecal samples, which stands out for its high specificity and non-invasive character.
Dias D., Fonseca C., Mendo S., Caetano T. (2022): First characterization of the faecal resistome of eurasian otter (Lutra lutra), a sentinel species for aquatic environments. Chemosphere 309: 136644.
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Antimicrobial resistance (AMR) is a global health concern. Nowadays, antibiotic resistance genes (ARGs) are considered emerging pollutants. This study followed the One Health framework, in which AMR surveillance in the environment, including in wild animals, is advisable to mitigate this problem. Here we investigated AMR associated with Eurasian otter, a semi-aquatic mammal considered an indicator of freshwater health. To do so, otter’s faecal resistome was characterized by a high-throughput qPCR array. This technique has a high-capacity of ARGs profiling. Additionally, we have assessed the antimicrobial susceptibility of two indicator bacteria, E. coli and Enterococcus spp, isolated from otter spraints and interpreted the results according to clinical and epidemiological cut-offs (ECOFFs).
Brandell, E. E., Jackson, M. K., Cross, P. C., Piaggio, A. J., Taylor, D. R., Smith, D. W., Boufana B., Stahler D. R., Hudson P. J. (2022): Evaluating noninvasive methods for estimating cestode prevalence in a wild carnivore population. Plos One 17: e0277420.
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Helminth infections are cryptic and can be difficult to study in wildlife species. Helminth research in wildlife hosts has historically required invasive animal handling and necropsy, while results from noninvasive parasite research, like scat analysis, may not be possible at the helminth species or individual host levels. To increase the utility of noninvasive sampling, individual hosts can be identified by applying molecular methods. This allows for longitudinal sampling of known hosts and can be paired with individual-level covariates. Here we evaluate a combination of methods and existing long-term monitoring data to identify patterns of cestode infections in gray wolves in Yellowstone National Park. Our goals were: (1) Identify the species and apparent prevalence of cestodes infecting Yellowstone wolves; (2) Assess the relationships between wolf biological and social characteristics and cestode infections; (3) Examine how wolf samples were affected by environmental conditions with respect to the success of individual genotyping. We collected over 200 wolf scats from 2018–2020 and conducted laboratory analyses including individual wolf genotyping, sex identification, cestode identification, and fecal glucocorticoid measurements. Wolf genotyping success rate was 45%, which was higher in the winter but decreased with higher precipitation and as more time elapsed between scat deposit and collection. One cestode species was detected in 28% of all fecal samples, and 38% of known individuals. The most common infection was Echinococcus granulosus sensu lato (primarily E. canadensis). Adult wolves had 4x greater odds of having a cestode infection than pups, as well as wolves sampled in the winter. Our methods provide an alternative approach to estimate cestode prevalence and to linking parasites to known individuals in a wild host system, but may be most useful when employed in existing study systems and when field collections are designed to minimize the time between fecal deposition and collection.
Tian J., Sanganyado E., Wang Z., Kong Z., Han J., Lu Z., Liu W. (2022): Spotted seals (Phoca largha) harbor unique gut microbiota shaped by their host habitat. Science of The Total Environment 832: 155015.
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Assessing the structure and composition of gut microbiota of sentinel species such as spotted seals (Phoca largha) is a potential tool for assessing the health of the marine mammals and their habitats. However, the link between the host microbiome and their habitat is poorly understood. In this study, microbial communities in the habitat (sea ice and water) and marine mammalian host (fecal matter from P. largha) were evaluated in samples obtained from the Liaodong Bay, China during population aggregation period. Results from high-throughput sequencing showed that the bacterial communities in P. largha fecal matter were less rich and diverse compared to those from the water and ice samples. Significant differences in the composition and function of bacterial communities were also found among the water, ice, and fecal samples, in which sample type and sampling site had the greatest impact on composition and function variations, respectively. Several potential pathogenic bacteria and bacteria with functions associated with human disease were significantly enhanced in the communities of P. largha feces compared to those of surrounding environments. The ratios of environmental microorganisms sourced from the P. largha fecal matter were estimated. The results showed that certain bacteria in P. largha-inhabited fecal matter were associated with sea ice and had specific antibiotic resistance and infectious capacity. These findings provide critical data for monitoring the health of marine mammals and their habitats, which is essential for predicting the impact of anthropogenic disturbances on marine ecosystems.
Barroso P., Palencia P. (2023): Camera traps reveal a high prevalence of sarcoptic mange in red foxes from northern Spain. Research in Veterinary Science 166: 105098.
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The mite Sarcoptes scabiei affects numerous mammal species causing the sarcoptic mange, a widespread disease with relevance for wildlife conservation, welfare, and management. The red fox (Vulpes vulpes) could become infested by direct and indirect routes leading to external skin lesions potentially recognizable by devices such as camera traps (CTs). In the present study, 86 randomly placed CTs were used to investigate the apparent prevalence and severity of S. scabiei in a red fox population from northern Spain. Their potential environmental and population-related drivers were also assessed. A total of 341 independent encounters were examined to visually identify mange-compatible lesions. The apparent prevalence was 19.16% (confidence interval (CI) 95%: 15.08–23.80) of which 82.81% (CI95%: 71.33–91.10) were severe. Our results revealed that habitat attributes such as lower altitudes, higher coverage of water-linked habitats and woodland predominance, were significant predictors of the apparent risk of mange. The models also suggested that the apparent prevalence of mange was associated with poor body condition and elevated frequencies of spatial coincidence among fox encounters, which facilitates indirect transmission. Interestingly, we did not observe mange-compatible lesions in other sympatric wild species (>15,000 encounters examined). This could be explained by the mite’s host specificity and the low probability that these other potential hosts use sites where transmission among foxes usually occurs, such as dens. This study illustrates how camera trapping can be used as an interesting tool for the surveillance of wildlife diseases, thus overcoming the logistic constraints derived from direct sampling and allowing the early detection and better management of pathogens in the riskiest areas.
Herrero-García G., Barroso P., Dashti A., González-Barrio D., Naves J., Fernández-Gil A., Ugarte-Ruiz M., Pérez-Sancho M., Royo L. J., Carmena D., De Miguel A. (2024): Non-invasive surveillance of shared pathogens in the Eurasian brown bear (Ursus arctos) human interface. One Health 18: 100746.
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Multi-host communities are perfect scenarios for the emergence and spread of pathogens, threatening the recovery of endangered, isolated, or inbred populations, such as the brown bear (Ursus arctos) in northwestern Spain. The population recovery in recent years has forced bears to occupy highly anthropized areas, increasing their interaction with human and domestic animals, with potential consequences for global health. During 2022–2023 a survey of parasites, bacteria and viruses shared between wildlife, domestic animals and humans was performed in this population using non-invasive surveillance, i.e., bear fecal samples (n = 73) and sponge-based sampling of trees (n = 42; 14 rubbed trees and 28 control trees). Pathogen detection rates were defined as the percentage of qPCR or culture-positive samples. Generalized linear models were fitted to assess their relationship with environmental variables including dispersion of the human population, and percentage of agricultural and periurban habitats in a 6 km-buffer around each sample. Canine Adenovirus type 1 (45.2%), Giardia spp. (15.1%), Salmonella spp. (12.3%), and extended-spectrum-beta-lactamases (ESBL) Escherichia coli (1.4%) were identified in fecal samples. In contrast, only five sponges from three rubbed and two control trees resulted positive to E. coli (14.3%). The results suggest that several pathogens are common in the Cantabrian brown bear population and that anthropization of the territory modulates their prevalence and richness. The effective design of management programs for bear conservation will require a one-health approach, in which genetic analysis of non-invasive samples can be key tools for the sanitary surveillance at the wildlife-livestock-human interface.
CETACEANS
Acevedo‐Whitehouse K., Rocha‐Gosselin A., Gendron D. (2010): A novel non‐invasive tool for disease surveillance of free‐ranging whales and its relevance to conservation programs. Animal Conservation 13: 217-225.
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The numbers of potentially pathogenic microorganisms that have been isolated from stranded cetaceans in the last three decades underscore the urgent need for methods of detection of microorganisms that might cause significant disease and increase the likelihood of population declines. We have designed and implemented two non-invasive techniques for the collection of exhaled breath condensate (blow) from free-ranging whales and demonstrated their suitability for the detection of respiratory bacteria. We successfully collected 22 individual blow samples from eight cetacean species. Using well-established molecular techniques we detected three bacterial genera (Haemophilus, Streptococcus and Staphylococcus). Haemophilus spp. was detected in fin whale Balaenoptera physalus, sperm whale Physeter macrocephalus, humpback whale Megaptera novaeangliae and gray whale Eschrichtius robustus blows, while unidentified β-hemolytic streptococci and Staphylococcus aureus were detected in gray whale and blue whale Balaenoptera musculus blows. The detection limit of the test was determined as 1 CFU mL−1. None of the identified bacteria were found in environmental (control) samples, suggesting that their presence in the blows was genuine and not due to inadvertent contamination. While the population-level relevance of these bacteria is as yet unclear and it is possible that they are commensal microorganisms, S. aureus has been identified previously as a high-risk pathogen to cetacean health, and streptococci have increasingly been associated with cetacean mortality events. We suggest that future cetacean monitoring programs of vulnerable or threatened species include blow sampling as a means to determine the prevalence of the respiratory bacteria in the populations and monitor spatiotemporal fluctuations as indicators of changes in cetacean health.
Lima N., Rogers T., Acevedo‐Whitehouse K., Brown M. V. (2012): Temporal stability and species specificity in bacteria associated with the bottlenose dolphins respiratory system. Environmental Microbiology Reports 4: 89-96.
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We demonstrate that the exhaled breath condensate, or ‘blow’, from marine mammals can be used to examine respiratory associated microbial communities using non-invasive sampling methods. Blow samples from two species of bottlenose dolphin, Tursiops truncatus and T. aduncus, along with hybrid offspring, were examined using molecular microbial ecology methods. A temporal analysis revealed that microbial community structure of each individual remained distinct from other individuals over a two-month period, indicting strong host specificity. The taxonomic composition of samples, based on pyrosequencing of the V1–V3 regions of the 16S rRNA gene, from 24 healthy individuals was dominated by the Cardiobacteraceae lineage of Gammaproteobacteria, comprising on average 52% of sequences in all samples. Sequences in this taxa were associated with novel clades that contain only sequences from dolphin respiratory tracts. Other genera that likely form part of the core biota include the Saccharospirillaceae (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria), Hydrogenimonaceae (Epsilonproteobacteria), Halotalea (Gammaproteobacteria), Aquimarina (Flavobacteria) and Helococcus (Clostridia). Significant differences between samples from different species were observed only at the species/ strain level, driven by the relative contributions of strains from the most common phylogenetic lineages. Analysis of communities associated with hybrid animals provides tentative evidence for a paternal role in community assembly. Clear overlap was observed with data collected by capture and swabbing of bottlenose dolphins blowholes, indicating this method provides a novel non-invasive alternative to monitoring marine mammal population health.
Cumeras R., Cheung W. H., Gulland F., Goley D., Davis C. E. (2014): Chemical analysis of whale breath volatiles: a case study for non-invasive field health diagnostics of marine mammals. Metabolites 4: 790-806.
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We explored the feasibility of collecting exhaled breath from a moribund gray whale (Eschrichtius robustus) for potential non-invasive health monitoring of marine mammals. Biogenic volatile organic compound (VOC) profiling is a relatively new field of research, in which the chemical composition of breath is used to non-invasively assess the health and physiological processes on-going within an animal or human. In this study, two telescopic sampling poles were designed and tested with the primary aim of collecting whale breath exhalations (WBEs). Once the WBEs were successfully collected, they were immediately transferred onto a stable matrix sorbent through a custom manifold system. A total of two large volume WBEs were successfully captured and pre-concentrated onto two Tenax®-TA traps (one exhalation per trap). The samples were then returned to the laboratory where they were analyzed using solid phase micro extraction (SPME) and gas chromatography/mass spectrometry (GC/MS). A total of 70 chemicals were identified (58 positively identified) in the whale breath samples. These chemicals were also matched against a database of VOCs found in humans, and 44% of chemicals found in the whale breath are also released by healthy humans. The exhaled gray whale breath showed a rich diversity of chemicals, indicating the analysis of whale breath exhalations is a promising new field of research.
Wu Q., Conway J., Phillips K. M., Stolen M., Durden W. N., Fauquier D., McFee W. E., Schwacke L. (2016): Detection of Brucella spp. in bottlenose dolphins Tursiops truncatus by a real-time PCR using blowhole swabs. Diseases of Aquatic Organisms 120: 241-244.
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Blowhole swabs are a simple and non-invasive method for collecting samples from cetaceans and can be used for screening large numbers of animals in the field. This study reports a real-time PCR assay for the detection of Brucella spp. using blowhole swab samples from bottlenose dolphins Tursiops truncatus stranded in the coastal region of Virginia, South Carolina and northern Florida, USA, between 2013 and 2015. We used real-time PCR results on lung samples from the same dolphins in order to estimate the relative sensitivity and specificity of real-time PCR of blowhole swabs. Brucella DNA was detected in lung tissue of 22% (18/81) and in blowhole swabs of 21% (17/81) of the sampled dolphins. The relative sensitivity and specificity of real-time PCR on blowhole swabs as compared to the real-time PCR on lung samples was 94% (17/18) and 100% (63/63), respectively. These results indicate that real-time PCR on blowhole swabs may be used as a non-invasive test for rapid detection of Brucella spp. in the respiratory tract of dolphins. To our knowledge, this is the first report on the use of blowhole swabs for detection of bacterial pathogens by real-time PCR in bottlenose dolphins.
Apprill A., Miller C. A., Moore M. J., Durban J. W., Fearnbach H., Barrett-Lennard L. G. (2017): Extensive core microbiome in drone-captured whale blow supports a framework for health monitoring. mSystems 2: e00119-17.
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The pulmonary system is a common site for bacterial infections in cetaceans, but very little is known about their respiratory microbiome. We used a small, unmanned hexacopter to collect exhaled breath condensate (blow) from two geographically distinct populations of apparently healthy humpback whales (Megaptera novaeangliae), sampled in the Massachusetts coastal waters off Cape Cod (n = 17) and coastal waters around Vancouver Island (n = 9). Bacterial and archaeal small-subunit rRNA genes were amplified and sequenced from blow samples, including many of sparse volume, as well as seawater and other controls, to characterize the associated microbial community. The blow microbiomes were distinct from the seawater microbiomes and included 25 phylogenetically diverse bacteria common to all sampled whales. This core assemblage comprised on average 36% of the microbiome, making it one of the more consistent animal microbiomes studied to date. The closest phylogenetic relatives of 20 of these core microbes were previously detected in marine mammals, suggesting that this core microbiome assemblage is specialized for marine mammals and may indicate a healthy, noninfected pulmonary system. Pathogen screening was conducted on the microbiomes at the genus level, which showed that all blow and few seawater microbiomes contained relatives of bacterial pathogens; no known cetacean respiratory pathogens were detected in the blow. Overall, the discovery of a shared large core microbiome in humpback whales is an important advancement for health and disease monitoring of this species and of other large whales.
Borras E., Aksenov A. A., Baird M., Novick B., Schivo M., Zamuruyev K. O., Pasamontes A., Parry C., Foutouhi S., Venn-Watson S., Weimer B. C., Davis C. E. (2017): Exhaled breath condensate methods adapted from human studies using longitudinal metabolomics for predicting early health alterations in dolphins. Analytical and Bioanalytical Chemistry 409: 6523-6536.
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Monitoring health conditions is essential to detect early asymptomatic stages of a disease. To achieve this, blood, urine and breath samples are commonly used as a routine clinical diagnostic. These samples offer the opportunity to detect specific metabolites related to diseases and provide a better understanding of their development. Although blood samples are commonly used routinely to monitor health, the implementation of a relatively noninvasive technique, such as exhaled breath condensate (EBC) analysis, may further benefit the well-being of both humans and other animals. EBC analysis can be used to track possible physical or biochemical alterations caused by common diseases of the bottlenose dolphin (Tursiops truncatus), such as infections or inflammatory-mediated processes. We have used an untargeted metabolomic method with liquid chromatography–mass spectrometry analysis of EBC samples to determine biomarkers related to disease development. In this study, five dolphins under human care were followed up for 1 year. We collected paired blood, physical examination information, and EBC samples. We then statistically correlated this information to predict specific health alterations. Three dolphins provided promising case study information about biomarkers related to cutaneous infections, respiratory infections, dental disease, or hormonal changes (pregnancy). The use of complementary liquid chromatography platforms, with hydrophilic interaction chromatography and reverse-phased columns, allowed us to detect a wide spectrum of EBC biomarker compounds that could be related to these health alterations. Moreover, these two analytical techniques not only provided complementary metabolite information but in both cases they also provided promising diagnostic information for these health conditions.
Pirotta V., Smith A., Ostrowski M., Russell D., Jonsen I. D., Grech A., Harcourt R. (2017): An economical custom-built drone for assessing whale health. Frontiers in Marine Science 4: 425.
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Drones or Unmanned Aerial Vehicles (UAVs) have huge potential to improve the safety and efficiency of sample collection from wild animals under logistically challenging circumstances. Here we present a method for surveying population health that uses UAVs to sample respiratory vapor, ‘whale blow,’ exhaled by free-swimming humpback whales (Megaptera novaeangliae), and coupled this with amplification and sequencing of respiratory tract microbiota. We developed a low-cost multirotor UAV incorporating a sterile petri dish with a remotely operated ‘blow’ to sample whale blow with minimal disturbance to the whales. This design addressed several sampling challenges: accessibility; safety; cost, and critically, minimized the collection of atmospheric and seawater microbiota and other potential sources of sample contamination. We collected 59 samples of blow from northward migrating humpback whales off Sydney, Australia and used high throughput sequencing of bacterial ribosomal gene markers to identify putative respiratory tract microbiota. Model-based comparisons with seawater and drone-captured air demonstrated that our system minimized external sources of contamination and successfully captured sufficient material to identify whale blow-specific microbial taxa. Whale-specific taxa included species and genera previously associated with the respiratory tracts or oral cavities of mammals (e.g., Pseudomonas, Clostridia, Cardiobacterium), as well as species previously isolated from dolphin or killer whale blowholes (Corynebacteria, others). Many examples of exogenous marine species were identified, including Tenacibaculum and Psychrobacter spp. that have been associated with the skin microbiota of marine mammals and fish and may include pathogens. This information provides a baseline of respiratory tract microbiota profiles of contemporary whale health. Customized UAVs are a promising new tool for marine megafauna research and may have broad application in cost-effective monitoring and management of whale populations worldwide.
Raverty S. A., Rhodes L. D., Zabek E., Eshghi A., Cameron C. E., Hanson M. B., Schroeder J. P. (2017): Respiratory microbiome of endangered southern resident killer whales and microbiota of surrounding sea surface microlayer in the Eastern North Pacific. Scientific Reports 7: 394.
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In the Salish Sea, the endangered Southern Resident Killer Whale (SRKW) is a high trophic indicator of ecosystem health. Three major threats have been identified for this population: reduced prey availability, anthropogenic contaminants, and marine vessel disturbances. These perturbations can culminate in significant morbidity and mortality, usually associated with secondary infections that have a predilection to the respiratory system. To characterize the composition of the respiratory microbiota and identify recognized pathogens of SRKW, exhaled breath samples were collected between 2006–2009 and analyzed for bacteria, fungi and viruses using (1) culture-dependent, targeted PCR-based methodologies and (2) taxonomically broad, non-culture dependent PCR-based methodologies. Results were compared with sea surface microlayer (SML) samples to characterize the respective microbial constituents. An array of bacteria and fungi in breath and SML samples were identified, as well as microorganisms that exhibited resistance to multiple antimicrobial agents. The SML microbes and respiratory microbiota carry a pathogenic risk which we propose as an additional, fourth putative stressor (pathogens), which may adversely impact the endangered SRKW population.
Flores-Cascante L., Gómez-Gutiérrez J., Del Prado M. D. C. G., Gendron D. (2019): Helminth load in feces of free-ranging blue and fin whales from the Gulf of California. Acta Parasitologica 64: 625-637.
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This is the first worldwide systematic and quantitative study to count and identify helminth parasites from 100 blue and 44 fin whale fecal samples collected in the Gulf of California during winter (1993–2014). Blue and fin whale feces had similar prevalence of adult acanthocephalans (Bolbosoma sp.) in feces (18.2% and 14.6%, respectively), but blue whales had significantly higher helminth egg prevalence in feces (100%) and mean intensity (443 ± 318 eggs/g) compared to fin whales (61%, 252 ± 327 eggs/g). Diphyllobothrium sp. eggs were identified in blue whale feces and Diphyllobothridae, Ogmogaster sp. and Crassicauda sp. eggs were identified in fin whale feces. We tested the hypothesis that egg intensity in blue whale’s feces varies as a function of age class, reproductive status, sex, preservation and sampling years using a Generalized Linear Model. This model explained 61% of the variance in the helminth egg intensity, but it was not significant. Eighteen blue whale individuals were resampled over time without significant difference between consecutive samples. Thus, all individual blue whales that migrate to the Gulf of California during winter are permanently parasitized with helminths, while the resident fin whales showed lower prevalence and intensity. This helminth load difference is likely due to their different diets during summer–fall, when blue whales feed on other krill species in the California Current System and fin whales shift to school fish prey types in the Gulf of California.
Horton T. W., Hauser N., Cassel S., Klaus K. F., Fettermann T., Key N. (2019): Doctor Drone: non-invasive measurement of humpback whale vital signs using unoccupied aerial system infrared thermography. Frontiers in Marine Science 6: 466.
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Measuring and monitoring the behavior and biomedical condition of free-ranging whales remains a fundamental challenge in cetacean science and conservation. Advances in unoccupied aerial systems (UAS) and infrared thermography (IRT) create unprecedented opportunities to fill these knowledge gaps and advance our understanding of how cetaceans interact with the environment. Here, we show that non-invasive UAS-IRT systems, deployed from shore-based positions in a humpback whale (Megaptera novaeangliae) calving ground, can be used to document rarely observed whale behaviors and to quantify biomedical vital signs, including blowhole and dorsal fin skin temperature, respiration rate, and heart rate. Our findings demonstrate: (1) prolonged (>3 h) logging behavior by a mother-calf pair located ∼550 m offshore; (2) that the calf’s respiration rate (∼3 breaths per minute) was six times higher than its mother’s (∼0.5 breaths per minute); (3) that the calf’s blowholes were ∼1.55°C warmer than adjacent ocean water and that the mother’s blowholes were ∼2.16°C warmer than adjacent ocean water; (4) that the mother’s dorsal fin included four infrared (IR) hot-spots, each separated by ∼20 cm in horizontal distance, that ranged between 1 and 2°C warmer than adjacent ocean water; (5) a significant (p <<0.05; wavelet analysis) temporal cyclicity in the hottest of the mother’s dorsal fin hot-spots consistent with cardiovascular blood flow pumped at an apneic heart rate of ∼9.3 beats per minute. Despite these novel results, there remain several key limitations to UAS-IRT, including its: sensitivity to environmental conditions and animal behavior; equipment costs and associated risks; potential regulatory restrictions; time-intensive nature of IR data processing; factors that can impact data quality, such as imaging angle and sensor accuracy. Future opportunities created by the UAS-IRT results we report center on the potential to couple non-invasive behavioral and physiological monitoring tools, quantify cetacean response to prolonged environmental change and acute disturbances, and extend UAS-IRT applications to cover a wider range of environmental and behavioral contexts. Considering the small sample size of the dataset we report, application of UAS-IRT to live-stranded and captive cetaceans, where environmental and cetacean conditions can be independently measured, is of paramount importance.
Vendl C., Slavich E., Nelson T., Acevedo‐Whitehouse K., Montgomery K., Ferrari B., Thomas T., Rogers T. (2020): Does sociality drive diversity and composition of airway microbiota in cetaceans?. Environmental Microbiology Reports 12: 324-333.
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The number of social contacts of mammals is positively correlated with the diversity of their gut microbes. There is some evidence that sociality also affects microbes in the respiratory tract. We tested whether the airway microbiota of cetacean species differ depending on the whales’ level of sociality. We sampled the blow of blue (Balaenoptera musculus), grey (Eschrichtius robustus), humpback (Megaptera novaeangliae) and long-finned pilot whales (PWs) (Globicephala melas) and analysed the blow microbiota by barcode tag sequencing targeting the V4 region of the bacterial 16S rRNA gene. Humpback whales (HWs) show higher levels of sociality than blue (BW) and grey (GW), while PWs are the most gregarious among the four species. The blow samples of the HWs showed the highest richness and diversity. HWs were also the only species with a species-specific clustering of their microbial community composition and a relatively large number of core taxa. Therefore, we conclude that it cannot be sociality alone shaping the diversity and composition of airway microbiota. We suggest the whale species’ lung volume and size of the plume of exhaled air as an additional factor impacting the transmission potential of blow microbiota from one individual whale to another.
Aoki K., Watanabe Y., Inamori D., Funasaka N., Sakamoto K. Q. (2021): Towards non-invasive heart rate monitoring in free-ranging cetaceans: a unipolar suction cup tag measured the heart rate of trained Risso’s dolphins. Philosophical Transactions of the Royal Society B 376: 20200225.
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Heart rate monitoring in free-ranging cetaceans to understand their behavioural ecology and diving physiology is challenging. Here, we developed a simple, non-invasive method to monitor the heart rate of cetaceans in the field using an electrocardiogram-measuring device and a single suction cup equipped with an electrode. The unipolar suction cup was placed on the left lateral body surface behind the pectoral fin of Risso’s dolphins (Grampus griseus) and a false killer whale (Pseudorca crassidens) in captivity; their heart rate was successfully monitored. We observed large heart rate oscillations corresponding to respiration in the motionless whales during surfacing (a false killer whale, mean 47 bpm, range 20–75 bpm; Risso’s dolphins, mean ± s.d. 61 ± 15 bpm, range 28–120 bpm, n = 4 individuals), which was consistent with the sinus arrhythmia pattern (eupneic tachycardia and apneic bradycardia) observed in other cetaceans. Immediately after respiration, the heart rate rapidly increased to approximately twice that observed prior to the breath. Heart rate then gradually decreased at around 20–50 s and remained relatively constant until the next breath. Furthermore, we successfully monitored the heart rate of a free-swimming Risso’s dolphin. The all-in-one suction cup device is feasible for field use without restraining animals and is helpful in further understanding the diving physiology of free-ranging cetaceans.
Atkinson S., Rogan A., Baker C. S., Dagdag R., Redlinger M., Polinski J., Urban J., Sremba A., Branson M., Mashburn K., Pallin L. (2021): Genetic, endocrine, and microbiological assessments of blue, humpback and killer whale health using unoccupied aerial systems. Wildlife Society Bulletin 45: 654-669.
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Unoccupied aerial system (UAS) technologies applied to health assessments of large whales can have positive implications for progressive management. We focused on the collection of cetacean respiratory blow samples for endocrine, DNA profiling, microbial metabarcoding, and metagenomics analyses, with the goal of improving management of large whale populations. Blow samples were collected from humpback (Megaptera novaeangliae, n = 109 samples analyzed), blue (Balaenoptera musculus, n = 21 samples analyzed), and killer whale (Orcinus orca, n = 1 sample analyzed) species, as well as the responses of the whales to the collection of their blow by UAS. Endocrine analyses were validated for 5 steroid hormones in humpback whales and 4 hormones in blue whales. For DNA profiling, we attempted to extract and amplify nuclear and mitochondrial DNA, resulting in sequencing of mtDNA haplotypes for 54% of samples, identification of sex for 39%, and individual identification by microsatellite genotyping for 17%. The DNA profiles of 2 of the blow samples from humpback whales were matched to a DNA register for this regional population. Metagenomic and microbial metabarcoding classifications found a diverse number of bacteria, eukaryotes, and viruses in humpback whale blow. Although a significant portion of classifications were found in both seawater and blow, several of the most abundant organisms were present only in blow samples, suggesting they are true members of the respiratory microbiome. A comprehensive integration of laboratory-based approaches using noninvasive UAS collection technologies could become an important management tool for health assessments of large cetaceans, especially for species listed as endangered. The addition of individual and population-level health assessments to currently practiced stewardship of large whales, renders them as excellent sentinels of ocean health.
Glaeser S. P., Silva L. M., Prieto R., Silva M. A., Franco A., Kämpfer P., Hermosilla C., Taubert A., Eisenberg T. (2021): A preliminary comparison on faecal microbiomes of free-ranging large baleen (Balaenoptera musculus, B. physalus, B. borealis) and toothed (Physeter macrocephalus) whales. Microbial Ecology 83: 18-33.
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Large baleen and toothed whales play crucial ecological roles in oceans; nonetheless, very little is known about their intestinal microbiomes. Based on striking differences in natural history and thus in feeding behaviours, it can be expected that intestinal microbiomes of large baleen whales and toothed whales are different. To test this hypothesis, the phylogenetic composition of faecal microbiomes was investigated by a 16S rRNA gene amplicon sequence–based approach for Bacteria and Archaea. Faecal samples from free-ranging large whales collected off the Azores Archipelago (Portugal) were used, comprising 13 individual baleen whales (one sei, two blue and ten fin whales) and four sperm whales. The phylogenetic composition of the Bacteria faecal microbiomes of baleen and toothed whales showed no significant differences at the phylum level. However, significant differences were detected at the family and genus levels. Most abundant phyla were Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes and Spirochaeta. Few highly abundant bacterial genera were identified as key taxa with a high contribution to differences among baleen and toothed whales microbiomes. Only few archaeal sequences were detected, primarily Methanomassiliicoccales representing potential methanogenic Archaea. This is the first study that directly compares the faecal bacterial and archaeal microbiomes of free-ranging baleen and toothed whales which represent the two parvorders of Cetacea which members are fully aquatic large mammals which were evolutionary split millions of years ago.
Groch K. R., Blazquez D. N., Marcondes M. C., Santos J., Colosio A., Díaz Delgado J., Catão‐Dias J. L. (2021): Cetacean morbillivirus in Humpback whales’ exhaled breath. Transboundary and Emerging Diseases 68: 1736-1743.
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The humpback whale (HW; Megaptera novaeangliae) population that seasonally resides along the Brazilian coast concentrates in the Abrolhos Bank (Bahia and Espírito Santo states) for breeding during austral winter and spring. Cetacean morbillivirus (CeMV, Paramyxoviridae family) is currently one of the most significant biological threats to cetaceans worldwide with high infection and mortality rates. CeMV is pleiotropic yet it has special tropism for the respiratory, lymphoid and nervous system and is primarily transmitted by the aerogenous route. A new lineage of CeMV, the Guiana dolphin morbillivirus (GDMV), is known to affect cetaceans off Brazil. GDMV was first detected in a Guiana dolphin (Sotalia guianensis) stranded in the Abrolhos Bank region, in 2010. In addition to pathologic examinations on stranded HW, pathogen survey of free-ranging HW may provide valuable insight into the epidemiology of diseases. We hypothesized that HW in the Brazilian breeding ground could be exposed to CeMV. Thus, in the present study, we investigated the presence of CeMV in exhaled breath condensates (EBC) of HW in the Abrolhos Bank. Overall, 73 samples of EBC from 48 groups of HW were collected during the breeding seasons of 2011 (n = 16) and 2012 (n = 57). One sample failed to have the reference gene amplified and was excluded from the study. CeMV was detected by a RT-qPCR method in 2 EBC samples, representing 2 whale groups. Phylogenetic analysis of partial morbillivirus phosphoprotein gene showed 100% homology to GDMV. Our results show that HW in Brazil are infected by CeMV with a relative prevalence of 4.3% (2/47) and demonstrate the suitability of using EBC and RT-qPCR as a non-invasive tool for CeMV survey in free-ranging whales. This pioneer study provides scientific basis for non-invasive CeMV monitoring of HW, suggests HW may play a role in the dynamics of CeMV and raises concern for potential conservation implications for this species.
Segura-Göthlin S., Fernández A., Arbelo M., Felipe-Jiménez I., Colom-Rivero A., Almunia J., Sierra E. (2021): The validation of a non-invasive skin sampling device for detecting cetacean poxvirus. Animals 11: 2814.
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Poxvirus-like lesions are widely used as a potential health indicator in cetaceans, although for this application, corroboration of Poxvirus skin disease is imperative. Aiming to address skin biopsies intrusiveness, a preliminary investigation of a non-invasive skin sampling procedure to molecularly detect CePV-1 in 12 tattoo-like-lesions from two free-ranging stranded cetaceans in the Canary Islands was performed. Skin lesions were brushed with cytology cell samplers (CCSs) and placed into 1.5 mL microcentrifuge tubes with 1 mL of RNAlaterTM Stabilization Solution. For factual comparisons, DNA extractions from sloughed skin obtained with CCS and biopsies from the same lesions were accomplished with DNA Tissue Kit STM (QuickGene, Kurabo, Japan). Moreover, a second DNA extraction from sloughed skin with DNeasyTM Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) was performed to ascertain kit suitability for CCS. Molecular detection of CePV-1 was performed through a real-time PCR. As a result, a 91.7% and 83.3% rates of positivity were obtained with biopsies and CCS through Quickgene, respectively, compared to the rate of 100% using CCS with Qiagen. Accordingly, CCS is a reliable non-invasive sampling device to obtain sufficient genetic material to be analyzed for CePV-1 in tattoo-skin-lesions as well as for other purposes in cetaceans under human care.
Le Net R., Larrat S., Michaud R., Lair S. (2022): Pathological and epidemiological investigation of skin lesions in belugas (Delphinapterus leucas) from the St. Lawrence Estuary, Quebec, Canada. Marine Mammal Science 38: 653-681.
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Following the evaluation of 26,020 photographs collected from 2003 to 2014 as part of a photo-identification program in St. Lawrence Estuary belugas, an atlas of cutaneous anomalies, composed of 18 skin lesions categories (SLCs), is proposed. At least one SLC was present in 51%, 97%, and 94% of neonates, gray, and white belugas, respectively. The most common SLC observed were “single linear fissure” (22%), “single linear scar” (19%), and “ulcer-like lesion” (17%) in neonates, and “rake mark” (77%; 70%), “single linear fissure” (31%; 24%) and “circular depression” (40%; 35%) in gray and white belugas, respectively. Logistic regression modeling revealed significant correlations between temporal and individual variables for most SLCs. Histological evaluation of cutaneous lesions from 111 belugas stranded between 1983 and 2017 were also performed. “Single linear fissure,” “single linear scar,” “crater-like scar,” “rake mark,” and “Morse code lesions” appear to be of traumatic origin. Results from pathological and epidemiological analyses suggest that some of these SLCs, such as “yellow patch,” “circular depression,” and “map depression” are associated with molting. Postnatal molting could account for “ulcer-like lesions” and “single linear fissures” in neonates. Urchin spines were found within “pinhole erosions” and a gamma-herpesvirus was detected by PCR in a wound.
Leslie M. S., Kant L., Perkins-Taylor C., Van Bressem M. F., Minton G., MacDonald D., Christiansen F., Willson M. S., Collins T., Baldwin R., Al Harthi S., Willson A. (2022): Remote and non-invasive quantification of ‘Tattoo Skin Disease-Like’ dermatopathy in endangered Arabian Sea humpback whales using drone photography. Mammalian Biology 102: 1605-1617.
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Arabian Sea humpback whales (Megaptera novaeangliae; ASHW) are listed as Endangered by the International Union for the Conservation of Nature (IUCN). The long-term presence and increased prevalence of tattoo skin disease-like (TSD-L) dermatopathy is a concern for this small non-migratory population. Characterized by irregular or rounded, light gray or whitish cutaneous lesions, this condition resembles tattoo skin disease, caused by cetacean poxviruses. Although the etiological agent and pathogenicity of TSD-L dermatopathy are unknown, previous studies have suggested that it is an indicator of population health. Until now, disease diagnosis had been based on photographs collected from survey vessels. In this study, we describe a novel method of identifying and quantifying TSD-L lesions in ASHW, using drone aerial photography. Aerial photos of the entire dorsum were selected for 18 whales with the same criteria applied for assessing body condition to quantify the percent coverage for each individual. We effectively diagnosed this condition from close-up aerial photos or good-quality photos of the lateral body surface taken from the research vessel in 13 whales. TSD-L dermatopathy coverage ranged from 2.34 to 57.00% and measurements were consistent between photographs of the same whale (SD = 1.86%). Drone aerial photography provided a useful and complimentary approach to identify and quantify TSD-L lesions. Continued monitoring using this non-invasive method should be combined with other population and health monitoring tools to increase our understanding of the characteristics and epidemiology of this condition, and to provide critical information for conservation efforts that ensure the recovery of this endangered population.
Rhodes L. D., Emmons C. K., Wisswaesser G., Wells A. H., Hanson M. B. (2022): Bacterial microbiomes from mucus and breath of southern resident killer whales (Orcinus orca). Conservation Physiology 10: coac014.
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Opportunities to assess odontocete health are restricted due to their limited time at the surface, relatively quick movements and large geographic ranges. For endangered populations such as the southern resident killer whales (SKRWs) of the northeast Pacific Ocean, taking advantage of non-invasive samples such as expelled mucus and exhaled breath is appealing. Over the past 12 years, such samples were collected, providing a chance to analyse and assess their bacterial microbiomes using amplicon sequencing. Based on operational taxonomic units, microbiome communities from SRKW and transient killer whales showed little overlap between mucus, breath and seawater from SRKW habitats and six bacterial phyla were prominent in expelled mucus but not in seawater. Mollicutes and Fusobacteria were common and abundant in mucus, but not in breath or seawater, suggesting these bacterial classes may be normal constituents of the SRKW microbiome. Out of 134 bacterial families detected, 24 were unique to breath and mucus, including higher abundances of Burkholderiaceae, Moraxellaceae and Chitinophagaceae. Although there were multiple bacterial genera in breath or mucus that include pathogenic species (e.g. Campylobacter, Hemophilus, Treponema), the presence of these bacteria is not necessarily evidence of disease or infection. Future emphasis on genotyping mucus samples to the individual animal will allow further assessment in the context of that animal’s history, including body condition index and prior contaminants burden. This study is the first to examine expelled mucus from cetaceans for microbiomes and demonstrates the value of analysing these types of non-invasive samples.
Richard J. T., Schultz K., Goertz C. E., Hobbs R. C., Romano T. A., Sartini B. L. (2022): Evaluating beluga (Delphinapterus leucas) blow samples as a potential diagnostic for immune function gene expression within the respiratory system. Conservation Physiology 10: coac045.
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Evaluating respiratory health is important in the management of cetaceans, which are vulnerable to respiratory diseases. Quantifying the expression of genes related to immune function within the respiratory tract could be a valuable tool for directly assessing respiratory health. Blow (exhale) samples allow DNA analysis, and we hypothesized that RNA could also be isolated from blow samples for gene expression studies of immune function. We evaluated the potential to extract RNA from beluga blow samples and tested whether transcripts associated with immune function could be detected with endpoint polymerase chain reaction. A total of 54 blow samples were collected from clinically healthy aquarium belugas (n = 3), and 15 were collected from wild belugas temporarily restrained for health assessment in Bristol Bay, Alaska (n = 9). Although RNA yield varied widely (range, 0–265.2 ng; mean = 85.8; SD = 71.3), measurable RNA was extracted from 97% of the samples. Extracted RNA was assessed in 1–6 PCR reactions targeting housekeeping genes (Rpl8, Gapdh or ActB) or genes associated with immune function (TNFα, IL-12p40 or Cox-2). Fifty of the aquarium samples (93%) amplified at least one transcript; overall PCR success for housekeeping genes (96/110, 87%) and genes associated with immune function (90/104, 87%) were similarly high. Both RNA yield and overall PCR success (27%) were lower for wild beluga samples, which is most likely due to the reduced forcefulness of the exhale when compared with trained or free-swimming belugas. Overall, the high detection rate with PCR suggests measuring gene expression in blow samples could provide diagnostic information about immune responses within the respiratory tract. While further study is required to determine if quantitative gene expression data from blow samples is associated with disease states, the non-invasive nature of this approach may prove valuable for belugas, which face increasing anthropogenic disturbances.
Lonati G. L., Hynes N., Klymentieva H., Warren A., Zitterbart D., Richter S., Moore M. J., Davies K. T. (2024): Drone-based infrared thermography to measure the intranasal temperature of baleen whales. International Journal of Remote Sensing 46: 148-176.
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Traditional methods for quantifying the internal temperature of marine mammals require handling live animals, which is not practical for free-swimming baleen whales. Developing a less invasive, more repeatable method would significantly improve our understanding of whale health and thermal physiology. Infrared thermography (IRT) devices compatible with remotely piloted aircraft systems (RPAS) have facilitated qualitative assessments of heat signatures from marine mammals at sea, but absolute temperatures derived using this approach are rare. The goal of this study was to develop a precise empirical method for estimating intranasal temperatures of baleen whales using RPAS-based IRT. We conducted controlled field experiments and flights over North Atlantic right whales (Eubalaena glacialis, NARWs) to develop and test the methodology. Two approaches were evaluated to estimate intranasal temperatures from IRT sensor intensities: a three-point empirical line regression calibrated per flight using known-temperature objects and a generalized linear model incorporating environmental variables. Controlled field experiments demonstrated that the former approach had a median bias of −0.6°C (interquartile range: 1.5ºC), while the latter approach had unexplained negative proportional bias with increasing true temperature of the target object. After accounting for bias, the former approach yielded an average intranasal temperature of 26.9 ± 1.7°C for 21 unique NARWs. The anatomy of the mysticete upper respiratory tract and physiological heat conservation strategies may explain why estimates were low compared to internal temperatures measured from baleen whales using other techniques (30–39ºC). Variability within whales was less than ± 2°C, supporting the use of these methods to monitor the health of individuals over time. However, variability among whales was greater (up to 7ºC). Improvements in our understanding of whale physiology and respiratory mechanics and advancements in RPAS-based IRT calibrations could make this technology more reliable for assessing individual body temperatures and monitoring populations in the future.
Russell J. P., Osborn S. D., Herrick K. E., Schmitt T. L., Robeck T. (2024): Infrared thermography of the blowhole as a potential diagnostic tool for health assessment in killer whales (Orcinus orca). Animals 14: 1867.
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Killer whales (Orcinus orca) are experiencing increasing environmental pressures, with some ecotypes being identified as endangered, and the development and validation of non-invasive health assessment tools is critical for assessing the well-being of individuals within these endangered populations. Infrared thermography of the blowhole is a non-contact method of temperature measurement that was recently investigated in killer whales in managed care. Two male killer whales presenting with clinical signs at separate institutions had veterinary clinical health assessments performed, which included infrared thermography of the blowhole as well as concurrent rectal temperature measurement. The current case report is aimed at describing the clinical use of infrared thermography of the blowhole as a method to detect elevated body temperature in two killer whales. Both animals exhibited blowhole temperatures above the previously reported values (36.4 °C and 37.6 °C; the mean in healthy whales is reported to be 34.21 ± 1.47 °C) with concurrently elevated rectal temperatures, as well as clinicopathologic findings consistent with a systemic inflammatory response (e.g., neutrophilia, increased fibrinogen and erythrocyte sedimentation rate, hypoferritinemia). Following veterinary intervention, both animals’ blowhole and rectal temperatures returned to baseline. Infrared thermography of the blowhole represents a promising tool for the identification of pyrexic animals and with further investigation may be considered as part of conservation health assessments for threatened free-ranging populations.
ELEPHANTS
Lynsdale C. L., Dos Santos D. J. F., Hayward A. D., Mar K. U., Htut W., Aung H. H., Soe A. T., Lummaa V. (2015): A standardised faecal collection protocol for intestinal helminth egg counts in Asian elephants, Elephas maximus. International Journal for Parasitology: Parasites and Wildlife 4: 307-315.
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The quantitative assessment of parasite infection is necessary to measure, manage and reduce infection risk in both wild and captive animal populations. Traditional faecal flotation methods which aim to quantify parasite burden, such as the McMaster egg counting technique, are widely used in veterinary medicine, agricultural management and wildlife parasitology. Although many modifications to the McMaster method exist, few account for systematic variation in parasite egg output which may lead to inaccurate estimations of infection intensity through faecal egg counts (FEC). To adapt the McMaster method for use in sampling Asian elephants (Elephas maximus), we tested a number of possible sources of error regarding faecal sampling, focussing on helminth eggs and using a population of over 120 semi-captive elephants distributed across northern Myanmar. These included time of day of defecation, effects of storage in 10% formalin and 10% formol saline and variation in egg distribution between and within faecal boluses. We found no significant difference in the distribution of helminth eggs within faecal matter or for different defecation times, however, storage in formol saline and formalin significantly decreased egg recovery. This is the first study to analyse several collection and storage aspects of a widely-used traditional parasitology method for helminth parasites of E. maximus using known host individuals. We suggest that for the modified McMaster technique, a minimum of one fresh sample per elephant collected from any freshly produced bolus in the total faecal matter and at any point within a 7.5 h time period (7.30am–2.55 pm) will consistently represent parasite load. This study defines a protocol which may be used to test pre-analytic factors and effectively determine infection load in species which produce large quantities of vegetative faeces, such as non-ruminant megaherbivores.
Abhijith T. V., Ashokkumar M., Dencin R. T., George C. (2018): Gastrointestinal parasites of Asian elephants (Elephas maximus L. 1798) in south Wayanad forest division, Kerala, India. Journal of Parasitic Diseases 42: 382-390.
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Microscopic-coprological examination of Asian Elephant (Elephas maximus L., 1798) dung piles (n = 55) in South Wayanad Forest Division from March to August, 2017 revealed 74.5% prevalence of parasites in elephants. Ancylostoma sp. Anoplocephala sp., Strongyle type egg and Strongyloides sp. were the major parasites recorded. Strongyloides sp. and Strongyle type egg were observed more frequently (58.1%). Ancylostoma sp. and Anoplocephala sp. were constituted 1.8% each; mixed parasitic species infections were recorded. The frequency distribution of parasitic load in elephants showed skewed distribution of propagules. Centrifugal sedimentation and floatation methods of fecal examination of outer and inner regions of dung did not show significant difference in number of propagules. The highest number of parasitic propagules was recorded in floatation method. The number of propagules varied among dung samples of different herds collected from different localities. There were no relation between the parasitic load and age of elephants. The mean density of parasite eggs was higher in solitary animals (214.3 ± 155.4 epg) than herd elephants (147.78 ± 111.1 epg). Though parasitic load was higher in solitary males, based on the occurrence of parasites using logistic regression it was found that females had 1.83 times higher occurrence for parasitic infection than males. Both length and width of parasite egg size classes were used to classify into different taxonomic groups using discriminate function analysis. Three distinct size clusters were identified. Nematode and Cestode eggs were classified correctly with 95.7% accuracy. Since, the egg size was similar in nematode group separation into genus was difficult. Further, inclusion of stages of development of egg and larvae enable better separation.
Budd K., Gunn J. C., Finch T., Klymus K., Sitati N., Eggert L. S. (2020): Effects of diet, habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis). Ecology and Evolution 10: 5637-5650.
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The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop-raiding versus noncrop-raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.
Parker J. M., Goldenberg S. Z., Letitiya D., Wittemyer G. (2020): Strongylid infection varies with age, sex, movement and social factors in wild African elephants. Parasitology 147: 348-359.
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Comparing parasitic infection among individuals of wildlife populations can provide insight into factors that influence wildlife disease ecology. Strongylids are parasitic worms that infect the intestinal tract of vertebrates, and infection with strongylids can be approximated by counting strongylid eggs in dung samples. Here we tested for correlations between strongylid egg counts and 18 different individual characteristics, environmental and social factors in individually known wild African elephants. We counted more eggs in the dung samples of younger elephants and females relative to mature elephants and males. We also found that elephants spending more time outside reserves shed more strongylid eggs than elephants that were more often within reserves. Elephants that were less socially integrated, as measured by how much aggression they received from other elephants, shed fewer strongylid eggs; relatedly, socially isolated orphan elephants that had left their family shed fewer strongylid eggs than elephants that remained with their family. Our results suggest that landscapes altered by livestock grazing and social disruption caused by humans may impact parasitic infection in wildlife.
Common S. M., Yun Y., Silva‐Fletcher A., Thitaram C., Janyamethakul T., Khammesri S., Molenaar F. M. (2021): Developing a non‐invasive method of detecting elephant endotheliotropic herpesvirus infections using faecal samples. Veterinary Record: e833.
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Elephant endotheliotropic herpesvirus (EEHV)-associated haemorrhagic disease (EEHV-HD) is a leading cause of death in Asian elephant calves across the world. Cases of EEHV-HD have been detected in free-living calves through post-mortem examination (PME) indicating the presence of the virus in the wild. In the absence of a non-invasive sampling method, little research into free-living populations has been possible. This study aimed to provide evidence that faeces can be used as a non-invasive sampling method for the detection of EEHV excretion using quantitative polymerase chain reaction. Serial saliva swabs and faecal samples were taken from five captive Asian elephants in Thailand over 12 weeks. To ensure the presence of detectable elephant DNA within the sample, qPCR was run for amplification of the Asian elephant tumour necrosis factor (TNF-α) gene, EEHV1 and EEHV4. Of 28 sample pairs, seven saliva samples were positive for EEHV, of which two had paired positive faecal samples. This study presents the first evidence that EEHV is excreted in faeces at detectable levels. This method may in future be used for improved understanding of the epidemiology of EEHV in free-living elephant populations, as well as detection of EEHV excretion in captive herds.
Moustafa M. A. M., Chel H. M., Thu M. J., Bawm S., Htun L. L., Win M. M., Oo Z. M., Ohsawa N., Lahdenperä M., Mohamed W. M. A., Ito K. (2021): Anthropogenic interferences lead to gut microbiome dysbiosis in Asian elephants and may alter adaptation processes to surrounding environments. Scientific Reports 11: 741.
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Human activities interfere with wild animals and lead to the loss of many animal populations. Therefore, efforts have been made to understand how wildlife can rebound from anthropogenic disturbances. An essential mechanism to adapt to environmental and social changes is the fluctuations in the host gut microbiome. Here we give a comprehensive description of anthropogenically induced microbiome alterations in Asian elephants (n = 30). We detected gut microbial changes due to overseas translocation, captivity and deworming. We found that microbes belonging to Planococcaceae had the highest contribution in the microbiome alterations after translocation, while Clostridiaceae, Spirochaetaceae and Bacteroidia were the most affected after captivity. However, deworming significantly changed the abundance of Flavobacteriaceae, Sphingobacteriaceae, Xanthomonadaceae, Weeksellaceae and Burkholderiaceae. These findings may provide fundamental ideas to help guide the preservation tactics and probiotic replacement therapies of a dysbiosed gut microbiome in Asian elephants. More generally, these results show the severity of anthropogenic activities at the level of gut microbiome, altering the adaptation processes to new environments and the subsequent capability to maintain normal physiological processes in animals.
HEDGEHOGS, SHREWS, AND DESMANS
Haigh A., Kelly M., Butler F., O’Riordan R. M. (2014): Non-invasive methods of separating hedgehog (Erinaceus europaeus) age classes and an investigation into the age structure of road kill. Acta Theriologica 59: 165-171.
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The separation of animals into age classes is often fraught with difficulties, but is essential for the interpretation of survival rates and population structure. This study investigated both live hedgehogs and road kill in order to develop techniques that could be utilised to separate age classes in the field. Body measurements were taken from 24 live hedgehogs caught at a site in County Cork, Ireland over a three year period. Additionally, carcasses from around Ireland were collected and measured. There was a significant correlation between the weight and hind foot length of both live and dead hedgehogs. The weight of individuals was also significantly correlated with the body length and jaw length of hedgehogs. In all of these correlations, the sample clustered clearly into two groups with ‘juveniles’ meeting all of the following criteria: a hind foot length of <3.6 cm, a body length of < 16 cm and a jaw length of below 4.5 cm. It is suggested that these parameters, combined with the presence of growing spines, could provide a way of separating age classes in the field. This would be of particular benefit to short term studies, allowing the population structure to be accurately assessed and the reproductive success and sustainability of a population to be investigated. Road kill were also aged using the dentary bone in order to investigate the age profiles of hedgehogs killed on the road. Amongst those killed, 54 % of hedgehogs were 1-year-old or less. The majority of hedgehogs (87 %) were between 0–3-years-old, the oldest females were nine (n = 2), and the oldest males were eight years old (n = 2), respectively. Road kill hedgehogs had an average longevity of 1.94 years, and the mean age of females was higher (2.10 years) than males (1.87 years).
Ripa A., Díaz-Caballero J. A., Palacios-González M. J., Zalba J., Espinosa A., García-Zapata J. L., Gómez-Martín A., Tkach V., Fernández-Garcia J. L. (2023): Non-invasive wildlife disease surveillance using real time PCR assays: the case of the endangered Galemys pyrenaicus populations from the Central System mountains (Extremadura, Spain). Animals 13: 1136.
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The Iberian desman (Galemys pyrenaicus) is a small semi-aquatic mammal that inhabits mountainous areas from the centre to the north of the Iberian Peninsula and the Pyrenees and is listed as endangered because it has suffered a serious decline. Since 1960, only three species of digeneans (Omphalometra flexuosa, Maritrema pyrenaica and Mathovius galemydis) and two nematodes (Aonchotheca galemydis and Paracuaria hispanica) have been reported from the desman, but no further information on health status and no data from Extremadura has been available. The aim of our study was to characterise the diversity and distribution of parasites and microbiomes of desmans in different areas of the Central System of Extremadura. Between 2019 and 2021 we collected 238 fecal samples and one tissue (intestine) sample that was obtained from a dead desman. DNA templates were processed by commercial or customised real-time PCR using TaqMan probes. Representative data were obtained for Cryptosporidium spp., Omphalometra spp., Eimeria spp., Salmonella spp., Staphylococcus spp. and Leptospira spp. Omphalometra spp. was studied using a newly developed PCR test. The screening of the dead desman allowed us to obtain, for the first time, a partial sequence of the 18SrDNA. This study is the most complete study of the desman, allowing us to identify parasites and the microbiome in populations of G. pyrenaicus using non-invasive sampling.
MARSUPIALS
Hing S., Northover A. S., Narayan E. J., Wayne A. F., Jones K. L., Keatley S., Thompson R. A., Godfrey S. S. (2017): Evaluating stress physiology and parasite infection parameters in the translocation of critically endangered woylies (Bettongia penicillata). EcoHealth 14: 128-138.
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Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1–3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.
Hernandez S. E., Strona A. L. S., Leiner N. O., Suzán G., Romano M. C. (2018): Seasonal changes of faecal cortisol metabolite levels in Gracilinanus agilis (Didelphimorphia: Didelphidae) and its association to life histories variables and parasite loads. Conservation Physiology 6: coy021.
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The aim of this study was to evaluate the role of environmental (dry versus wet season) and individual (sex, body mass and reproductive status) factors in the levels of faecal cortisol metabolites (FGCs) in Gracilinanus agilis faecal samples as an index of stress levels in this species; as well as its association with abundance of Eimeria spp, as an indicator of immunocompetence against parasites. Our study found that FGCFGCs are a reliable indicator of adrenal activity in G. agilis. We found that FGCFGCs increase considerably by environmental stressors like the dry season. Moreover, the observed positive association between FGCs and body mass is the result of the effect of season and reproduction in both variables. We also demonstrated that an increase in FGC levels among G. agilis during the dry season is associated with a rise in the probability of being infected by Eimeria spp. Hence, our finding supports the corticosteroid-fitness hypothesis, which predicts that increased glucocorticoids as a response to stressors usually results in decreased fitness of individuals, translated into low future survival and reproductive success, and higher parasite infection. To our knowledge, this is the first study that integrates environmental changes, hormone responses and parasite loads in a US marsupial in both empirical and experimental approaches.
Brice K. L., Trivedi P., Jeffries T. C., Blyton M. D., Mitchell C., Singh B. K., Moore B. D. (2019): The koala (Phascolarctos cinereus) faecal microbiome differs with diet in a wild population. PeerJ 7: e6534.
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The diet of the koala (Phascolarctos cinereus) is comprised almost exclusively of foliage from the genus Eucalyptus (family Myrtaceae). Eucalyptus produces a wide variety of potentially toxic plant secondary metabolites which have evolved as chemical defences against herbivory. The koala is classified as an obligate dietary specialist, and although dietary specialisation is rare in mammalian herbivores, it has been found elsewhere to promote a highly-conserved but low-diversity gut microbiome. The gut microbes of dietary specialists have been found sometimes to enhance tolerance of dietary PSMs, facilitating competition-free access to food. Although the koala and its gut microbes have evolved together to utilise a low nutrient, potentially toxic diet, their gut microbiome has not previously been assessed in conjunction with diet quality. Thus, linking the two may provide new insights in to the ability of the koala to extract nutrients and detoxify their potentially toxic diet. The 16S rRNA gene was used to characterise the composition and diversity of faecal bacterial communities from a wild koala population (n = 32) comprising individuals that predominately eat either one of two different food species, one the strongly preferred and relatively nutritious species Eucalyptus viminalis, the other comprising the less preferred and less digestible species Eucalyptus obliqua. Alpha diversity indices indicated consistently and significantly lower diversity and richness in koalas eating E. viminalis. Assessment of beta diversity using both weighted and unweighted UniFrac matrices indicated that diet was a strong driver of both microbial community structure, and of microbial presence/absence across the combined koala population and when assessed independently. Further, principal coordinates analysis based on both the weighted and unweighted UniFrac matrices for the combined and separated populations, also revealed a separation linked to diet. During our analysis of the OTU tables we also detected a strong association between microbial community composition and host diet. We found that the phyla Bacteroidetes and Firmicutes were co-dominant in all faecal microbiomes, with Cyanobacteria also co-dominant in some individuals; however, the E. viminalis diet produced communities dominated by the genera Parabacteroides and/or Bacteroides, whereas the E. obliqua-associated diets were dominated by unidentified genera from the family Ruminococcaceae. We show that diet differences, even those caused by differential consumption of the foliage of two species from the same plant genus, can profoundly affect the gut microbiome of a specialist folivorous mammal, even amongst individuals in the same population. We identify key microbiota associated with each diet type and predict functions within the microbial community based on 80 previously identified Parabacteroides and Ruminococcaceae genomes.
Cristescu R. H., Miller R. L., Schultz A. J., Hulse L., Jaccoud D., Johnston S., Hanger J., Booth R., Frère C. H. (2019): Developing noninvasive methodologies to assess koala population health through detecting Chlamydia from scats. Molecular Ecology Resources 19: 957-969.
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Wildlife diseases are a recognized driver of global biodiversity loss, have substantial economic impacts, and are increasingly becoming a threat to human health. Disease surveillance is critical but remains difficult in the wild due to the substantial costs and potential biases associated with most disease detection methods. Noninvasive scat surveys have been proposed as a health monitoring methodology to overcome some of these limitations. Here, we use the known threat of Chlamydia disease to the iconic, yet vulnerable, koala Phascolarctos cinereus to compare three methods for Chlamydia detection in scats: multiplex quantitative PCR, next generation sequencing, and a detection dog specifically trained on scats from Chlamydia-infected koalas. All three methods demonstrated 100% specificity, while sensitivity was variable. Of particular interest is the variable sensitivity of these diagnostic tests to detect sick individuals (i.e., not only infection as confirmed by Chlamydia-positive swabs, but with observable clinical signs of the disease); for koalas with urogenital tract disease signs, sensitivity was 78% with quantitative PCR, 50% with next generation genotyping and 100% with the detection dog method. This may be due to molecular methods having to rely on high-quality DNA whereas the dog most likely detects volatile organic compounds. The most appropriate diagnostic test will vary with disease prevalence and the specific aims of disease surveillance. Acknowledging that detection dogs might not be easily accessible to all, the future development of affordable and portable “artificial noses” to detect diseases from scats in the field might enable cost-effective, rapid and large-scale disease surveillance.
Emami-Khoyi A., Agnew T. W., Adair M. G., Murphy E. C., Benmazouz I., Monsanto D. M., Parbhu S. P., Main D. C., Le Roux R., Golla T. R., Schnelle C. (2021): A new non-invasive method for collecting DNA from small mammals in the field, and its application in simultaneous vector and disease monitoring in brushtail possums. Frontiers in Environmental Science 9: 701033.
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Large-scale monitoring of wild populations in remote areas using traditional live-capturing methods is logistically and financially challenging. Devices that can be used to obtain biological material remotely and store it for an extended period have considerable potential to monitor population densities and health status, but their applicability remains largely unexplored. The present study describes a device that collects trace amounts of DNA from the saliva of small mammals that is deposited on the surface of a collection medium (WaxTags®). The device’s performance was evaluated on Australian brushtail possums (Trichosurus vulpecula), an invasive pest species and the most significant vector of bovine tuberculosis infective agent (Mycobacterium bovis), under field conditions in Canterbury, New Zealand. The retrieved DNA was used to amplify eight possum-specific microsatellite markers and bacterial 16S rRNA. The design is mechanically robust, and the quality of the recovered DNA was adequate for microsatellite-based identification of individual possums, estimation of population density, and partial reconstruction of their oral microbiomes as a potential indicator of health. Several medically important bacteria, including strains of environmental Mycobacterium sp., were detected. The design can be refined to monitor other animals’ populations proactively and provide different levels of information necessary to manage wild populations.
Okoh G. S. R., Ariel E., Whitmore D., Horwood P. F. (2023): Metagenomic and molecular detection of novel fecal viruses in free-ranging agile wallabies. EcoHealth 20: 427-440.
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The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1–2, agile wallaby polyomavirus 1–2, agile wallaby associated picobirnavirus 1–9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.
PRIMATES
Amato K. R., Yeoman C. J., Kent A., Righini N., Carbonero F., Estrada A., Gaskins H. R., Stumpf R. M., Yildirim S., Torralba M., Gillis M. (2013): Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. The ISME Journal 7: 1344-1353.
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The gastrointestinal (GI) microbiome contributes significantly to host nutrition and health. However, relationships involving GI microbes, their hosts and host macrohabitats remain to be established. Here, we define clear patterns of variation in the GI microbiomes of six groups of Mexican black howler monkeys (Alouatta pigra) occupying a gradation of habitats including a continuous evergreen rainforest, an evergreen rainforest fragment, a continuous semi-deciduous forest and captivity. High throughput microbial 16S ribosomal RNA gene sequencing indicated that diversity, richness and composition of howler GI microbiomes varied with host habitat in relation to diet. Howlers occupying suboptimal habitats consumed less diverse diets and correspondingly had less diverse gut microbiomes. Quantitative real-time PCR also revealed a reduction in the number of genes related to butyrate production and hydrogen metabolism in the microbiomes of howlers occupying suboptimal habitats, which may impact host health.
Smiley Evans T., Gilardi K. V., Barry P. A., Ssebide B. J., Kinani J. F., Nizeyimana F., Noheri J. B., Byarugaba D. K., Mudakikwa A., Cranfield M. R., Mazet J. A. (2016): Detection of viruses using discarded plants from wild mountain gorillas and golden monkeys. American Journal of Primatology 78: 1222-1234.
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Infectious diseases pose one of the most significant threats to the survival of great apes in the wild. The critically endangered mountain gorilla (Gorilla beringei beringei) is at high risk for contracting human pathogens because approximately 60% of the population is habituated to humans to support a thriving ecotourism program. Disease surveillance for human and non-human primate pathogens is important for population health and management of protected primate species. Here, we evaluate discarded plants from mountain gorillas and sympatric golden monkeys (Cercopithecus mitis kandti), as a novel biological sample to detect viruses that are shed orally. Discarded plant samples were tested for the presence of mammalian-specific genetic material and two ubiquitous DNA and RNA primate viruses, herpesviruses, and simian foamy virus. We collected discarded plant samples from 383 wild human-habituated mountain gorillas and from 18 habituated golden monkeys. Mammalian-specific genetic material was recovered from all plant species and portions of plant bitten or chewed by gorillas and golden monkeys. Gorilla herpesviral DNA was most consistently recovered from plants in which leafy portions were eaten by gorillas. Simian foamy virus nucleic acid was recovered from plants discarded by golden monkeys, indicating that it is also possible to detect RNA viruses from bitten or chewed plants. Our findings show that discarded plants are a useful non-invasive sampling method for detection of viruses that are shed orally in mountain gorillas, sympatric golden monkeys, and potentially other species. This method of collecting specimens from discarded plants is a new non-invasive sampling protocol that can be combined with collection of feces and urine to evaluate the most common routes of viral shedding in wild primates.
Springer A., Kappeler P.M. (2016): Intestinal parasite communities of six sympatric lemur species at Kirindy Forest, Madagascar. Primate Biology 3: 51-63.
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Intestinal parasites impact host health, survival and reproductive success and therefore exert selective pressures on hosts’ ecology and behavior. Thus, characterizing and comparing the parasitic fauna of different wildlife hosts sharing the same habitat can provide insights into the mechanisms underlying variation in parasitism, as well as the role of parasites as possible conservation threats. Several host traits have been proposed to generate differences in parasite diversity among different host species, including phylogeny, host body mass, host longevity, diet, and differences in ranging and social behavior. Here, we provide an overview of intestinal helminths and protozoa detected by fecal microscopy in six sympatric lemur species in Kirindy Forest, western Madagascar. The described patterns indicate that host phylogeny and diet may play an important role in shaping intestinal parasite assemblages in this system, as the closely related, omnivorous cheirogaleids showed the strongest overlap in parasite communities. No indication was found for an effect of body mass or longevity on parasite species richness. Regarding the effect of sociality, the two group-living lemur species, Propithecus verreauxi and Eulemur rufifrons, harbored directly transmitted parasites at higher prevalence than solitary foragers, but not at higher diversity. Effects of season and sex on parasite prevalence confirm the results of previous studies, with higher prevalence in the energetically demanding dry season and a male bias in parasitism. We highlight the opportunities of exploring the parasitic fauna of wildlife from a community ecology and evolutionary perspective, and identify prospects for future research on lemur parasitism.
Perofsky A. C., Lewis R. J., Abondano L. A., Di Fiore A., Meyers L. A. (2017): Hierarchical social networks shape gut microbial composition in wild Verreaux’s sifaka. Proceedings of the Royal Society B: Biological Sciences 284: 20172274.
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In wild primates, social behaviour influences exposure to environmentally acquired and directly transmitted microorganisms. Prior studies indicate that gut microbiota reflect pairwise social interactions among chimpanzee and baboon hosts. Here, we demonstrate that higher-order social network structure—beyond just pairwise interactions—drives gut bacterial composition in wild lemurs, which live in smaller and more cohesive groups than previously studied anthropoid species. Using 16S rRNA gene sequencing and social network analysis of grooming contacts, we estimate the relative impacts of hierarchical (i.e. multilevel) social structure, individual demographic traits, diet, scent-marking, and habitat overlap on bacteria acquisition in a wild population of Verreaux’s sifaka (Propithecus verreauxi) consisting of seven social groups. We show that social group membership is clearly reflected in the microbiomes of individual sifaka, and that social groups with denser grooming networks have more homogeneous gut microbial compositions. Within social groups, adults, more gregarious individuals, and individuals that scent-mark frequently harbour the greatest microbial diversity. Thus, the community structure of wild lemurs governs symbiotic relationships by constraining transmission between hosts and partitioning environmental exposure to microorganisms. This social cultivation of mutualistic gut flora may be an evolutionary benefit of tight-knit group living.
Pouillevet H., Dibakou S. E., Ngoubangoye B., Poirotte C., Charpentier M. J. (2017): A comparative study of four methods for the detection of nematode eggs and large protozoan cysts in mandrill faecal material. Folia Primatologica 88: 344-357.
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Coproscopical methods like sedimentation and flotation techniques are widely used in the field for studying simian gastrointestinal parasites. Four parasites of known zoonotic potential were studied in a free-ranging, non-provisioned population of mandrills (Mandrillus sphinx): 2 nematodes (Necatoramericanus/Oesophagostomum sp. complex and Strongyloides sp.) and 2 protozoan species (Balantidium coli and Entamoeba coli). Different coproscopical techniques are available but they are rarely compared to evaluate their efficiency to retrieve parasites. In this study 4 different field-friendly methods were compared. A sedimentation method and 3 different McMaster methods (using sugar, salt, and zinc sulphate solutions) were performed on 47 faecal samples collected from different individuals of both sexes and all ages. First, we show that McMaster flotation methods are appropriate to detect and thus quantify large protozoan cysts. Second, zinc sulphate McMaster flotation allows the retrieval of a higher number of parasite taxa compared to the other 3 methods. This method further shows the highest probability to detect each of the studied parasite taxa. Altogether our results show that zinc sulphate McMaster flotation appears to be the best technique to use when studying nematodes and large protozoa.
Schneider-Crease I., Griffin R. H., Gomery M. A., Dorny P., Noh J. C., Handali S., Chastain H. M., Wilkins P. P., Nunn C. L., Snyder-Mackler N., Beehner J. C. (2017): Identifying wildlife reservoirs of neglected taeniid tapeworms: non-invasive diagnosis of endemic Taenia serialis infection in a wild primate population. Plos Neglected Tropical Diseases 11: e0005709.
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Despite the global distribution and public health consequences of Taenia tapeworms, the life cycles of taeniids infecting wildlife hosts remain largely undescribed. The larval stage of Taenia serialis commonly parasitizes rodents and lagomorphs, but has been reported in a wide range of hosts that includes geladas (Theropithecus gelada), primates endemic to Ethiopia. Geladas exhibit protuberant larval cysts indicative of advanced T. serialis infection that are associated with high mortality. However, non-protuberant larvae can develop in deep tissue or the abdominal cavity, leading to underestimates of prevalence based solely on observable cysts. We adapted a non-invasive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect circulating Taenia spp. antigen in dried gelada urine. Analysis revealed that this assay was highly accurate in detecting Taenia antigen, with 98.4% specificity, 98.5% sensitivity, and an area under the curve of 0.99. We used this assay to investigate the prevalence of T. serialis infection in a wild gelada population, finding that infection is substantially more widespread than the occurrence of visible T. serialis cysts (16.4% tested positive at least once, while only 6% of the same population exhibited cysts). We examined whether age or sex predicted T. serialis infection as indicated by external cysts and antigen presence. Contrary to the female-bias observed in many Taenia-host systems, we found no significant sex bias in either cyst presence or antigen presence. Age, on the other hand, predicted cyst presence (older individuals were more likely to show cysts) but not antigen presence. We interpret this finding to indicate that T. serialis may infect individuals early in life but only result in visible disease later in life. This is the first application of an antigen ELISA to the study of larval Taenia infection in wildlife, opening the doors to the identification and description of infection dynamics in reservoir populations.
Löhrich T., Behringer V., Wittig R. M., Deschner T., Leendertz F. H. (2018): The use of neopterin as a noninvasive marker in monitoring diseases in wild chimpanzees. EcoHealth 15: 792-803.
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Pathogen analysis in wild great apes is both time- and resource-consuming. Therefore, we examined the potential use of urinary neopterin, a sensitive marker of cell-mediated immune system activation, as a disease marker and unspecific screening tool to facilitate informed pathogen analysis in great ape health monitoring. To test this, urinary neopterin was correlated to other disease markers such as sickness behaviors, fever, and urine parameters. Seasonal variation in urinary neopterin levels was investigated as well. The study encompassed noninvasively collected longitudinal data of young wild chimpanzees from the Taï National Park, Côte d´Ivoire. Relationships between disease markers were examined using a linear mixed model and a case study approach. Seasonal variation in urinary neopterin was tested using a linear mixed model. While the linear mixed model found no obvious relationship between urinary neopterin levels and other disease markers, the case study approach revealed a pattern resembling those found in humans. Urinary neopterin levels indicated seasonal immune system activation peaking in times of low ambient temperatures. We suggest the use of urinary neopterin as an unspecific screening tool in great ape health monitoring to identify relevant samples, individuals, and time periods for selective pathogen analysis and zoonotic risk assessment.
Villabona‐Arenas C. J., Ayouba A., Esteban A., D’arc M., Mpoudi Ngole E., Peeters M. (2018): Noninvasive western lowland gorilla’s health monitoring: a decade of simian immunodeficiency virus surveillance in southern Cameroon. Ecology and Evolution 8: 10698-10710.
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Simian immunodeficiency virus (SIVgor) causes persistent infection in critically endangered western lowland gorillas (Gorilla gorilla gorilla) from west central Africa. SIVgor is closely related to chimpanzee and human immunodeficiency viruses (SIVcpz and HIV‐1, respectively). We established a noninvasive method that does not interfere with gorillas’ natural behaviour to provide wildlife pathogen surveillance and health monitoring for conservation. A total of 1,665 geo‐referenced fecal samples were collected at regular intervals from February 2006 to December 2014 (123 sampling days) in the Campo‐Ma’an National Park (southwest Cameroon). Host genotyping was performed using microsatellite markers, SIVgor infection was identified by serology and genetic amplification was attempted on seropositive individuals. We identified at least 125 distinct gorillas, 50 were resampled (observed 3.5 times in average) and 38 were SIVgor+ (seven individuals were seroconverters). Six groups of gorillas were identified based on the overlapping occurrence of individuals with apparent high rates of gene flow. We obtained SIVgor genetic sequences from 25 of 38 seropositive genotyped gorillas and showed that the virus follows exponential growth dynamics under a strict molecular clock. Different groups shared SIVgor lineages demonstrating intergroup viral spread and recapture of positive individuals illustrated intra‐host viral evolution. Relatedness and relationship genetic analysis of gorillas together with Bayesian phylogenetic inference of SIVgor provided evidence suggestive of vertical transmission. In conclusion, we provided insights into gorilla social dynamics and SIVgor evolution and emphasized the utility of noninvasive sampling to study wildlife health populations. These findings contribute to prospective planning for better monitoring and conservation.
Wu D. F., Behringer V., Wittig R. M., Leendertz F. H., Deschner T. (2018): Urinary neopterin levels increase and predict survival during a respiratory outbreak in wild chimpanzees (Taï National Park, Côte d’Ivoire). Scientific Reports 8: 13346.
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Monitoring immune system activation of wild animals has garnered increasing interest within the field of ecological immunology, leading to an urgent need for non-invasive biomarkers measuring these changes. Urinary neopterin, a marker of the cell-mediated immune response, is validated as an immune-related biomarker in captive and laboratory animals. However, wild animals naturally host higher and chronic pathogen loads. Therefore, detection and quantification of additional infections via neopterin might not be possible against the background of a chronically challenged immune system. To assess the suitability of urinary neopterin in wild animals, we measured neopterin corrected for specific gravity with an enzyme immunoassay in 185 samples collected before, during and after a respiratory disease outbreak in 28 individuals from a group of wild chimpanzees (Taï National Park, Côte d’Ivoire). Urinary neopterin levels were significantly higher during periods when individuals showed respiratory symptoms versus before and after the outbreak. Furthermore, urinary neopterin levels were significantly higher in individuals that died, with higher levels already apparent before the outbreak, suggesting individuals may have an already activated immune system. Measuring urinary neopterin levels, with other biomarkers of energetic condition, stress challenges, and reproduction will contribute towards a deeper understanding of life-history trade-offs in wild animals.
Hernández‐Rodríguez D., Vásquez‐Aguilar A. A., Serio‐Silva J. C., Rebollar E. A., Azaola‐Espinosa A. (2019): Molecular detection of Bifidobacterium spp. in faeces of black howler monkeys (Alouatta pigra). Journal of Medical Primatology 48: 99-105.
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Bifidobacterium genus are considered to be beneficial bacteria for their hosts; however, knowledge about the specific species that are part of the gut microbiome of howler monkeys is scarce. Polymerase chain reaction (PCR) is a useful technique for the identification of non-cultivable or difficult to grow bacterial species. With the goal of detecting species of the genus Bifidobacterium in black howler monkeys, we used PCR on DNA derived from faecal samples. We collected and extracted DNA from 40 faecal samples. Using specific primers, we performed PCR and nested PCR to detect members of the Bifidobacterium genus and a subset of species: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium animalis subsp. animalis. 97.5% (39/40) of the samples were positive for Bifidobacterium spp. We found B. longum in 100% of the analysed samples. This is the first report of B. longum in black howler monkey faeces.
Lacroux C., Guma N., Krief S. (2019): Facial dysplasia in wild forest olive baboons (Papio anubis) in Sebitoli, Kibale National Park, Uganda: Use of camera traps to detect health defects. Journal of Medical Primatology 48: 143-153.
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Primate populations are in decline, mainly affected by agriculture leading to habitat loss, fragmentation but also chemical pollution. Kibale National Park (Uganda), Sebitoli forest, surrounded by tea and crop fields, is the home range of chimpanzees presenting congenital facial dysplasia. This study aimed to identify to what extent the same phenotypical features are observed in baboons (Papio anubis) of this area. A total of 25 390 clips recorded by 14 camera traps between January 2017 and April 2018 were analyzed. We identified 30 immature and adult baboons of both sexes with nose and lip deformities. They were more frequently observed in the northwestern part of the area. A possible effect of pesticides used in crops at the border of their habitat is suspected to alter the embryonic development. This study emphasizes the importance of non-invasive methods to detect health problems in wild primates that can act as sentinels for human health.
Gaillard C. M., Pion S. D., Hamou H., Sirima C., Bizet C., Lemarcis T., Rodrigues J., Esteban A., Peeters M., Mpoudi Ngole E., Mombo I. (2020): Detection of DNA of filariae closely related to Mansonella perstans in faecal samples from wild non-human primates from Cameroon and Gabon. Parasites & Vectors 13: 313.
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The Onchocercidae is a family of filarial nematodes with several species of medical or veterinary importance. Microfilariae are found in the blood and/or the dermis and are usually diagnosed in humans by microscopy examination of a blood sample or skin biopsy. The main objectives of this study were to evaluate whether filariae DNA can be detected in faecal samples of wild non-human primates (NHPs), whether the detected parasites were closely related to those infecting humans and whether filarial DNA detection in faeces is associated with co-infections with nematodes (Oesophagostumum sp. and Necator sp.) known to cause blood loss while feeding on the host intestinal mucosa. A total of 315 faecal samples from 6 species of NHPs from Cameroon and Gabon were analysed. PCRs targeted DNA fragments of cox1 and 12S rDNA genes, to detect the presence of filariae, and the internal transcribed spacer 2 (ITS2), to detect the presence of Oesophagostomum sp. and Necator sp. infections. Among the 315 samples analysed, 121 produced sequences with > 90% homology with Onchocercidae reference sequences. However, 63% of the 12S rDNA and 78% of the cox1 gene sequences were exploitable for phylogenetic analyses and the amplification of the 12S rDNA gene showed less discriminating power than the amplification of the cox1 fragment. Phylogenetic analyses showed that the cox1 sequences obtained from five chimpanzee DNA faecal samples from Gabon and two from Cameroon cluster together with Mansonella perstans with high bootstrap support. Most of the remaining sequences clustered together within the genus Mansonella, but the species could not be resolved. Among the NHP species investigated, a significant association between filarial DNA detection and Oesophagostomum sp. and Necator sp. infection was observed only in gorillas. To our knowledge, this is the first study reporting DNA from Mansonella spp. in faecal samples. Our results raise questions about the diversity and abundance of these parasites in wildlife, their role as sylvatic reservoirs and their potential for zoonotic transmission. Future studies should focus on detecting variants circulating in both human and NHPs, and improve the molecular information to resolve or support taxonomy classification based on morphological descriptions.
Behringer V., Müller‐Klein N., Strube C., Schülke O., Heistermann M., Ostner J. (2021): Responsiveness of fecal immunoglobulin A to HPA‐axis activation limits its use for mucosal immunity assessment. American Journal of Primatology 83: e23329.
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The assessment of mucosal immunity as a component of animal health is an important aspect for the understanding of variation in host immunity, and its tradeoff against other life-history traits. We investigated immunoglobulin A (IgA), the major type of antibody associated with mucosal immunity, in relation to changes in parasitic burden following anthelminthic treatment in noninvasively collected fecal samples in a semi-free ranging group of Barbary macaques (Macaca sylvanus). We measured IgA in 340 fecal samples of fourteen females and nine males. As IgA has been found to be responsive to stressors, we also related fecal IgA (fIgA) levels to fecal glucocorticoid metabolites (fGCM) measured in the same samples as part of a previous study. We found a high variability within and between individual fIgA levels over time. Running generalized additive mixed models, we found that fIgA levels were higher in males than in females, but did not change in response to the anthelmintic treatment and the resulting reduction in worm burden. Instead, fIgA level changes were significantly correlated to changes in fGCM levels. Our findings indicate that due to the strong responsiveness of fIgA to HPA-axis activity, the measurement of fIgA may have certain limitations with respect to reflecting gastrointestinal parasitic burden. Moreover, the responsiveness of fIgA to stressors interferes with the interpretation of IgA levels in fecal samples as a measure of mucosal immunity, at least in our study population of the Barbary macaques.
Shutt-Phillips K., Pafčo B., Heistermann M., Kasim A., Petrželková K. J., Profousová-Pšenková I., Modrý D., Todd A., Fuh T., Dicky J.-F., Bopalanzognako J.-B., Setchell J. M. (2021): Fecal glucocorticoids and gastrointestinal parasite infections in wild western lowland gorillas (Gorilla gorilla gorilla) involved in ecotourism. General and Comparative Endocrinology 312: 113859.
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Wildlife ecotourism can offer a source of revenue which benefits local development and conservation simultaneously. However, habituation of wildlife for ecotourism can cause long-term elevation of glucocorticoid hormones, which may suppress immune function and increase an animal’s vulnerability to disease. We have previously shown that western lowland gorillas (Gorilla gorilla gorilla) undergoing habituation in Dzanga-Sangha Protected Areas, Central African Republic, have higher fecal glucocorticoid metabolite (FGCM) levels than both habituated and unhabituated gorillas. Here, we tested the relationship between FGCM levels and strongylid infections in the same gorillas. If high FGCM levels suppress the immune system, we predicted that FGCM levels will be positively associated with strongylid egg counts and that gorillas undergoing habituation will have the highest strongylid egg counts, relative to both habituated and unhabituated gorillas. We collected fecal samples over 12 months in two habituated gorilla groups, one group undergoing habituation and completely unhabituated gorillas. We established FGCM levels and fecal egg counts of Necator/Oesophagostomum spp. and Mammomonogamus sp. Controlling for seasonal variation and age-sex category in strongylid infections we found no significant relationship between FGCMs and Nectator/Oesophagostomum spp. or Mammomonogamus sp. egg counts in a within group comparison in either a habituated group or a group undergoing habituation. However, across groups, egg counts of Nectator/Oesophagostomum spp. were lowest in unhabituated animals and highest in the group undergoing habituation, matching the differences in FGCM levels among these gorilla groups. Our findings partially support the hypothesis that elevated glucocorticoids reduce a host’s ability to control the extent of parasitic infections, and show the importance of non-invasive monitoring of endocrine function and parasite infection in individuals exposed to human pressure including habituation process and ecotourism.
Mason B., Piel A. K., Modrý D., Petrželková K. J., Stewart F. A., Pafčo B. (2022): Association of human disturbance and gastrointestinal parasite infection of yellow baboons in western Tanzania. Plos One 17: e0262481.
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Human disturbance is an ongoing threat to many wildlife species, manifesting as habitat destruction, resource overuse, or increased disease exposure, among others. With increasing human: non-human primate (NHP) encounters, NHPs are increasingly susceptible to human-introduced diseases, including those with parasitic origins. As such, epidemiology of parasitic disease is becoming an important consideration for NHP conservation strategies. To investigate the relationship between parasite infections and human disturbance we studied yellow baboons (Papio cynocephalus) living outside of national park boundaries in western Tanzania, collecting 135 fresh faecal samples from nine troops occupying areas with varying levels of human disturbance. We fixed all samples in 10% formalin and later evaluated parasite prevalence and abundance (of isotrichid ciliates and Strongylida). We identified seven protozoan and four helminth taxa. Taxa showed varied relationships with human disturbance, baboon troop size and host age. In four taxa, we found a positive association between prevalence and troop size. We also report a trend towards higher parasite prevalence of two taxa in less disturbed areas. To the contrary, high levels of human disturbance predicted increased abundance of isotrichid ciliates, although no relationship was found between disturbance and Strongylida abundance. Our results provide mixed evidence that human disturbance is associated with NHP parasite infections, highlighting the need to consider monitoring parasite infections when developing NHP conservation strategies.
Mallott E. K., Skovmand L. H., Garber P. A., Amato K. R. (2022): The fecal metabolome of black howler monkeys (Alouatta pigra) varies in response to seasonal dietary changes. Molecular Ecology 31: 4146– 4161.
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Mammals rely on the metabolic functions of their gut microbiota to meet their energetic needs and digest potentially toxic components in their diet. The gut microbiome plastically responds to shifts in host diet and may buffer variation in energy and nutrient availability. However, it is unclear how seasonal differences in the gut microbiome influence microbial metabolism and nutrients available to hosts. In this study, we examine seasonal variation in the gut metabolome of black howler monkeys (Alouatta pigra) to determine whether those variations are associated with differences in gut microbiome composition and nutrient intake, and if plasticity in the gut microbiome buffers shortfalls in energy or nutrient intake. We integrated data on the metabolome of 81 faecal samples from 16 individuals collected across three distinct seasons with gut microbiome, nutrient intake and plant metabolite consumption data from the same period. Faecal metabolite profiles differed significantly between seasons and were strongly associated with changes in plant metabolite consumption. However, microbial community composition and faecal metabolite composition were not strongly associated. Additionally, the connectivity and stability of faecal metabolome networks varied seasonally, with network connectivity being highest during the dry, fruit-dominated season when black howler monkey diets were calorically and nutritionally constrained. Network stability was highest during the dry, leaf-dominated season when most nutrients were being consumed at intermediate rates. Our results suggest that the gut microbiome buffers seasonal variation in dietary intake, and that the buffering effect is most limited when host diet becomes calorically or nutritionally restricted.
Mason B., Petrzelkova K. J., Kreisinger J., Bohm T., Cervena B., Fairet E., Fuh T., Gomez A., Knauf S., Maloueki U., Modry D. (2022): Gastrointestinal symbiont diversity in wild gorilla: a comparison of bacterial and strongylid communities across multiple localities. Molecular Ecology 31: 4127– 4145.
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Western lowland gorillas (Gorilla gorilla gorilla) are Critically Endangered and show continued population decline. Consequently, pressure is mounting to better understand their conservation threats and ecology. Gastrointestinal symbionts, such as bacterial and eukaryotic communities, are believed to play vital roles in the physiological landscape of the host. Gorillas host a broad spectrum of eucaryotes, so called parasites, with strongylid nematodes being particularly prevalent. While these communities are partially consistent, they are also shaped by various ecological factors, such as diet or habitat type. To investigate gastrointestinal symbionts of wild western lowland gorillas, we analysed 215 faecal samples from individuals in five distinct localities across the Congo Basin, using high-throughput sequencing techniques. We describe the gut bacterial microbiome and genetic diversity of strongylid communities, including strain-level identification of amplicon sequence variants (ASVs). We identified strongylid ASVs from eight genera and bacterial ASVs from 20 phyla. We compared these communities across localities, with reference to varying environmental factors among populations, finding differences in alpha diversity and community compositions of both gastrointestinal components. Moreover, we also investigated covariation between strongylid nematodes and the bacterial microbiome, finding correlations between strongylid taxa and Prevotellaceae and Rikenellaceae ASVs that were consistent across multiple localities. Our research highlights the complexity of the bacterial microbiome and strongylid communities in several gorilla populations and emphasizes potential interactions between these two symbiont communities. This study provides a framework for ongoing research into strongylid nematode diversity, and their interactions with the bacterial microbiome, among great apes.
Negrey J. D., Emery Thompson M., Dunn C. D., Otali E., Wrangham R. W., Mitani J. C., Machanda Z. P., Muller M. N., Langergraber K. E., Goldberg T. L. (2022): Female reproduction and viral infection in a long‐lived mammal. Journal of Animal Ecology 91: 1999-2009.
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For energetically limited organisms, life-history theory predicts trade-offs between reproductive effort and somatic maintenance. This is especially true of female mammals, for whom reproduction presents multifarious energetic and physiological demands. Here, we examine longitudinal changes in the gut virome (viral community) with respect to reproductive status in wild mature female chimpanzees Pan troglodytes schweinfurthii from two communities, Kanyawara and Ngogo, in Kibale National Park, Uganda. We used metagenomic methods to characterize viromes of individual chimpanzees while they were cycling, pregnant and lactating. Females from Kanyawara, whose territory abuts the park’s boundary, had higher viral richness and loads (relative quantity of viral sequences) than females from Ngogo, whose territory is more energetically rich and located farther from large human settlements. Viral richness (total number of distinct viruses per sample) was higher when females were lactating than when cycling or pregnant. In pregnant females, viral richness increased with estimated day of gestation. Richness did not vary with age, in contrast to prior research showing increased viral abundance in older males from these same communities. Our results provide evidence of short-term physiological trade-offs between reproduction and infection, which are often hypothesized to constrain health in long-lived species.
Patrono L. V., Röthemeier C., Kouadio L., Couacy‐Hymann E., Wittig R. M., Calvignac‐Spencer S., Leendertz F. H. (2022): Non‐invasive genomics of respiratory pathogens infecting wild great apes using hybridisation capture. Influenza and Other Respiratory Viruses 16: 858-861.
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Human respiratory pathogens have repeatedly caused lethal outbreaks in wild great apes across Africa, leading to population declines. Nonetheless, our knowledge of potential genomic changes associated with pathogen introduction and spread at the human-great ape interface remains sparse. Here, we made use of target enrichment coupled with next generation sequencing to non-invasively investigate five outbreaks of human-introduced respiratory disease in wild chimpanzees living in Taï National Park, Ivory Coast. By retrieving 34 complete viral genomes and three distinct constellations of pneumococcal virulence factors, we provide genomic insights into these spillover events and describe a framework for non-invasive genomic surveillance in wildlife.
Yang L., Wang W., Wronski T., Sun P., Jin K., Tang W. (2022): Community structure and environmental determinants of the bacterial and fungal gut microflora in Hainan gibbons (Nomascus hainanus). Global Ecology and Conservation 36: e02114.
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High-throughput sequencing technology was used to establish the OTU (Operational Taxonomic Units) composition and diversity of bacteria and fungi in the gut of three family groups of Hainan gibbons (Nomascus hainanus) in the Bawangling National Nature Reserve on Hainan Island, southern China. Firmicutes (47.23%) and Bacteroidetes (36.54%) predominated at the phylum level, while the predominant genera were Prevotella-7 (15.30%), Lachnospiraceae_NK3A20_group (12.49%), and uncultured_bacterium_f_Erysipelotrichaceae (11.79%). At the phylum level, the dominant fungi were Ascomycota (66.41%), as well as Hanseniaspora (23.33%), Schwanniomyces (13.44%), and Pichia (5.43%) at the genus level. We found significant differences in the bacterial OTU diversity (Shannon index) between family groups living in Tropical Lowland or Tropical Montane Rain Forest, compared to those inhabiting Tropical Montane Evergreen Forest at higher altitudes. Bacterial OTU community composition also differed between family groups, unraveling significant differences among the 30 most dominant bacterial core taxa. Furthermore, we unraveled a significant difference of OTU richness in the fungal microbiome (Chao 1) between family groups living at lower altitudes, i.e., Tropical Lowland or Tropical Montane Rain Forest and those inhabiting the Tropical Montane Evergreen Forest. Our analysis further indicated significant differences in the fungal OTU community composition between the three family groups, especially regarding the three most dominant fungal core taxa. Subsequently, two habitat factors, and nine environmental and anthropogenic variables were used to explore possible causes of disparity in the microbial flora of gibbon groups. A factor reduction procedure resulted into three principal components which were correlated to bacterial and fungal OTU richness and diversity using Spearman’s rank-order correlations. Bacterial and fungal OTU diversity was high in areas of high altitude, steep slopes, high tree density, but low tree height, while high fungal OTU richness corresponded to high altitude habitats, i.e., in the Tropical Montane Evergreen Forest. Distance to human settlements and to the next water body showed no significant relation with bacterial and fungal richness and diversity.
Michel A., Minocher R., Niehoff P. P., Li Y., Nota K., Gadhvi M. A., Su J., Iyer N., Porter A., Ngobobo-As-Ibungu U., Binyinyi E., Pekeyake R. N., Parducci L., Caillaud D., Guschanski K. (2023): Isolated Grauer’s gorilla populations differ in diet and gut microbiome. Molecular Ecology 32: 6523-6542.
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The animal gut microbiome has been implicated in a number of key biological processes, ranging from digestion to behaviour, and has also been suggested to facilitate local adaptation. Yet studies in wild animals rarely compare multiple populations that differ ecologically, which is the level at which local adaptation may occur. Further, few studies simultaneously characterize diet and gut microbiome from the same sample, despite their probable interdependence. Here, we investigate the interplay between diet and gut microbiome in three geographically isolated populations of the critically endangered Grauer’s gorilla (Gorilla beringei graueri), which we show to be genetically differentiated. We find population- and social group-specific dietary and gut microbial profiles and covariation between diet and gut microbiome, despite the presence of core microbial taxa. There was no detectable effect of age, and only marginal effects of sex and genetic relatedness on the microbiome. Diet differed considerably across populations, with the high-altitude population consuming a lower diversity of plants compared to low-altitude populations, consistent with plant availability constraining dietary choices. The observed pattern of covariation between diet and gut microbiome is probably a result of long-term social and environmental factors. Our study suggests that the gut microbiome is sufficiently plastic to support flexible food selection and hence contribute to local adaptation.
Bambi M., Galla G., Donati C., Rovero F., Hauffe H. C., Barelli C. (2024): Gut microbiota variations in wild yellow baboons (Papio cynocephalus) are associated with sex and habitat disturbance. Scientific Reports 14: 869.
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Although male and female mammals differ in biological traits and functional needs, the contribution of this sexual dimorphism to variations in gut bacteria and fungi (gut microbiota) in relation to habitat type has not been fully examined. To understand whether the combination of sex and habitat affects gut microbiota variation, we analyzed 40 fecal samples of wild yellow baboons (Papio cynocephalus) living in contrasting habitat types (intact, well-protected vs. fragmented, less protected forests) in the Udzungwa Mountains of Tanzania. Sex determination was performed using the marker genes SRY (Sex-determining Region Y) and DDX3X-DDX3Y (DEAD-Box Helicase 3). Samples were attributed to 34 individuals (19 females and 15 males) belonging to five social groups. Combining the results of sex determination with two amplicon sequencing datasets on bacterial (V1–V3 region of the 16S rRNA gene) and fungal (ITS2) gut communities, we found that overall, baboon females had a significantly higher gut bacterial richness compared to males. Beta diversity estimates indicated that bacterial composition was significantly different between males and females, and this was true for individuals from both well- and less protected forests. Our results highlight the combined role of sex and habitat type in shaping variation in gut microbial communities in wild non-human primates.
Guimaraes Sales N., da Cruz Kaizer M., Browett S. S., Gabriel S. I., McDevitt A. D. (2024): Assessing the gut microbiome and the influence of host genetics on a critically endangered primate, the northern muriqui (Brachyteles hypoxanthus). Environmental DNA 6: e559.
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The Northern muriqui (Brachyteles hypoxanthus) is one of the world’s most critically endangered primates, with only ~1000 mature individuals remaining in the wild. Habitat loss and hunting have led to its sharp decline, making conservation efforts crucial. Analyses of gut microbiomes in wild populations can provide valuable information on host health and vulnerability, and ultimately, contribute to baseline knowledge toward improving conservation programs and reintroduction efforts. In this study, we analyzed the microbiome (16S rRNA metabarcoding) of fecal samples belonging to 53 uniquely genotyped individuals from three social groups from the Caparaó National Park, aiming to provide the first assessment of the microbiome diversity and composition for this species. Our results showed the muriqui gut microbiome was predominantly composed of the phyla Bacteroidetes and Firmicutes, with the dominant classes represented by Bacteroidia and Clostridia. High similarity in bacterial diversity and composition was found for individuals from distinct groups, suggesting a negligible geographical effect at the fine spatial scale analyzed. No significant effect of host genotype heterozygosity levels on microbiota diversity was recovered, but a significant influence of genetic distance on microbiota community structure and composition was demonstrated. Our findings stress the importance of considering associations between host genetics and the microbiome and suggest that the analyzed populations host a similar microbiome composition. This detailed microbiome assessment can aid conservation actions, including future anthropogenic impact assessments and animal reintroductions.
Sereme Y., Zarza S. M., Medkour H., Mezouar S., Dotras L., Barciela A., Hernandez-Aguilar R. A., Vitte J., Šmajs D., Louni M., Mulot B. (2024): Treponematosis in critically endangered Western chimpanzees (Pan troglodytes verus) in Senegal. One Health 18: 100694.
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Treponematoses encompass a group of chronic and debilitating bacterial diseases transmitted sexually or by direct contact and attributed to Treponema pallidum. Despite being documented since as far back as 1963, the epidemiology of treponematoses in wild primates has remained an uninvestigated territory due to the inherent challenges associated with conducting examinations and obtaining invasive biological samples from wild animals. The primary aim of this study was to investigate the presence of treponemal infections in the critically endangered Western chimpanzees in Senegal, utilizing an innovative non-invasive stool serology method. We provide compelling evidence of the existence of anti-Treponema-specific antibodies in 13 out of 29 individual chimpanzees. Our study also underscores the significant potential of stool serology as a valuable non-invasive tool for monitoring and surveilling crucial emerging diseases in wild animals. We recognize two major implications: (1) the imperative need to assess the risks of treponematosis in Western chimpanzee populations and (2) the necessity to monitor and manage this disease following a holistic One Health approach.
Wang D., Eckert J., Teague S., Al-Naji A., Haun D., Chahl J. (2024): Estimating the cardiac signals of chimpanzees using a digital camera: validation and application of a novel non-invasive method for primate research. Behavior Research Methods 56: 2064-2082.
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Cardiac measures such as heart rate measurements are important indicators of both physiological and psychological states. However, despite their extraordinary potential, their use is restricted in comparative psychology because traditionally cardiac measures involved the attachment of sensors to the participant’s body, which, in the case of undomesticated animals such as nonhuman primates, is usually only possible during anesthesia or after extensive training. Here, we validate and apply a camera-based system that enables contact-free detection of animals’ heart rates. The system automatically detects and estimates the cardiac signals from cyclic change in the hue of the facial area of a chimpanzee. In Study 1, we recorded the heart rate of chimpanzees using the new technology, while simultaneously measuring heart rate using classic PPG (photoplethysmography) finger sensors. We found that both methods were in good agreement. In Study 2, we applied our new method to measure chimpanzees’ heart rate in response to seeing different types of video scenes (groupmates in an agonistic interaction, conspecific strangers feeding, nature videos, etc.). Heart rates changed during video presentation, depending on the video content: Agonistic interactions and conspecific strangers feeding lead to accelerated heart rate relative to baseline, indicating increased emotional arousal. Nature videos lead to decelerated heart rate relative to baseline, indicating a relaxing effect or heightened attention caused by these stimuli. Our results show that the new contact-free technology can reliably assess the heart rate of unrestrained chimpanzees, and most likely other primates. Furthermore, our technique opens up new avenues of research within comparative psychology and facilitates the health management of captive individuals.
RABBITS, HARES, AND PIKAS
Kohl K. D., Varner J., Wilkening J. L., Dearing M. D. (2018): Gut microbial communities of American pikas (Ochotona princeps): evidence for phylosymbiosis and adaptations to novel diets. Journal of Animal Ecology 87: 323-330.
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Gut microbial communities provide many physiological functions to their hosts, especially in herbivorous animals. We still lack an understanding of how these microbial communities are structured across hosts in nature, especially within a given host species. Studies on laboratory mice have demonstrated that host genetics can influence microbial community structure, but that diet can overwhelm these genetic effects. We aimed to test these ideas in a natural system, the American pika (Ochotona princeps). First, pikas are high-elevation specialists with significant population structure across various mountain ranges in the USA, allowing us to investigate whether similarities in microbial communities match host genetic differences. Additionally, pikas are herbivorous, with some populations exhibiting remarkable dietary plasticity and consuming high levels of moss, which is exceptionally high in fibre and low in protein. This allows us to investigate adaptations to an herbivorous diet, as well as to the especially challenging diet of moss. Here, we inventoried the microbial communities of pika caecal pellets from various populations using 16S rRNA sequencing to investigate structuring of microbial communities across various populations with different natural diets. Microbial communities varied significantly across populations, and differences in microbial community structure were congruent with genetic differences in host population structure, a pattern known as “phylosymbiosis.” Several microbial members (Ruminococcus, Prevotella, Oxalobacter and Coprococcus) were detected across all samples, and thus likely represent a “core microbiome.” These genera are known to perform a number of services for herbivorous hosts such as fibre fermentation and the degradation of plant defensive compounds, and thus are likely important for herbivory in pikas. Moreover, pikas that feed on moss harboured microbial communities highly enriched in Melainabacteria. This uncultivable candidate phylum has been proposed to ferment fibre for herbivores, and thus may contribute to the ability of some pika populations to consume high amounts of moss. These findings demonstrate that both host genetics and diet can influence the microbial communities of the American pika. These animals may be novel sources of fibre-degrading microbes. Last, we discuss the implications of population-specific microbial communities for conservation efforts in this species.
Funosas G., Triadó-Margarit X., Castro F., Villafuerte R., Delibes-Mateos M., Rouco C., Casamayor E. O. (2021): Individual fate and gut microbiome composition in the European wild rabbit (Oryctolagus cuniculus). Scientific Reports 11: 766.
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Studies connecting microbiome composition and functional performance in wildlife have received little attention and understanding their connections with wildlife physical condition are sorely needed. We studied the variation in gut microbiota (hard fecal pellets) between allopatric subspecies of the European wild rabbit in wild populations and in captured individuals studied under captivity. We evaluated the influence of environmental and host-specific factors. The microbiome of wild rabbit populations reduced its heterogeneity under controlled conditions. None of the host-specific factors tested correlated with the microbiota composition. We only observed significant intra-group dispersion for the age factor. The most diverse microbiomes were rich in Ruminococcaceae potentially holding an enriched functional profile with dominance of cellulases and xylanases, and suggesting higher efficiency in the digestion of fiber-rich food. Conversely, low diversity gut microbiomes showed dominance of Enterobacteriaceae potentially rich in amylases. We preliminary noticed geographical variations in field populations with higher dominance of Ruminococcaceae in south-western than in north-eastern Spain. Spatial differences appeared not to be subspecies driven, since they were lost in captivity, but environmentally driven, although differences in social structure and behavior may also play a role that deserve further investigations. A marginally significant relationship between the Ruminococcaceae/Enterobacteriaceae ratio and potential life expectancy was observed in captive rabbits. We hypothesize that the gut microbiome may determine the efficiency of feeding resource exploitation, and can also be a potential proxy for life expectancy, with potential applications for the management of declining wild herbivorous populations. Such hypotheses remain to be explored in the future.
Montes-Carreto L. M., Aguirre-Noyola J. L., Solís-García I. A., Ortega J., Martinez-Romero E., Guerrero J. A. (2021): Diverse methanogens, bacteria and tannase genes in the feces of the endangered volcano rabbit (Romerolagus diazi). PeerJ 9: e11942.
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The volcano rabbit is the smallest lagomorph in Mexico, it is monotypic and endemic to the Trans-Mexican Volcanic Belt. It is classified as endangered by Mexican legislation and as critically endangered by the IUCN, in the Red List. Romerolagus diazi consumes large amounts of grasses, seedlings, shrubs, and trees. Pines and oaks contain tannins that can be toxic to the organisms which consume them. The volcano rabbit microbiota may be rich in bacteria capable of degrading fiber and phenolic compounds. We obtained the fecal microbiome of three adults and one young rabbit collected in Coajomulco, Morelos, Mexico. Taxonomic assignments and gene annotation revealed the possible roles of different bacteria in the rabbit gut. We searched for sequences encoding tannase enzymes and enzymes associated with digestion of plant fibers such as cellulose and hemicellulose. The most representative phyla within the Bacteria domain were: Proteobacteria, Firmicutes and Actinobacteria for the young rabbit sample (S1) and adult rabbit sample (S2), which was the only sample not confirmed by sequencing to correspond to the volcano rabbit. Firmicutes, Actinobacteria and Cyanobacteria were found in adult rabbit samples S3 and S4. The most abundant phylum within the Archaea domain was Euryarchaeota. The most abundant genera of the Bacteria domain were Lachnoclostridium (Firmicutes) and Acinetobacter (Proteobacteria), while Methanosarcina predominated from the Archaea. In addition, the potential functions of metagenomic sequences were identified, which include carbohydrate and amino acid metabolism. We obtained genes encoding enzymes for plant fiber degradation such as endo 1,4 β-xylanases, arabinofuranosidases, endoglucanases and β-glucosidases. We also found 18 bacterial tannase sequences.
Fu H., Li W. (2022): Metagenomic comparisons between soft and hard feces of plateau pikas (Ochotona curzoniae). Animals 12: 149.
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The division of hard and soft feces is an effective digestion strategy in the order Lagomorpha. Although previous studies have reported that hard and soft feces differ in morphology and component, the discrepancy in the microbiome remains unclear. This study explored the microbiomes of hard and soft feces in plateau pikas by sequencing the V3 and V4 regions of 16S rDNA. We found that hard feces harbored higher Firmicutes, while soft feces harbored higher Akkermansia. Increased rare bacterial taxa were observed in hard feces compared with soft feces. Moreover, hard and soft feces displayed a greater difference in terms of core operational taxonomy units (OTUs) compared to the total OTUs. The soft feces showed enhancements in all predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functions, indicating an advancing microbial metabolism compared to hard feces. The significantly upregulated pathways in soft feces were mainly enriched in metabolism of energy and carbohydrate, glycan biosynthesis, cofactors and vitamins, and amino acids—all of which are associated with increased contents of microbial proteins, vitamins, and short-chain fatty acids. Our study reports, for the first time, the differential microbiomes between hard and soft feces of pikas and provides direction for the future studies on cecotrophy.
RODENTS
Everest D. J., Tolhurst-Cherriman D. A. R., Davies H., Dastjerdi A., Ashton A., Blackett T., Meredith A. L., Milne E., Mill A., Shuttleworth C. M. (2019): Assessing a potential non-invasive method for viral diagnostic purposes in European squirrels. Hystrix 30: 44-50.
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Viral infections globally threaten wild and captive mammal populations, with surveillance options limited by a lack of non-invasive diagnostics; especially when infection is asymptomatic in nature. We explored the potential for hair samples collected from red (Sciurus vulgaris) and grey (Sciurus carolinensis) squirrels to provide a means of screening for adenovirus (ADV) and squirrelpox virus (SQPV) using evolving polymerase chain reaction (PCR) assays. An initial pilot study phase utilised samples opportunistically harvested from grey squirrels controlled in Gwynedd, United Kingdom (UK). The screening of 319 grey squirrel carcasses revealed 58% spleen ADV DNA qPCR and 69% SQPV antibody enzyme linked immunosorbent assay (ELISA) positives. We developed new nested ADV and SQPV qPCRs and examined tail hair samples from a sub-set of 80 of these 319 sampled squirrels and these assays amplified ADV and SQPV DNA in a higher proportion of animals than the original qPCR (94% and 21% respectively). Tail hair samples obtained from six Cumbrian red squirrels which had died from squirrelpox disease also revealed 100% SQPV and 50% ADV DNA positive by the nested qPCR assays. These findings indicate enhanced sensitivity for the new platform. The integration of this non-invasive approach in assessing viral infection has wide application in epidemiological studies of wild mammal populations, in particular, during conservation translocations, where asymptomatic infections are of concern.
Srivathsan A., Nagarajan N., Meier R. (2019): Boosting natural history research via metagenomic clean-up of crowdsourced feces. Plos Biology 17: e3000517.
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Biodiversity is in crisis due to habitat destruction and climate change. The conservation of many noncharismatic species is hampered by the lack of data. Yet, natural history research—a major source of information on noncharismatic species—is in decline. We here suggest a remedy for many mammal species, i.e., metagenomic clean-up of fecal samples that are “crowdsourced” during routine field surveys. Based on literature data, we estimate that this approach could yield natural history information for circa 1,000 species within a decade. Metagenomic analysis would simultaneously yield natural history data on diet and gut parasites while enhancing our understanding of host genetics, gut microbiome, and the functional interactions between traditional and new natural history data. We document the power of this approach by carrying out a “metagenomic clean-up” on fecal samples collected during a single night of small mammal trapping in one of Alfred Wallace’s favorite collecting sites.
Catalano S., Symeou A., Marsh K. J., Borlase A., Léger E., Fall C. B., Sène M., Diouf N. D., Ianniello D., Cringoli G., Rinaldi L. (2019): Mini-FLOTAC as an alternative, non-invasive diagnostic tool for Schistosoma mansoni and other trematode infections in wildlife reservoirs. Parasites & Vectors 12: 439.
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Schistosomiasis and food-borne trematodiases are not only of major public health concern, but can also have profound implications for livestock production and wildlife conservation. The zoonotic, multi-host nature of many digenean trematodes is a significant challenge for disease control programmes in endemic areas. However, our understanding of the epidemiological role that animal reservoirs, particularly wild hosts, may play in the transmission of zoonotic trematodiases suffers a dearth of information, with few, if any, standardised, reliable diagnostic tests available. We combined qualitative and quantitative data derived from post-mortem examinations, coprological analyses using the Mini-FLOTAC technique, and molecular tools to assess parasite community composition and the validity of non-invasive methods to detect trematode infections in 89 wild Hubert’s multimammate mice (Mastomys huberti) from northern Senegal. Parasites isolated at post-mortem examination were identified as Plagiorchis sp., Anchitrema sp., Echinostoma caproni, Schistosoma mansoni, and a hybrid between Schistosoma haematobium and Schistosoma bovis. The reports of E. caproni and Anchitrema sp. represent the first molecularly confirmed identifications for these trematodes in definitive hosts of sub-Saharan Africa. Comparison of prevalence estimates derived from parasitological analysis at post-mortem examination and Mini-FLOTAC analysis showed non-significant differences indicating comparable results between the two techniques (P = 1.00 for S. mansoni; P = 0.85 for E. caproni; P = 0.83 for Plagiorchis sp.). A Bayesian model, applied to estimate the sensitivities of the two tests for the diagnosis of Schistosoma infections, indicated similar median posterior probabilities of 83.1% for Mini-FLOTAC technique and 82.9% for post-mortem examination (95% Bayesian credible intervals of 64.0–94.6% and 63.7–94.7%, respectively). Our results showed that the Mini-FLOTAC could be applied as an alternative diagnostic technique for the detection of the zoonotic S. mansoni and other trematodes in rodent reservoirs. The implementation of non-invasive diagnostics in wildlife would offer numerous advantages over lethal sampling methodologies, with potential impact on control strategies of zoonotic helminthiases in endemic areas of sub-Saharan Africa and on fostering a framework of animal use reduction in scientific practice.
Fackelmann G., Gillingham M. A., Schmid J., Heni A. C., Wilhelm K., Schwensow N., Sommer S. (2021): Human encroachment into wildlife gut microbiomes. Communications Biology 4: 800.
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In the Anthropocene, humans, domesticated animals, wildlife, and their environments are interconnected, especially as humans advance further into wildlife habitats. Wildlife gut microbiomes play a vital role in host health. Changes to wildlife gut microbiomes due to anthropogenic disturbances, such as habitat fragmentation, can disrupt natural gut microbiota homeostasis and make animals vulnerable to infections that may become zoonotic. However, it remains unclear whether the disruption to wildlife gut microbiomes is caused by habitat fragmentation per se or the combination of habitat fragmentation with additional anthropogenic disturbances, such as contact with humans, domesticated animals, invasive species, and their pathogens. Here, we show that habitat fragmentation per se does not impact the gut microbiome of a generalist rodent species native to Central America, Tome’s spiny rat Proechimys semispinosus, but additional anthropogenic disturbances do. Indeed, compared to protected continuous and fragmented forest landscapes that are largely untouched by other human activities, the gut microbiomes of spiny rats inhabiting human-disturbed fragmented landscapes revealed a reduced alpha diversity and a shifted and more dispersed beta diversity. Their microbiomes contained more taxa associated with domesticated animals and their potential pathogens, suggesting a shift in potential metagenome functions. On the one hand, the compositional shift could indicate a degree of gut microbial adaption known as metagenomic plasticity. On the other hand, the greater variation in community structure and reduced alpha diversity may signal a decline in beneficial microbial functions and illustrate that gut adaption may not catch up with anthropogenic disturbances, even in a generalist species with large phenotypic plasticity, with potentially harmful consequences to both wildlife and human health.
Khalifeh A., Blumstein D. T., Fontenele R. S., Schmidlin K., Richet C., Kraberger S., Varsani A. (2021): Diverse cressdnaviruses and an anellovirus identified in the fecal samples of yellow-bellied marmots. Virology 554: 89-96.
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Over that last decade, coupling multiple strand displacement approaches with high throughput sequencing have resulted in the identification of genomes of diverse groups of small circular DNA viruses. Using a similar approach but with recovery of complete genomes by PCR, we identified a diverse group of single-stranded viruses in yellow-bellied marmot (Marmota flaviventer) fecal samples. From 13 fecal samples we identified viruses in the family Genomoviridae (n = 7) and Anelloviridae (n = 1), and several others that ware part of the larger Cressdnaviricota phylum but not within established families (n = 19). There were also circular DNA molecules identified (n = 4) that appear to encode one viral-like gene and have genomes of <1545 nts. This study gives a snapshot of viruses associated with marmots based on fecal sampling.
Wood R., Bangura U., Mariën J., Douno M., Fichet-Calvet E. (2021): Detection of Lassa virus in wild rodent feces: Implications for Lassa fever burden within households in the endemic region of Faranah, Guinea. One Health 13: 100317.
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Lassa arenavirus (LASV) is the cause of Lassa Fever in humans in West Africa. The multimammate mouse (Mastomys natalensis) is a reservoir host of LASV and the primary source of human infections. Humans are assumed to become infected due to contact with this animal or its excretions. Thus far, the available literature does not describe the sampling of feces as a means to detect LASV in M. natalensis populations. More evidence is needed to know if feces of naturally infected M. natalensis can be LASV-positive and an exposure risk to humans. This study sampled feces deposits in households from three villages in the LASV-endemic region of Faranah, Guinea. PCR analysis found 10 out of 88 samples to be positive for LASV, and sequencing showed clustering to previously identified Yarawelia and Dalafilani strains. We conclude that feces sampling is a viable, non-invasive method for the determination and sequencing of LASV strains.
Petrullo L., Ren T., Wu M., Boonstra R., Palme R., Boutin S., McAdam A. G., Dantzer B. (2022): Glucocorticoids coordinate changes in gut microbiome composition in wild North American red squirrels. Scientific Reports 12: 2605.
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The gut microbiome impacts host health and fitness, in part through the diversification of gut metabolic function and pathogen protection. Elevations in glucocorticoids (GCs) appear to reduce gut microbiome diversity in experimental studies, suggesting that a loss of microbial diversity may be a negative consequence of increased GCs. However, given that ecological factors like food availability and population density may independently influence both GCs and microbial diversity, understanding how these factors structure the GC-microbiome relationship is crucial to interpreting its significance in wild populations. Here, we used an ecological framework to investigate the relationship between GCs and gut microbiome diversity in wild North American red squirrels (Tamiasciurus hudsonicus). As expected, higher GCs predicted lower gut microbiome diversity and an increase in metabolic taxa. Surprisingly, but in line with prior empirical studies on wild animals, gastrointestinal pathogens decreased as GCs increased. Both dietary heterogeneity and an upcoming food pulse exhibited direct effects on gut microbiome diversity, whereas conspecific density and reproductive activity impacted diversity indirectly via changes in host GCs. Our results provide evidence of a gut–brain axis in wild red squirrels and highlight the importance of situating the GC-gut microbiome relationship within an ecological framework.
Chua K. O., Fatima I., Lau Y. Y., Hong K. W., Yin W. F., Mardaryev A., Chan K. G., Chang C. Y. (2022): Bacterial microbiome of faecal samples of naked mole-rat collected from the toilet chamber. BMC Research Notes 15: 107.
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The naked mole rats (NMRs, Heterocephalus glaber) are subterranean rodents that belong to the family Bathyergidae. They gained the attention of the scientific community for their exceptionally long lifespan of up to 30 years and have become an animal model of biomedical research on neurodegenerative diseases, aging and cancer. NMRs dig and survive in a maze of underground tunnels and chambers and demarcate toilet chambers for defecation and urination. Due to their coprophagic behaviours, we believed that the toilet chamber might play a role in maintaining optimal health of the NMRs. A 16S rRNA gene amplicon sequencing was performed to characterize the bacterial microbiome of faecal samples collected from the toilet chamber of a laboratory NMR colony. Four faecal samples were collected at different time points from the same toilet chamber of a laboratory NMR colony for analysis. The 16S rRNA gene amplicon sequencing revealed that bacterial phyla Firmicutes and Bacteroidetes were the dominant taxa in the bacterial microbiome of NMRs. The relative abundance of the bacterial taxa shifted substantially between time points, indicating a dynamic microbiome in the toilet chamber. The data provided an insight to the faecal microbiome of NMRs in the toilet chamber.
Scholier T., Lavrinienko A., Kallio E. R., Watts P. C., Mappes T. (2024): Effects of past and present habitat on the gut microbiota of a wild rodent. Proceedings of the Royal Society B 291: 20232531.
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The response of the gut microbiota to changes in the host environment can be influenced by both the host’s past and present habitats. To quantify their contributions for two different life stages, we studied the gut microbiota of wild bank voles (Clethrionomys glareolus) by performing a reciprocal transfer experiment with adults and their newborn offspring between urban and rural forests in a boreal ecosystem. Here, we show that the post-transfer gut microbiota in adults did not shift to resemble the post-transfer gut microbiota of animals ‘native’ to the present habitat. Instead, their gut microbiota appear to be structured by both their past and present habitat, with some features of the adult gut microbiota still determined by the past living environment (e.g. alpha diversity, compositional turnover). By contrast, we did not find evidence of the maternal past habitat (maternal effects) affecting the post-transfer gut microbiota of the juvenile offspring, and only a weak effect of the present habitat. Our results show that both the contemporary living environment and the past environment of the host organism can structure the gut microbiota communities, especially in adult individuals. These data are relevant for decision-making in the field of conservation and wildlife translocations.
UNGULATES
de Carvalho Ferreira H. C., Weesendorp E., Quak S., Stegeman J. A., Loeffen W. L. A. (2014): Suitability of faeces and tissue samples as a basis for non-invasive sampling for African swine fever in wild boar. Veterinary Microbiology 172: 449-454.
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A challenging aspect of ASFV control in wild boar populations is the design and implementation of effective surveillance and monitoring programmes, both for early warning, and to determine the ongoing epidemiological situation in an infected population. Testing blood samples requires invasive sampling strategies like hunting or capture of wild boar. Besides being biased towards healthy animals, such strategies are also linked to further spread of the virus. Non-invasive sampling strategies would increase the reliability of surveillance of ASFV in wild boar populations, without the negative side effects. This study evaluates the potential of faeces and tissue samples as a basis for non-invasive sampling strategies for ASFV in wild boar. In the acute phase (0–21 days after infection), in comparison with virus detection in blood, virus can be detected in faeces 50–80% of the time. This percentage decreases to below 10% for the subacute/chronic phase. ASFV DNA is quite stable in faeces. Half-lives range from more than 2 years at temperature up to 12 °C, to roughly 15 days at temperatures of 30 °C. In tissue samples, stored at 20 °C, half-lives mostly range from 1.7 to 7.4 days. The sample of preference is the spleen, where the highest titres and highest half-life of ASFV DNA are observed. The level and duration of excretion of ASFV in the faeces, combined with the stability of the DNA, suggest that sampling of faeces could be the basis for a non-invasive sampling strategy to monitor ASFV in wild boar.
Mouchantat S., Haas B., Böhle W., Globig A., Lange E., Mettenleiter T. C., Depner K. (2014): Proof of principle: non-invasive sampling for early detection of foot-and-mouth disease virus infection in wild boar using a rope-in-a-bait sampling technique. Veterinary Microbiology 172: 329-333.
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In this study we describe the use of a rope-in-a-bait sampling method (“pSWAB”: pathogen sampling wild animals with baits) for non-invasive saliva sampling aimed at the detection of foot-and-mouth disease (FMD) viral genome in wild boar. The pSWABs are produced in the form of a standardized product by embedding a 10 cm long cotton rope in a cereal-based bait matrix. To assess the general suitability of this novel sampling technique an animal experiment was conducted to detect FMD viral genome in saliva of infected wild boar. Two juvenile animals were inoculated in the bulb of the heel with a recent wild boar FMD virus isolate and kept together with three noninoculated wild boar of the same age. Over a period of 29 days, the animals were sampled by using five pSWABs per day in addition to the collection of blood and conventional saliva swabs taken every three to four days. Viral RNA in pSWABs was identified already 24 h after infection during the incubation period and until 23 dpi. Comparison of the results of pSWAB sampling with those of conventional saliva swabs or serum samples showed satisfactory sensitivity. These experimental data demonstrate the suitability of non-invasive sampling of wild boar by using pSWABs as a sensitive, cheap and feasible sample collection technique independent of hunting activities. In addition, the use of non-invasive sampling in an appropriate surveillance strategy is discussed.
Budischak S. A., Hoberg E. P., Abrams A., Jolles A. E., Ezenwa V. O. (2015): A combined parasitological molecular approach for noninvasive characterization of parasitic nematode communities in wild hosts. Molecular Ecology Resources 15: 1112-1119.
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Most hosts are concurrently or sequentially infected with multiple parasites; thus, fully understanding interactions between individual parasite species and their hosts depends on accurate characterization of the parasite community. For parasitic nematodes, noninvasive methods for obtaining quantitative, species-specific infection data in wildlife are often unreliable. Consequently, characterization of gastrointestinal nematode communities of wild hosts has largely relied on lethal sampling to isolate and enumerate adult worms directly from the tissues of dead hosts. The necessity of lethal sampling severely restricts the host species that can be studied, the adequacy of sample sizes to assess diversity, the geographic scope of collections and the research questions that can be addressed. Focusing on gastrointestinal nematodes of wild African buffalo, we evaluated whether accurate characterization of nematode communities could be made using a noninvasive technique that combined conventional parasitological approaches with molecular barcoding. To establish the reliability of this new method, we compared estimates of gastrointestinal nematode abundance, prevalence, richness and community composition derived from lethal sampling with estimates derived from our noninvasive approach. Our noninvasive technique accurately estimated total and species-specific worm abundances, as well as worm prevalence and community composition when compared to the lethal sampling method. Importantly, the rate of parasite species discovery was similar for both methods, and only a modest number of barcoded larvae (n = 10) were needed to capture key aspects of parasite community composition. Overall, this new noninvasive strategy offers numerous advantages over lethal sampling methods for studying nematode–host interactions in wildlife and can readily be applied to a range of study systems.
Stahl R. S., Ellis C. K., Nol P., Waters W. R., Palmer M., VerCauteren K. C. (2015): Fecal volatile organic ccompound profiles from white-tailed deer (Odocoileus virginianus) as indicators of Mycobacterium bovis exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccination. Plos One 10: e0129740.
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White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95–1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non-invasive monitoring system for the detection of tuberculosis in captive deer and other livestock.
Cheng Y. C., Hannaoui S., John T. R., Dudas S., Czub S., Gilch S. (2016): Early and non-invasive detection of chronic wasting disease prions in elk feces by real-time quaking induced conversion. Plos One 11: e0166187.
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Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.
Peterson R. O., Hoy S. R., Vucetich L. M., Vucetich J. A., Kraus V. B., Huebner J. L. (2018): Urinary biomarkers to non-invasively evaluate health in wild moose (Alces alces). Osteoarthritis and Cartilage 26: S196-S197.
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Our goal was to use non-invasive techniques to evaluate health and nutritional status of the moose population in Isle Royale National Park, Michigan, a 544-km2 island in Lake Superior, North America. More specifically, we evaluated the feasibility of using urinary biomarkers as indicators of skeletal integrity in a free-ranging moose population where osteoarthritis (OA) is a major pathology that predisposes older individuals (>10 years old) to death by wolf predation. We collected frozen urine deposited in the snow by moose of unknown sex and health status in winter. Moose were classified as either 9-month-old juveniles or non-juveniles based on the size of tracks left in the snow. We then quantified collagen type II (CTXII) and collagen type I (CTXI) C-telopeptide fragments by IDS ELISA; urea nitrogen (UN) and creatinine (Cr) were determined by spectrophotometry. Concentrations of all urinary biomarkers were corrected for dilution by Cr levels. Simple linear regression was used to assess the relationship between CTXI and UN (normalized to Cr). Both CTXI and CTXII were detectable in free-ranging moose using immunoassays specific for humans. We found that CTXII levels were significantly higher in juvenile moose (nine months old) than in older moose (Fig. 1), a result expected because juvenile moose exhibit open epiphyseal growth plates. Levels of CTXI (but not CTXII) were generally positively correlated (Fig. 2) with ratio of urinary urea nitrogen corrected for dilution by creatinine levels (UN:C). High UN:C values reflect active muscle catabolism that is often necessary for survival through winter for old moose with compromised health (including OA, osteoporosis, and periodontal disease). Finally, we found differences in CTXI levels between moose residing on opposite ends of Isle Royale that mirror other findings (UN:C, stable nitrogen isotope ratios, and proportion of balsam fir in the diet) that suggest fundamental spatial differences in population musculoskeletal status, possibly arising from differences in age structure, demography, or foraging ecology. It is feasible to determine CTXI and II levels in wild moose from frozen snow-urine corrected for dilution using urinary creatinine levels using the same methods developed for humans. Further evaluation is recommended using moose of known health status to gain a better understanding of CTXI and CTXII levels in free-ranging animals. Additionally, longitudinal sampling of wild moose (where age and sex can be determined using fecal DNA) could provide a better understanding of OA development in wild moose and what factors might contribute to an increased risk of OA. Finally, there could be an evaluation of how changing population age structure is related to CTXI and CTXII levels and how age structure and skeletal health might be linked to predation rates. Previously, we could monitor health status of this free-ranging population only by examining carcasses of deceased moose. Using this method, we found a steep decline in the prevalence of OA over the past four decades that was linked to a climate-induced decline in body size and ultimately shorter average lifespans. However, this approach was limited by the substantial length of time (decades) needed to collect a sufficient number of carcasses. In contrast, these biomarkers represent a valuable opportunity to gain insights about the health of the population in real time.
Montillo M., Caslini C., Peric T., Prandi A., Netto P., Tubaro F., Pedrotti L., Bianchi A., Mattiello S. (2019): Analysis of 19 minerals and cortisol in red deer hair in two different areas of the Stelvio National Park: a preliminary study. Animals 9: 492.
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The aim of the study was to perform an investigation on the concentration of 19 minerals and cortisol in red deer (Cervus elaphus) hair, a matrix that is easy to collect with non-invasive and painless sampling, able to represent an integrative values of long-term substance concentrations, and able to give useful information, also when performed on dead animals, given its extreme stability over time. In the study thirty-five animals were included, coming from two different sides of a valley in the Stelvio National Park, where official water analysis had pointed out elevated concentrations of As in one of the two orographic sides. Hair cortisol concentrations were measured using a RIA (Radio Immuno Assay), while minerals were detected using ICP-MS (Inductively Coupled Plasma- Mass Spectrometry). Results showed a negative relationship between cortisol and some mineral concentrations (Li, Co, As, Cd, Cr and Tl) and significant differences in some mineral concentrations between park areas (Al, Co, Cu, Cd and Ni). As, Cr and cortisol differences approached statistical significance. This preliminary study represents a step forward in the study of wildlife allostatic load and a valid method for applications in wildlife management programs, in environmental studies and in public health programs.
Muneza A. B., Ortiz-Calo W., Packer C., Cusack J. J., Jones T., Palmer M. S., Swanson A., Kosmala M., Dickman A. J., Macdonald D. W., Montgomery R. A. (2019): Quantifying the severity of giraffe skin disease via photogrammetry analysis of camera trap data. Journal of Wildlife Diseases 55: 770-781.
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Developing techniques to quantify the spread and severity of diseases afflicting wildlife populations is important for disease ecology, animal ecology, and conservation. Giraffes (Giraffa camelopardalis) are in the midst of a dramatic decline, but it is not known whether disease is playing an important role in the broad-scale population reductions. A skin disorder referred to as giraffe skin disease (GSD) was recorded in 1995 in one giraffe population in Uganda. Since then, GSD has been detected in 13 populations in seven African countries, but good descriptions of the severity of this disease are not available. We photogrammetrically analyzed camera trap images from both Ruaha and Serengeti National parks in Tanzania to quantify GSD severity. Giraffe skin disease afflicts the limbs of giraffes in Tanzania, and we quantified severity by measuring the vertical length of the GSD lesion in relation to the total leg length. Applying the Jenks natural breaks algorithm to the lesion proportions that we derived, we classified individual giraffes into disease categories (none, mild, moderate, and severe). Scaling up to the population level, we predicted the proportion of the Ruaha and Serengeti giraffe populations with mild, moderate, and severe GSD. This study serves to demonstrate that camera traps presented an informative platform for examinations of skin disease ecology.
Seeber P. A., Dayaram A., Sicks F., Osterrieder N., Franz M., Greenwood A. D. (2019): Noninvasive detection of equid herpesviruses in fecal samples. Applied and Environmental Microbiology 85: e02234-18.
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Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grévy’s zebra (Equus grevyi) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened (n = 18 [Equus quagga and E. zebra]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids.
Harvey A. M., Beausoleil N. J., Ramp D., Mellor D. J. (2020): A ten-stage protocol for assessing the welfare of individual non-captive wild animals: free-roaming horses (Equus ferus caballus) as an example. Animals 10: 148.
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Knowledge of the welfare status of wild animals is vital for informing debates about the ways in which we interact with wild animals and their habitats. Currently, there is no published information about how to scientifically assess the welfare of free-roaming wild animals during their normal day-to-day lives. Using free-roaming horses as an example, we describe a ten-stage protocol for systematically and scientifically assessing the welfare of individual non-captive wild animals. The protocol starts by emphasising the importance of readers having an understanding of animal welfare in a conservation context and also of the Five Domains Model for assessing welfare. It goes on to detail what species-specific information is required to assess welfare, how to identify measurable and observable indicators of animals’ physical states and how to identify which individuals are being assessed. Further, it addresses how to select appropriate methods for measuring/observing physical indicators of welfare, the scientific validation of these indicators and then the grading of animals’ welfare states, along with assigning a confidence score. Finally, grading future welfare risks and how these can guide management decisions is discussed. Applying this ten-stage protocol will enable biologists to scientifically assess the welfare of wild animals and should lead to significant advances in the field of wild animal welfare.
Nol P., Ionescu R., Geremariam Welearegay T., Barasona J. A., Vicente J., de Jesus Beleño-Sáenz K., Barrenetxea I., Jose Torres M., Ionescu F., Rhyan J. (2020): Evaluation of volatile organic compounds obtained from breath and feces to detect Mycobacterium tuberculosis complex in wild boar (Sus scrofa) in Doñana National Park, Spain. Pathogens 9: 346.
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The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donaña National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12–24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an α value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.
Carrau T., Martínez-Carrasco C., Garijo M. M., Alonso F., de Ybáñez R. R., Tizzani P. (2021): Evaluation of the Baermann–Wetzel method for detecting lungworm larvae in wild ruminants from faecal samples. Journal of Helminthology 95: e13.
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Lungworms can exert a negative impact on wild ruminant fitness; for this reason, the diagnosis of the associated diseases is an important prevention measure. The Baerman–Wetzel technique is the most usual method for the diagnosis of bronchopulmonary nematodes and is based on the active migration and movement of their first-stage larvae. Pulmonary tissue samples are frequently used for the post-mortem diagnosis of these parasites, but this kind of sample is not always available and easy to obtain. Faecal samples represent a more accessible choice for parasite monitoring. This work aimed to evaluate the agreement between the results obtained by the Baermann–Wetzel technique when samples of lung parenchyma or faeces from wild ruminants are used. A good level of agreement as well as a similar sensitivity between the two types of sample were observed, validating the use of faecal samples as a less invasive and cost-effective alternative for the monitoring of lungworm in wild ruminant populations.
Clavadetscher I., Bond M., Martin L., Schiffmann C., Hatt J. M., Clauss M. (2021): Development of an image-based body condition score for giraffes Giraffa camelopardalis and a comparison of zoo-housed and free-ranging individuals. Journal of Zoo and Aquarium Research 9: 170-185.
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Historically, giraffes (Giraffa spp.) in zoos are known to have a high prevalence of deaths associated with serous fat atrophy, which has been linked to the impression that as browsers, they are more difficult to feed appropriately compared to grazing ruminants. Therefore, one could expect zoo-housed giraffes to be peculiar in that they might have, on average, a lower body condition than their free-ranging conspecifics. We collected photographs of free-ranging and zoo-housed individuals and used information on sex, age, body mass, and height for a subset of the zoo animals to develop and validate a body condition score (BCS). We developed an overview BCS for the shoulder area (4 levels) and hip area (7 levels), and validated and applied the score to 532 free-ranging and 232 zoo-housed giraffes. The rib area was not useful for BCS; skin folds in the rib area occurred across all BCS hip levels but were particularly prominent at higher BCS hip. The BCS hip was positively correlated with body mass index for adult female giraffes, suggesting it reflects body condition. The BCS hip differentiated better between age, gender, season in free-ranging animals, and habitat (zoo vs. free-ranging). The BCS shoulder was less precise, most likely because the visibility of the shoulder does not only reflect subcutaneous adipose tissue, but also muscle tissue and skin thickness, especially in males as an adaptation to the giraffe’s typical mode of intraspecific combat. Using the shoulder score or the presence of skin folds on the side of the thorax/abdomen for routine BCS application is therefore not recommended. Juvenile animals had the highest BCS hip (6.04±0.69 for free-ranging, 6.00±1.07 for zoo), which decreased with age in both populations. Adult males in the wild generally had higher BCS hip (3.52±1.35) scores than the females in the wild (3.31±1.31), indicating the increased energetic strain on females due to reproduction. This was not the case in zoo animals. In contrast to our prediction, zoo-housed animals had higher scores than the free-ranging population (4.53±1.54; 3.74±1.55; P<0.001), especially compared to the free-ranging population scores from the end of the dry season (3.46±1.56; P<0.001). This indicates that on average, zoo-housed giraffe are less constrained by dietary resources than their free-ranging counterparts, and corresponds to reports of an improvement of zoo diets in European zoos and the subjective impression of a reduced incidence of serous fat atrophy in recent years. Nevertheless, the highest score, often equated with obesity in other BCS systems, was observed in both zoos and in the wild, possibly excluding obesity as a pathological condition in this zoo-housed giraffe population. While low scores should be avoided in zoo-housed giraffes, there are no indications so far that high scores in our BCS are detrimental. The hip-based BCS is easily applicable to free-ranging animals by direct observation or based on photographs, and allows for reproducible data collection to monitor giraffes at a population level.
Davey M. L., Utaaker K. S., Fossøy F. (2021): Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding. Parasites & Vectors 14: 422.
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Gastrointestinal parasitic nematodes can impact fecundity, development, behaviour, and survival in wild vertebrate populations. Conventional monitoring of gastrointestinal parasitic nematodes in wild populations involves morphological identification of eggs, larvae, and adults from faeces or intestinal samples. Adult worms are typically required for species-level identification, meaning intestinal material from dead animals is needed to characterize the nematode community with high taxonomic resolution. DNA metabarcoding of environmental samples is increasingly used for time- and cost-effective, high-throughput biodiversity monitoring of small-bodied organisms, including parasite communities. Here, we evaluate the potential of DNA metabarcoding of faeces and soil samples for non-invasive monitoring of gastrointestinal parasitic nematode communities in a wild ruminant population. Faeces and intestines were collected from a population of wild reindeer, and soil was collected both from areas showing signs of animal congregation, as well as areas with no signs of animal activity. Gastrointestinal parasitic nematode faunas were characterized using traditional morphological methods that involve flotation and sedimentation steps to concentrate nematode biomass, as well as using DNA metabarcoding. DNA metabarcoding was conducted on bulk samples, in addition to samples having undergone sedimentation and flotation treatments. DNA metabarcoding and morphological approaches were largely congruent, recovering similar nematode faunas from all samples. However, metabarcoding provided higher-resolution taxonomic data than morphological identification in both faeces and soil samples. Although concentration of nematode biomass by sedimentation or flotation prior to DNA metabarcoding reduced non-target amplification and increased the diversity of sequence variants recovered from each sample, the pretreatments did not improve species detection rates in soil and faeces samples. DNA metabarcoding of bulk faeces samples is a non-invasive, time- and cost-effective method for assessing parasitic nematode populations that provides data with comparable taxonomic resolution to morphological methods that depend on parasitological investigations of dead animals. The successful detection of parasitic gastrointestinal nematodes from soils demonstrates the utility of this approach for mapping distribution and occurrences of the free-living stages of gastrointestinal parasitic nematodes.
Jiang F., Gao H., Qin W., Song P., Wang H., Zhang J., Liu D., Wang D., Zhang T. (2021): Marked seasonal variation in structure and function of gut microbiota in forest and alpine musk deer. Frontiers in Microbiology 12: 699797.
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Musk deer (Moschus spp.) is a globally endangered species due to excessive hunting and habitat fragmentation. Captive breeding of musk deer can efficiently relieve the hunting pressure and contribute to the conservation of the wild population and musk supply. However, its effect on the gut microbiota of musk deer is unclear. Recent studies have indicated that gut microbiota is associated with host health and its environmental adaption, influenced by many factors. Herein, high-throughput sequencing of the 16S rRNA gene was used based on 262 fecal samples from forest musk deer (M. berezovskii) (FMD) and 90 samples from alpine musk deer (M. chrysogaster) (AMD). We sought to determine whether seasonal variation can affect the structure and function of gut microbiota in musk deer. The results demonstrated that FMD and AMD had higher α-diversity of gut microbiota in the cold season than in the warm season, suggesting that season change can affect gut microbiota diversity in musk deer. Principal coordinate analysis (PCoA) also revealed significant seasonal differences in the structure and function of gut microbiota in AMD and FMD. Particularly, phyla Firmicutes and Bacteroidetes significantly dominated the 352 fecal samples from captive FMD and AMD. The relative abundance of Firmicutes and the ratio of Firmicutes to Bacteroidetes were significantly decreased in summer than in spring and substantially increased in winter than in summer. In contrast, the relative abundance of Bacteroidetes showed opposite results. Furthermore, dominant bacterial genera and main metabolic functions of gut microbiota in musk deer showed significant seasonal differences. Overall, the abundance of main gut microbiota metabolic functions in FMD was significantly higher in the cold season. WGCNA analysis indicated that OTU6606, OTU5027, OTU7522, and OTU3787 were at the core of the network and significantly related with the seasonal variation. These results indicated that the structure and function in the gut microbiota of captive musk deer vary with seasons, which is beneficial to the environmental adaptation and the digestion and metabolism of food. This study provides valuable insights into the healthy captive breeding of musk deer and future reintroduction programs to recover wild populations.
Liu R., Shi J., Shultz S., Guo D., Liu D. (2021): Fecal bacterial community of allopatric przewalski’s gazelles and their sympatric relatives. Frontiers in Microbiology 12: 737042.
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Mammal gastrointestinal tracts harbor diverse bacterial communities that play important roles in digestion, development, behavior, and immune function. Although, there is an increasing understanding of the factors that affect microbial community composition in laboratory populations, the impact of environment and host community composition on microbiomes in wild populations is less understood. Given that the composition of bacterial communities can be shaped by ecological factors, particularly exposure to the microbiome of other individuals, inter-specific interactions should impact on microbiome community composition. Here, we evaluated inter-population and inter-specific similarity in the fecal microbiota of Przewalski’s gazelle (Procapra przewalskii), an endangered endemic ruminant around Qinghai Lake in China. We compared the fecal bacterial communities of three Przewalski’s gazelle populations, with those of two sympatric ruminants, Tibetan gazelle (Procapra picticaudata) and Tibetan sheep (Ovis aries). The fecal bacterial community richness (Chao1, ACE) did not vary across the three Przewalski’s gazelle populations, nor did the composition vary between species. In contrast, the managed Przewalski’s gazelle population had higher bacterial diversity (Shannon and Simpson) and was more similar to its sympatric Tibetan sheep in beta diversity than the wild Przewalski’s gazelle populations. These results suggest that ecological factors like host community composition or diet affect Przewalski’s gazelle’s gastrointestinal bacterial community. The role of bacterial community composition in maintaining gastrointestinal health should be assessed to improve conservation management of endangered Przewalski’s gazelle. More broadly, captive breeding and reintroduction efforts may be impeded, where captive management results in dysbiosis and introduction of pathogenic bacteria. In free ranging populations, where wildlife and livestock co-occur, infection by domestic pathogens and diseases may be an underappreciated threat to wild animals.
Yanez-Montalvo A., Gaona O., Águila B., Arias-Domínguez N., Falcón L. I., Pérez-Flores J. (2021): Tapirus bairdii-associated fecal microbiome from a critical conservation area: Calakmul, México. Current Microbiology 78: 2648-2659.
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Baird’s tapir (Tapirus bairdii) is the largest native terrestrial mammal in the Neotropics, which is endangered primarily as a consequence of habitat loss and overhunting. Baird’s tapir is predominantly nocturnal and exists at low densities which complicates field studies. Baird’s tapir is a large-bodied herbivore that plays a key role in maintaining healthy tropical forests through seed dispersal in its feces. Studies of gut microbiome are essential and valuable to assess the health status of the host and the interaction with the environment. In this study, we collected fresh fecal samples of T. bairdii to analyze its gut microbiome during the rainy and dry seasons in the Calakmul region, which is a critical rainforest conservation area in Mexico. The results of a high-throughput 16S rDNA gene sequencing approach suggest that the fecal microbiome of Baird’s tapir has no significant differences in composition among seasons. The most common phyla were Firmicutes, Bacteroidetes, Proteobacteria, Kiritimatiellaeota, and Spirochaetes. This study suggests that the stability of the fecal microbiome is related to similar feeding strategies throughout the year, and emphasizes the value of tapir in seed dispersal (and associated microbes) to the well-conserved forests of the Greater Calakmul region as biodiversity hotspots for conservation.
Jutha N., Jardine C., Schwantje H., Mosbacher J., Kinniburgh D., Kutz S. (2022): Evaluating the use of hair as a non-invasive indicator of trace mineral status in woodland caribou (Rangifer tarandus caribou). Plos One 17: e0269441.
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Trace mineral imbalances can have significant effects on animal health, reproductive success, and survival. Monitoring their status in wildlife populations is, therefore, important for management and conservation. Typically, livers and kidneys are sampled to measure mineral status, but biopsies and lethal-sampling are not always possible, particularly for Species at Risk. We aimed to: 1) determine baseline mineral levels in Northern Mountain caribou (Rangifer tarandus caribou; Gmelin, 1788) in northwestern British Columbia, Canada, and 2) determine if hair can be used as an effective indicator of caribou mineral status by evaluating associations between hair and organ mineral concentrations. Hair, liver, and kidney samples from adult male caribou (nHair = 31; nLiver, nKidney = 43) were collected by guide-outfitters in 2016–2018 hunting seasons. Trace minerals and heavy metals were quantified using inductively-coupled plasma mass spectrometry, and organ and hair concentrations of same individuals were compared. Some organ mineral concentrations differed from other caribou populations, though no clinical deficiency or toxicity symptoms were reported in our population. Significant correlations were found between liver and hair selenium (rho = 0.66, p<0.05), kidney and hair cobalt (rho = 0.51, p<0.05), and liver and hair molybdenum (rho = 0.37, p<0.10). These findings suggest that hair trace mineral assessment may be used as a non-invasive and easily-accessible way to monitor caribou selenium, cobalt, and molybdenum status, and may be a valuable tool to help assess overall caribou health.
Liu D., Li Z., Hou Z., Bao H., Luan X., Zhang P., Liang X., Gong S., Tian Y., Zhang D., She W., Yang F., Chen S., Roberts N. J., Jiang G. (2022): Ecological relationships among habitat type, food nutrients, parasites and hormones in wild boar Sus scrofa during winter. Wildlife Biology: e01020.
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Habitat quality and parasite assembly influence wildlife health, and they are key indicators of health and survivability of wildlife populations. To investigate the potential ecological relationships among habitat type, food nutrients, parasites and hormones in wild boar Sus scrofa, we collected samples of wild boar feces and available plants in their habitat by line transects during winter. Along transects, we identified the composition of plants foraged by wild boar and measured the content of nutrients in available plants to estimate nutrient intake. We also quantified parasites and hormones in wild boar fecal samples. We compared food nutrients among different forest types and explored possible relationships among estimated nutrient intake, parasites and hormones. We found coniferous forest had positive effects on estimated fat intake and negative effects on estimated protein and fiber intake by wild boar. Furthermore, we revealed that estimated fat intake was negatively correlated with Metastrongylus elongatus parasites and positively correlated with triiodothyronine (T3). In contrast, estimated protein intake was positively correlated with M. elongatus and negatively correlated with T3. Finally, we found negative relationships between T3 concentrations and loads of Ascaris suum parasites and between cortisol (COR) and loads of Trichuris suis parasites. These insights on ecological relationships help identify potential dietary parameters in winter that could help predict and manage parasite and hormone responses for wild boar population recovery.
Pannoni S. B., Proffitt K. M., Holben W. E. (2022): Non‐invasive monitoring of multiple wildlife health factors by fecal microbiome analysis. Ecology and Evolution 12: e8564.
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Fecal microbial biomarkers represent a less invasive alternative for acquiring information on wildlife populations than many traditional sampling methodologies. Our goal was to evaluate linkages between fecal microbiome communities in Rocky Mountain elk (Cervus canadensis) and four host factors including sex, age, population, and physical condition (body-fat). We paired a feature-selection algorithm with an LDA-classifier trained on elk differential bacterial abundance (16S-rRNA amplicon survey) to predict host health factors from 104 elk microbiomes across four elk populations. We validated the accuracy of the various classifier predictions with leave-one-out cross-validation using known measurements. We demonstrate that the elk fecal microbiome can predict the four host factors tested. Our results show that elk microbiomes respond to both the strong extrinsic factor of biogeography and simultaneously occurring, but more subtle, intrinsic forces of individual body-fat, sex, and age-class. Thus, we have developed and described herein a generalizable approach to disentangle microbiome responses attributed to multiple host factors of varying strength from the same bacterial sequence data set. Wildlife conservation and management presents many challenges, but we demonstrate that non-invasive microbiome surveys from scat samples can provide alternative options for wildlife population monitoring. We believe that, with further validation, this method could be broadly applicable in other species and potentially predict other measurements. Our study can help guide the future development of microbiome-based monitoring of wildlife populations and supports hypothetical expectations found in host-microbiome theory.
Davey M. L., Kamenova S., Fossøy F., Solberg E. J., Davidson R., Mysterud A., Rolandsen C. M. (2023): Faecal metabarcoding provides improved detection and taxonomic resolution for non-invasive monitoring of gastrointestinal nematode parasites in wild moose populations. Parasites & Vectors 16: 19.
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Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume.
Pannoni S. B., Holben W. E. (2024): Wildlife fecal microbiota exhibit community stability across a longitudinal semi-controlled non-invasive sampling experiment. Frontiers in Microbiomes 3: 1274277.
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Wildlife microbiome studies are being used to assess microbial links with animal health and habitat. The gold standard of sampling microbiomes directly from captured animals is ideal for limiting potential abiotic influences on microbiome composition, yet fails to leverage the many benefits of non-invasive sampling. Application of microbiome-based monitoring for rare, endangered, or elusive species creates a need to non-invasively collect scat samples shed into the environment. Since controlling sample age is not always possible, the potential influence of time-associated abiotic factors was assessed. To accomplish this, we analyzed partial 16S rRNA genes of fecal metagenomic DNA sampled non-invasively from Rocky Mountain elk (Cervus canadensis) near Yellowstone National Park. We sampled pellet piles from four different elk, then aged them in a natural forest plot for 1, 3, 7, and 14 days, with triplicate samples at each time point (i.e., a blocked, repeat measures (longitudinal) study design). We compared fecal microbiota of each elk through time with point estimates of diversity, bootstrapped hierarchical clustering of samples, and a version of ANOVA–simultaneous components analysis (ASCA) with PCA (LiMM-PCA) to assess the variance contributions of time, individual and sample replication. Our results showed community stability through days 0, 1, 3 and 7, with a modest but detectable change in abundance in only 2 genera (Bacteroides and Sporobacter) at day 14. The total variance explained by time in our LiMM-PCA model across the entire 2-week period was not statistically significant (p>0.195) and the overall effect size was small (<10% variance) compared to the variance explained by the individual animal (p<0.0005; 21% var.). We conclude that non-invasive sampling of elk scat collected within one week during winter/early spring provides a reliable approach to characterize fecal microbiota composition in a 16S rDNA survey and that sampled individuals can be directly compared across unknown time points with minimal bias. Further, point estimates of microbiota diversity were not mechanistically affected by sample age. Our assessment of samples using bootstrap hierarchical clustering produced clustering by animal (branches) but not by sample age (nodes). These results support greater use of non-invasive microbiome sampling to assess ecological patterns in animal systems.
Varzandi A. R., Zanet S., Seano P. B., Occhibove F., Vada R., Benatti F., Mayordomo P. P., Ferroglio E. (2024): Detection of African swine fever virus and wild boar eDNA in soil and turbid water samples: towards environmental surveillance. European Journal of Wildlife Research 70: 4.
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Since 2007, an ongoing African swine fever (ASF) pandemic has significantly impacted Eurasia. Extensive field evidence and modeling confirm the central role of wild boar in ASF epidemiology. To effectively control and eradicate the infection, rapid detection of the ASF virus (ASFV) is crucial for prompt intervention in areas of recent viral introduction or ongoing outbreaks. Environmental DNA (eDNA) is a cost-effective and non-invasive technique that has shown promising results in monitoring animal species and their pathogens and has the potential to be used for wildlife disease surveillance. In this study, we designed and evaluated an eDNA sampling method for highly turbid water and soil samples to detect ASFV and wild boar (Sus scrofa) DNA as a control using qPCR while ensuring biosafety measures and evaluating ASF epidemiology. To validate our method, we obtained samples from La Mandria Regional Park (LMRP) in northwestern Italy, an area free of ASFV, and spiked them in a laboratory setting with an ASFV’s synthetic DNA template. Our findings highlight the potential of eDNA monitoring as a reliable, rapid, and safe method for early detection of ASFV from soil and turbid water samples.
MULTIPLE SPECIES
Kraberger S., Waits K., Ivan J., Newkirk E., VandeWoude S., Varsani A. (2018): Identification of circular single-stranded DNA viruses in faecal samples of Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus) inhabiting the Colorado San Juan Mountains. Infection, Genetics and Evolution 64: 1-8.
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The San Juan Mountains of southern Colorado provide subalpine habitat for a suite of mammalian species including Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus). In the winter field season of 2016 five faecal samples from lynx, and one each from moose and snowshoe hare were collected to identify small single-stranded DNA viruses associated with these three prominent species. Thirty-two novel viruses were identified and classified as members of two well established ssDNA families Genomoviridae (n = 22) and Microviridae (n = 10) and one recently proposed new family, Smacoviridae (n = 1). In addition one highly novel circular ssDNA virus was identified which at present does not group with any known family. A high level of genomovirus diversity was identified from faeces collected between and across the three mammal species, with full genome-wide pairwise comparisons showing 57%–97% identity. Twenty genomoviruses can be assigned to the genus Gemycircularvirus and represent 11 species, and two into a distinct species in the genus Gemykolovirus. The single smacovirus identified from moose also represents a distinct smacovirus species. Ten microviruses, seven from moose, one from snowshoe hare and two from lynx, all are part of the Gokushovirinae subfamily. The two from lynx are highly similar to a microvirus previously detected in domestic cat (sharing 88%–90% genome-wide identity), indicating this may be a common felid gut microbiome associated virus. Our findings highlight the broad range of diverse ssDNA viruses present in three mammals inhabiting the San Juan Mountains.
Kartzinel T. R., Hsing J. C., Musili P. M., Brown B. R., Pringle R. M. (2019): Covariation of diet and gut microbiome in African megafauna. Proceedings of the National Academy of Sciences 116: 23588-23593.
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A major challenge in biology is to understand how phylogeny, diet, and environment shape the mammalian gut microbiome. Yet most studies of nonhuman microbiomes have relied on relatively coarse dietary categorizations and have focused either on individual wild populations or on captive animals that are sheltered from environmental pressures, which may obscure the effects of dietary and environmental variation on microbiome composition in diverse natural communities. We analyzed plant and bacterial DNA in fecal samples from an assemblage of 33 sympatric large-herbivore species (27 native, 6 domesticated) in a semiarid East African savanna, which enabled high-resolution assessment of seasonal variation in both diet and microbiome composition. Phylogenetic relatedness strongly predicted microbiome composition (r = 0.91) and was weakly but significantly correlated with diet composition (r = 0.20). Dietary diversity did not significantly predict microbiome diversity across species or within any species except kudu; however, diet composition was significantly correlated with microbiome composition both across and within most species. We found a spectrum of seasonal sensitivity at the diet−microbiome nexus: Seasonal changes in diet composition explained 25% of seasonal variation in microbiome composition across species. Species’ positions on (and deviations from) this spectrum were not obviously driven by phylogeny, body size, digestive strategy, or diet composition; however, domesticated species tended to exhibit greater diet−microbiome turnover than wildlife. Our results reveal marked differences in the influence of environment on the degree of diet−microbiome covariation in free-ranging African megafauna, and this variation is not well explained by canonical predictors of nutritional ecology.
Brealey J. C., Leitão H. G., van der Valk T., Xu W., Bougiouri K., Dalén L., Guschanski K. (2020): Dental calculus as a tool to study the evolution of the mammalian oral microbiome. Molecular Biology and Evolution 37: 3003-3022.
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Dental calculus, the calcified form of the mammalian oral microbial plaque biofilm, is a rich source of oral microbiome, host, and dietary biomolecules and is well preserved in museum and archaeological specimens. Despite its wide presence in mammals, to date, dental calculus has primarily been used to study primate microbiome evolution. We establish dental calculus as a valuable tool for the study of nonhuman host microbiome evolution, by using shotgun metagenomics to characterize the taxonomic and functional composition of the oral microbiome in species as diverse as gorillas, bears, and reindeer. We detect oral pathogens in individuals with evidence of oral disease, assemble near-complete bacterial genomes from historical specimens, characterize antibiotic resistance genes, reconstruct components of the host diet, and recover host genetic profiles. Our work demonstrates that metagenomic analyses of dental calculus can be performed on a diverse range of mammalian species, which will allow the study of oral microbiome and pathogen evolution from a comparative perspective. As dental calculus is readily preserved through time, it can also facilitate the quantification of the impact of anthropogenic changes on wildlife and the environment.
Dib L. V., Palmer J. P. S., de Souza Carvalho Class C., Pinheiro J. L., Ramos R. C. F., Dos Santos C. R., Fonseca A. B. M., Rodriguez-Castro K. G., Goncalves C. F., Galetti P. M., da Silva Barbosa A. (2020): Non-invasive sampling in Itatiaia National Park, Brazil: wild mammal parasite detection. BMC Veterinary Research 16: 295.
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Non-invasive sampling through faecal collection is one of the most cost-effective alternatives for monitoring of free-living wild mammals, as it provides information on animal taxonomy as well as the dynamics of the gastrointestinal parasites that potentially infect these animals. In this context, this study aimed to perform an epidemiological survey of gastrointestinal parasites using non-invasive faecal samples from carnivores and artiodactyls identified by stool macroscopy, guard hair morphology and DNA sequencing in Itatiaia National Park. Between 2017 and 2018, faeces from carnivores and artiodactyls were collected along trails in the park. The host species were identified through macroscopic and trichological examinations and molecular biology. To investigate the parasites, the Faust, Lutz and modified Ritchie and Sheather techniques and enzyme immunoassays to detect Cryptosporidium sp. antigens were used. A total of 244 stool samples were collected. The species identified were Chrysocyon brachyurus, Leopardus guttulus, Canis familiaris, Cerdocyon thous, Puma yagouaroundi, Leopardus pardalis, Puma concolor and Sus scrofa. There were 81.1% samples that were positive for parasites distributed mainly in the high part of the park. Helminths, especially eggs of the family Ascarididae, were more frequently detected in carnivore faeces (70.9%). Protozoa, especially Cryptosporidium sp., represented the highest frequency of infection in artiodactyl faeces (87.1%). This zoonotic protozoon was detected in eight mammalian species, including in a wild boar. High values of structural richness and Shannon and Simpson diversity indices were observed for the parasites, especially in the faeces of C. brachyurus. Significant differences in parasite diversity were observed between wild and domestic animals, such as C. brachyurus and C. familiaris, respectively, and between taxonomically distant species, such as C. brachyurus and S. scrofa. The highest values for parasite similarity were found among the species that frequented similar areas of the park, such as C. brachyurus and L. guttulus. The animals and parasite infections were identified through the combination of three techniques. High frequency parasite structures were diagnosed. Zoonotic protozoa were found and mainly occurred in samples from introduced species.
Sepalage C. S., Rajakaruna R. S. (2020): Gastrointestinal helminth and protozoan infections of wild mammals in four major national parks in Sri Lanka. Journal of Threatened Taxa 12: 17093-17104.
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A cross-sectional, coprological survey of gastrointestinal (GI) parasites of wild mammals in four major National Parks in Sri Lanka: Wilpattu, Udawalawe, Wasgamuwa, and Horton Plains was carried out during November 2016 to August 2017. Fresh fecal samples were collected and analyzed using sedimentation technique, iodine & saline smears, and Sheather’s sucrose flotation for morphological identification parasite eggs, cysts, and larvae. A modified salt flotation was carried out for egg counts. Seventy samples from 10 mammal species: Asian Elephant, Spotted Deer, Water Buffalo, Sambar, Indian Hare, Asian Palm Civet, Sloth Bear, Wild Boar, Grey Langur, Leopard, and four unknown mammals (two carnivores, one herbivore and one omnivore) were analyzed. Most were infected (94.3%) with more than one GI parasites. The highest prevalence of infection was recorded in Horton Plains (100%), followed by Wasgamuwa (92.8%), Wilpattu (90.4%) and Udawalawe (75.0%) with a significant difference among four parks (Chi square test; χ2=35.435; df=3; p<0.001). Nineteen species of GI parasites were recorded, of which Entamoeba, Isospora, Balantidium, Fasciola, Moniezia, Dipylidium, strongyles, Toxocara, Trichiurus and hookworms were the most common. Strongyles (62.1%) and Entamoeba (80.3%) were the most prevalent helminth and protozoan infections, respectively. Overall, there was no difference in the prevalence of protozoans (84.3%) and helminths (87.1%; χ2=1.0; df=1; p=0.317). In carnivores, Entamoeba, Balantidium, Moniezia, strongyles and Strongyloides were common and in herbivores, Entamoeba, strongyles, Strongyloides and Toxocara were common. The quantitative analysis showed strongyles (17.639 EPG) and Isospora (18,743 OPG) having the highest infection intensity among helminthes and protozoans, respectively. This study provides baseline information of GI parasites and their distribution in wild mammals in the four national parks. Although the prevalence of GI infections was high, their intensity shows that they could be incidental infections. When the prevalence of an infection is high but the intensity is low, it is unlikely to be a major health problem leading to the endangerment of a species. Parasitic diseases can not only affect conservation efforts, but they are also natural selection agents and drive biological diversification, through influencing host reproductive isolation and speciation.
Alfano N., Dayaram A., Axtner J., Tsangaras K., Kampmann M. L., Mohamed A., Wong S. T., Gilbert M. T. P., Wilting A., Greenwood A. D. (2021): Non‐invasive surveys of mammalian viruses using environmental DNA. Methods in Ecology and Evolution 12: 1941-1952.
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Environmental DNA (eDNA) and invertebrate-derived DNA (iDNA) are used to survey biodiversity non-invasively to mitigate difficulties in obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA were used to monitor known wildlife pathogens; however, most wildlife pathogens are unknown and often evolutionarily divergent. To detect and identify known and novel mammalian viruses from eDNA/iDNA, we used a curated set of RNA oligonucleotides as viral baits in a hybridization capture system coupled with high-throughput sequencing. We detected multiple known and novel mammalian RNA and DNA viruses from multiple viral families from both waterhole eDNA and leech-derived iDNA. Congruence was found between detected hosts and viruses identified in leeches and waterholes. Our results demonstrate that eDNA/iDNA samples represent an effective non-invasive resource for studying wildlife viral diversity and for detecting novel potentially zoonotic viruses prior to their emergence.
Rojas C. A., Ramírez-Barahona S., Holekamp K. E., Theis K. R. (2021): Host phylogeny and host ecology structure the mammalian gut microbiota at different taxonomic scales. Animal Microbiome 3: 33.
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The gut microbiota is critical for host function. Among mammals, host phylogenetic relatedness and diet are strong drivers of gut microbiota structure, but one factor may be more influential than the other. Here, we used 16S rRNA gene sequencing to determine the relative contributions of host phylogeny and host diet in structuring the gut microbiotas of 11 herbivore species from 5 families living sympatrically in southwest Kenya. Herbivore species were classified as grazers, browsers, or mixed-feeders and dietary data (% C4 grasses in diet) were compiled from previously published sources. We found that herbivore gut microbiotas were highly species-specific, and that host taxonomy accounted for more variation in the gut microbiota (30%) than did host dietary guild (10%) or sample month (8%). Overall, similarity in the gut microbiota increased with host phylogenetic relatedness (r = 0.74) across the 11 species of herbivores, but among 7 closely related Bovid species, dietary %C4 grass values more strongly predicted gut microbiota structure (r = 0.64). Additionally, within bovids, host dietary guild explained more of the variation in the gut microbiota (17%) than did host species (12%). Lastly, while we found that the gut microbiotas of herbivores residing in southwest Kenya converge with those of distinct populations of conspecifics from central Kenya, fine-scale differences in the abundances of bacterial amplicon sequence variants (ASVs) between individuals from the two regions were also observed. Overall, our findings suggest that host phylogeny and taxonomy strongly structure the gut microbiota across broad host taxonomic scales, but these gut microbiotas can be further modified by host ecology (i.e., diet, geography), especially among closely related host species.
Webster A. B., Callealta F. J., Ganswindt A., Bennett N. C. (2021): A non-invasive assessment of essential trace element utilization at different trophic levels in African wildlife. Journal of Environmental Management 293: 112820.
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The complex relationships that exist between terrestrial mammals and their habitats make African ecosystems highly interactive environments. Anthropogenic activities including climate change have altered geochemical cycles, which influence nutrient availability and deficiency at local, regional and global scales. As synergistic and antagonistic interactions occur between essential elements at both deficiency and excess concentrations, the differences in feeding strategy between trophically distinct groups of terrestrial vertebrates are likely to influence the degree to which overall nutrient needs are met or may be deficient. The overall aim of this study was to investigate and compare quantitative differences of nine essential elements in terrestrial vertebrates occupying different trophic levels within two protected areas; Tswalu Kalahari Reserve (TKR) and Manyeleti Nature Reserve (MNR) South Africa, using faeces as an analytical matrix. Results from linear mixed effects models highlight that concentrations varied widely between individuals. Overall, measured concentrations above their respective means were evident for B and Mn in herbivores, Fe in omnivores and Cu, Co, Fe, Se and Zn in carnivores. Measured concentrations of Mo and Ni did not differ significantly between trophic groups. Although site-specific differences were evident for specific elements, measured mean concentrations of B, Co, Cu, Fe, Mo, Ni, Se and Zn were significantly higher overall at the MNR study site compared to the TKR site. This is the first study to non-invasively assess essential element concentrations across trophic levels in free ranging African wildlife species within protected areas of the savannah biome. Combined with the assessment of environmental matrices, this approach can be used as an effective diagnostic tool for the assessment of animal welfare and the management of protected areas globally.
Titcomb G. C., Pansu J., Hutchinson M. C., Tombak K. J., Hansen C. B., Baker C. C., Kartzinel T. R., Young H. S., Pringle R. M. (2022): Large-herbivore nemabiomes: patterns of parasite diversity and sharing. Proceedings of the Royal Society B 289: 20212702.
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Amidst global shifts in the distribution and abundance of wildlife and livestock, we have only a rudimentary understanding of ungulate parasite communities and parasite-sharing patterns. We used qPCR and DNA metabarcoding of fecal samples to characterize gastrointestinal nematode (Strongylida) community composition and sharing among 17 sympatric species of wild and domestic large mammalian herbivore in central Kenya. We tested a suite of hypothesis-driven predictions about the role of host traits and phylogenetic relatedness in describing parasite infections. Host species identity explained 27–53% of individual variation in parasite prevalence, richness, community composition and phylogenetic diversity. Host and parasite phylogenies were congruent, host gut morphology predicted parasite community composition and prevalence, and hosts with low evolutionary distinctiveness were centrally positioned in the parasite-sharing network. We found no evidence that host body size, social-group size or feeding height were correlated with parasite composition. Our results highlight the interwoven evolutionary and ecological histories of large herbivores and their gastrointestinal nematodes and suggest that host identity, phylogeny and gut architecture—a phylogenetically conserved trait related to parasite habitat—are the overriding influences on parasite communities. These findings have implications for wildlife management and conservation as wild herbivores are increasingly replaced by livestock.
Wu X., Wei Q., Wang X., Shang Y., Zhang H. (2022): Evolutionary and dietary relationships of wild mammals based on the gut microbiome. Gene 808: 145999.
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Gut microbiome influence the health and evolution of mammals and multiple factors modulate the structure and function of gut microbiome. However, the specific changes of the diets and phylogeny on the gut microbiome were unclear. Here, we compared the gut microbiome of 16 rare wild mammals. All data (>200G 16S rRNA gene sequences) were generated using a high-throughput sequencing platform. Firmicutes and Bacteroidetes were the most predominant phyla in all mammals. However, Proteobacteria was an additionally dominant phylum specifically detected in the microbiome of carnivores and omnivores. Moreover, the dominant phyla in canids were Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria. Phylogenetic reconstructions based on the gut microbiome and mitochondrial genome of these mammals were similar. The impact of the host on the microbiome community composition was most evident when considering conspecific and congeneric relationships. Similarity clustering showed that the gut microbiome of herbivores was clustered together, and the other clade comprised both omnivores and carnivores. Collectively, these results revealed that phylogenetic relationships and diet have an important impact on the gut microbiome, and thus the gut microbiome community composition may reflect both the phylogenetic relationships and diets. This study provides valuable basic data to facilitate future efforts related to animal conservation and health.
Rzucidlo C. L., Curry E., Shero M. R. (2023): Non-invasive measurements of respiration and heart rate across wildlife species using Eulerian Video Magnification of infrared thermal imagery. BMC Biology 21: 63.
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An animal’s metabolic rate, or energetic expenditure, both impacts and is impacted by interactions with its environment. However, techniques for obtaining measurements of metabolic rate are invasive, logistically difficult, and costly. Red–green–blue (RGB) imaging tools have been used in humans and select domestic mammals to accurately measure heart and respiration rate, as proxies of metabolic rate. The purpose of this study was to investigate if infrared thermography (IRT) coupled with Eulerian video magnification (EVM) would extend the applicability of imaging tools towards measuring vital rates in exotic wildlife species with different physical attributes. We collected IRT and RGB video of 52 total species (39 mammalian, 7 avian, 6 reptilian) from 36 taxonomic families at zoological institutions and used EVM to amplify subtle changes in temperature associated with blood flow for respiration and heart rate measurements. IRT-derived respiration and heart rates were compared to ‘true’ measurements determined simultaneously by expansion of the ribcage/nostrils and stethoscope readings, respectively. Sufficient temporal signals were extracted for measures of respiration rate in 36 species (85% success in mammals; 50% success in birds; 100% success in reptiles) and heart rate in 24 species (67% success in mammals; 33% success in birds; 0% success in reptiles) using IRT-EVM. Infrared-derived measurements were obtained with high accuracy (respiration rate, mean absolute error: 1.9 breaths per minute, average percent error: 4.4%; heart rate, mean absolute error: 2.6 beats per minute, average percent error: 1.3%). Thick integument and animal movement most significantly hindered successful validation. The combination of IRT with EVM analysis provides a non-invasive method to assess individual animal health in zoos, with great potential to monitor wildlife metabolic indices in situ.
Buigues J., Vinals A., Martinez-Recio R., Monrós J. S., Sanjuan R., Cuevas J. M. (2024): Complete genomes of DNA viruses in fecal samples from small terrestrial mammals in Spain. Viruses 16: 1885.
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Viromics studies are allowing us to understand not only the enormous diversity of the virosphere, but also the potential threat posed by the emerging viruses. Regarding the latter, the main concern lies in monitoring the presence of RNA viruses, but the zoonotic potential of some DNA viruses, on which we have focused in the present study, should also be highlighted. For this purpose, we analyzed 160 fecal samples from 14 species of small terrestrial mammals, 9 of them belonging to the order Rodentia. This allowed us to identify a total of 25 complete or near-complete genomes belonging to the families Papillomaviridae, Polyomaviridae, Adenoviridae, Circoviridae, and Genomoviridae, 18 of which could be considered new species or types. Our results provide a significant increase in the number of complete genomes of DNA viruses of European origin with zoonotic potential in databases, which are at present under-represented compared to RNA viruses. In addition, the characterization of whole genomes is of relevance for the further study of the evolutionary forces governing virus adaptation, such as recombination, which may play an important role in cross-species transmission.