GUT MICROBIOME DIVERSITY OF THREE RHINOCEROS SPECIES IN EUROPEAN ZOOS.

The wild rhinoceros populations have declined drastically in the past decades because the rhinoceros are heavily hunted for their horns. Zoological institutions aim to conserve rhinoceros populations in captivity, but one of the challenges of ex situ conservation is to provide food sources that resemble those available in the wild. Considering that the mammalian gut microbiota is a pivotal player in their host's health, the gut microbiota of rhinoceros may also play a role in the bioavailability of nutrients. Therefore, this study aims to characterize the fecal microbiome composition of grazing white rhinoceros (WR; Ceratotherium simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) as well as the browsing black rhinoceros (BR; Diceros bicornis) kept in European zoos. Over the course of 1 yr, 166 fecal samples in total were collected from 9 BR (n = 39), 10 GOHR (n = 56), and 14 WR (n = 71) from 23 zoological institutions. The bacterial composition in the samples was determined using 16S rRNA gene Illumina sequencing. The fecal microbiomes of rhinoceros clustered by species, with BR clustering more distantly from GOHR and WR. Furthermore, the data report clustering of rhinoceros microbiota according to individual rhinoceros and institutional origin, showing that zoological institutions play a significant role in shaping the gut microbiome of rhinoceros species. In addition, BR exhibit a relatively higher microbial diversity than GOHR and WR. BR seem more susceptible to microbial gut changes and appear to have a more diverse microbiome composition among individuals than GOHR and WR. These data expand on the role of gut microbes and can provide baseline data for continued efforts in rhinoceros conservation and health status.

Antemortem detection of Mycobacterium bovis in nasal swabs from African rhinoceros.

Mycobacterium bovis (M. bovis) infection has been identified in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros populations in Kruger National Park, South Africa. However, it is unknown whether M. bovis infected rhinoceros, like humans and cattle, can shed mycobacteria in respiratory secretions. Limited studies have suggested that rhinoceros with subclinical M. bovis infection may present minimal risk for transmission. However, recent advances that have improved detection of Mycobacterium tuberculosis complex (MTBC) members in paucibacillary samples warranted further investigation of rhinoceros secretions. In this pilot study, nasal swab samples from 75 rhinoceros with defined infection status based on M. bovis antigen-specific interferon gamma release assay (IGRA) results were analysed by GeneXpert MTB/RIF Ultra, BACTEC MGIT and TiKa-MGIT culture. Following culture, speciation was done using targeted PCRs followed by Sanger sequencing for mycobacterial species identification, and a region of difference (RD) 4 PCR. Using these techniques, MTBC was detected in secretions from 14/64 IGRA positive rhinoceros, with viable M. bovis having been isolated in 11 cases, but not in any IGRA negative rhinoceros (n = 11). This finding suggests the possibility that MTBC/M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment.

Histopathological Investigations and Molecular Confirmation Reveal Mycobacterium bovis in One-Horned Rhinoceros (Rhinoceros unicorns).

Mycobacterium bovis causes tuberculosis in dairy and wild animals. Presence of tuberculosis in animals poses a threat not only to their herd mates but also for public. No reports are available about the clinical, pathological, and molecular investigation of naturally occurring tuberculosis (TB) due to M. bovis in one-horned rhinoceros. One-horned female rhinoceros (Rhinoceros unicorns) at the age of 41 years died in a public park in Pakistan. Postmortem and other investigations were carried out to know the cause of death. The present study describes necropsy, histopathology, and molecular-based confirmation of TB in a captive female rhinoceros that died of this infection. Clinically, the rhinoceros showed nonspecific clinical signs including anorexia, lethargy, dyspnoea, coughing, and sudden death. At necropsy, the trachea exhibited mild congestion and contained catarrhal exudate at the bronchial bifurcation. Macroscopic examination revealed characteristic tubercles on all parenchymatous organs. The lungs showed consolidation, grey hepatization, and contained granulomatous lesions packed with cheesy exudate. Histopathological examination showed severe pneumonic changes in the form of granulomatous inflammation consisting of lymphocytes, multinucleated giant cells, caseous materials, and mineralized foci surrounded by a fibrous capsule. PCR amplicon of 500 bp confirmed the presence of M. bovis in multiple hepatic and pulmonary tissue samples, as well as in uterine exudates. It was concluded that the presence of tuberculosis in rhinoceros may pose potential transmission risk to other animals and the application of practical tools to determine TB status in the rhinoceros is crucial.

Epidemiology of Mycobacterium bovis infection in free-ranging rhinoceros in Kruger National Park, South Africa.

Mycobacterium bovis infection, which is a prominent cause of bovine tuberculosis, has been confirmed by mycobacterial culture in African rhinoceros species in Kruger National Park (KNP), South Africa. In this population-based study of the epidemiology of M. bovis in 437 African rhinoceros (Diceros bicornis, Ceratotherium simum), we report an estimated prevalence of 15.4% (95% CI: 10.4 to 21.0%), based on results from mycobacterial culture and an antigen-specific interferon gamma release assay from animals sampled between 2016 and 2020. A significant spatial cluster of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo (Syncerus caffer) herds in the vicinity of the rhinoceros sampling location. Since African buffaloes are important maintenance hosts for M. bovis in KNP, spillover of infection from these hosts to white rhinoceros sharing the environment is suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016­2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. These study findings provide a foundation for further epidemiological investigation of M. bovis, a multihost pathogen, in a complex ecosystem that includes susceptible species that are threatened and endangered.

Gut microbiome architecture of wild greater one-horned rhinoceros:a vulnerable species from Kaziranga National Park, India.

Rhinoceros unicornis, also known as the greater one-horned rhinoceros (GoHR), is a vulnerable wildlife species found in the Indian subcontinent with an estimated global population of 3582, of which an estimated 2995 resides in India. The Kaziranga National Park of Assam is the home to ~80.56% of the GoH population in India. Recent advances in genetics and microbial studies underscored the importance of gut microbial symbiosis as a crucial factor for host metabolic health and environmental interaction, particularly for higher mammals. Alteration of the normal microbiome can also be an indicator of chronic disease and infection. Freshly voided dung samples from nine dung heaps of free ranging or wild GoH rhinoceros were collected from Kaziranga National Park for mapping the gut microbial architecture through 16S-metagenomic approach. In our sample, the GoH gut harbours 168.8±12.55 (SE) bacteria-specific OTUs belonging to 21 phyla of which the gram-negative Proteobacteria is the most abundant phyla. Other abundant phylas found in the GoH gut are Firmicutes and Bacteroidetes. Although the GoH rhinoceros gut can utilize fibrous plant by microbial fermentation, the aerobic, nonfermenting Acinetobacter (20.7%), Stenotrophomonas (17.8%) and Brevundimonas (9.1%) constitute about 50% of all identified genus. Functional prediction of the GoH microbiome reveals that>50% of the bacteria present are involved in metabolism followed by cellular processes and information processing. A significant proportion (>1%) are associated with different diseases. In summary, our study characterized bacterial communities of nine wild GoH to identify some unique features and its implication in disease and survival of GoH.

CLINICAL FINDINGS, PATHOLOGY, BIOSECURITY, AND SEROSURVEILLANCE OF COXIELLOSIS IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) AT A CONSERVATION CENTER: TWO CASES.

A primiparous white rhinoceros (Ceratotherium simum) gave birth to a calf overnight after approximately 16 mo of gestation. The calf was found dead in the morning. Necrosuppurative placentitis with bacterial inclusions suggestive of coxiellosis was diagnosed histologically, and Coxiella burnetii was identified in fetal tissues and placenta by polymerase chain reaction and immunohistochemistry. Another primiparous female from the same herd aborted later that year after approximately 15 mo of gestation, and coxiellosis was similarly diagnosed in fetal tissues and on vaginal shedding. Estimates of exposure time, duration of vaginal shedding, and phase I and phase II antibody dynamics were determined retrospectively and prospectively for the two confirmed cases. Biosecurity measures were put in place to prevent guests, staff, and conspecific exposure to the organism. No other confirmed cases have occurred in the collection 3 yr after the initial cases. Coxiellosis outbreaks could represent an emerging threat to conservation efforts and ex situ white rhinoceros breeding programs.

ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI AND ENTEROCOCCUS SPP. ISOLATED FROM UNGULATES AT A ZOOLOGICAL COLLECTION IN THE UNITED KINGDOM.

Increase of antimicrobial resistance (AMR) is a global threat to health. The AMR profile of bacteria isolated from domesticated animals and free-ranging wildlife has been studied, but there are relatively few studies of bacteria isolated from captive wild animals. Understanding the dynamics of AMR in different populations is key to minimizing emergence of resistance and to preserve the efficacy of antimicrobials. In this study, fecal samples were collected from 17 species of healthy ungulates from a zoological collection in southeast England, which yielded 39 Escherichia coli and 55 Enterococcus spp. isolates for further analysis. Antibiotic sensitivity was investigated using agar disk diffusion. Escherichia coli isolates were resistant to a range of antibiotics, with resistance to ampicillin being the most common (28%). All E. coli isolates were susceptible to apramycin, enrofloxacin, chloramphenicol, and florfenicol. None tested positive for extended-spectrum beta-lactamase or AmpC activity. Seven of 39 (18%) E. coli isolates were resistant to three or more antibiotic classes. The E. coli isolates were further analyzed using multilocus sequence typing, which identified four pairs of identical sequence type isolates and 27 diverse strains. The Enterococcus spp. isolates were resistant to a range of antibiotics, with resistance to cefpodoxime seen in 95% of isolates. All Enterococcus spp. isolates were susceptible to ampicillin, gentamicin, chloramphenicol, and vancomycin. This study identified multidrug-resistant phenotypes in enterobacterial isolates that were like those commonly found in domestic ungulates. There was no apparent spatial clustering of the resistance profiles within the zoo. Review of the medical records of individual animals showed no direct relation to the AMR profiles observed. Observed resistance to antibiotics rarely or never used may have been due to coselection or directly acquired from other sources.

Rickettsia amblyommatis isolated from Amblyomma mixtum (Acari: Ixodida) from two sites in Panama.

Rickettsia amblyommatis is widely distributed in the Americas, and has been reported to infect different species of ticks within its distribution. In Panama, R. amblyommatis is the most common Rickettsia and its presence was molecularly detected in nine species of ticks and one flea species. This work described the isolation of R. amblyommatis in Vero cells by shell vial technique, from Amblyomma mixtum ticks collected from a captive tapir from Gamboa (Colon province), and a horse from El Valle de Antón (Cocle province). These represent the first isolations of R. amblyommatis in Panama.

Environmental Factors Influencing the Occurrence of Unhealthy Tapirs in the Southern Yucatan Peninsula.

Information about the effects of environmental degradation on the health of terrestrial forest wildlife is limited, especially for rare species. In this study, we analyse the influence of ecological factors such as landscape characteristics and seasonality on the health status of Baird's tapirs in Calakmul, Mexico. We collected georeferenced photographic records of healthy (n = 32) and unhealthy (n = 22) tapirs from 2008 to 2019 and characterized landscape composition around each record at three spatial scales (circular buffers of 1, 2 and 3-km radii according to Baird's tapir home ranges). Our logistic model building process consisted in selecting the best spatial scale for each landscape cover class, before including them along with distance to human settlements and seasonality in a full model. The model that best explained the occurrence of unhealthy tapirs included the percentage of agriculture within a 1-km radius. This study hints at the negative effect that land-use change to agriculture occurring in Calakmul might have on tapir health, with 95.45% of unhealthy tapirs recorded in such landscapes. Further studies should investigate the proximate determinants of tapir health in anthropogenic landscapes, which might be linked to stress or to contact with domestic animals.

The Xpert MTB/RIF Ultra assay detects Mycobacterium tuberculosis complex DNA in white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana).

The study describes the novel use of the Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis complex (MTBC) DNA in samples from white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana). Culture negative respiratory sample matrices were spiked to determine if the Ultra could detect MTBC DNA in rhinoceros and elephant samples. Rhinoceros bronchial alveolar lavage fluid (BALF) was found to have an inhibitory effect on the Ultra. In this study, the limit of detection (LOD) of M. tuberculosis H37Rv in all spiked animal samples were 2 CFU/ml compared to 15.6 CFU/ml for humans, while the LOD for M. bovis SB0121 was 30 CFU/ml compared to 143.4 CFU/ml for M. bovis BCG in humans. Screening was performed on stored tissue and respiratory samples from known MTBC-infected animals and MTBC DNA was detected in 92% of samples collected from six rhinoceros and two elephants. Conversely, 83% of culture-negative tissue and respiratory samples from uninfected animals tested negative on the Ultra. In conclusion, the Ultra assay appears to be a sensitive and rapid diagnostic test for the detection of MTBC DNA from tissue and respiratory samples collected from African elephants and rhinoceros. Furthermore, the Ultra assay could provide a new tool for the detection of MTBC in various sample types from other wildlife species.

The VetMAX™ M. tuberculosis complex PCR kit detects MTBC DNA in antemortem and postmortem samples from white rhinoceros (Ceratotherium simum), African elephants (Loxodonta africana) and African buffaloes (Syncerus caffer).

BACKGROUND: Bovine tuberculosis and tuberculosis are chronic infectious diseases caused by the Mycobacterium tuberculosis complex members, Mycobacterium bovis and Mycobacterium tuberculosis, respectively. Infection with M. bovis and M. tuberculosis have significant implications for wildlife species management, public health, veterinary disease control, and conservation endeavours. RESULTS: Here we describe the first use of the VetMAX™ Mycobacterium tuberculosis complex (MTBC) DNA quantitative real-time polymerase chain reaction (qPCR) detection kit for African wildlife samples. DNA was extracted from tissues harvested from 48 African buffaloes and MTBC DNA was detected (test-positive) in all 26 M. bovis culture-confirmed animals with an additional 12 PCR-positive results in culture-negative buffaloes (originating from an exposed population). Of six MTBC-infected African rhinoceros tested, MTBC DNA was detected in antemortem and postmortem samples from five animals. The PCR was also able to detect MTBC DNA in samples from two African elephants confirmed to have M. bovis and M. tuberculosis infections (one each). Culture-confirmed uninfected rhinoceros and elephants' samples tested negative in the PCR assay. CONCLUSIONS: These results suggest this new detection kit is a sensitive screening test for the detection of MTBC-infected African buffaloes, African elephants and white rhinoceros.

TUBERCULOSIS CAUSED BY MYCOBACTERIUM ORYGIS IN A GREATER ONE-HORNED RHINOCEROS (RHINOCEROS UNICORNIS): FIRST REPORT IN THE WESTERN HEMISPHERE.

Mycobacterium orygis, a newly identified member of the Mycobacterium tuberculosis complex, has been isolated predominantly from hoofstock in eastern Africa and the Arabian Peninsula, and sporadically in cattle (Bos taurus indicus), rhesus monkeys (Macaca mulatta), humans, and a greater one-horned rhinoceros (Rhinoceros unicornis) in South Asia. In rhinoceros, tuberculosis typically presents as a chronic progressive respiratory disease. The report describes the postmortem diagnosis of tuberculosis caused by Mycobacterium orygis in a greater one-horned rhinoceros with hind limb paresis due to neural granulomatosis. Serologic assays for detection of antibodies to M. tuberculosis complex proteins before culture results allowed for appropriate herd management protocols to be initiated. Mycobacterium genus-specific polymerase chain reaction assays with direct sequencing allowed timely confirmation of the serologic results. This is the first isolation of M. orygis in the western hemisphere, showing the need for mycobacterial testing of rhinoceros before international shipments and the urgency for validated antemortem M. tuberculosis complex screening assays in rhinoceros species.

Antibiotic Resistance of Escherichia coli from Humans and Black Rhinoceroses in Kenya.

Upsurge of antibiotic resistance in wildlife poses unprecedented threat to wildlife conservation. Surveillance of antibiotic resistance at the human-wildlife interface is therefore needed. We evaluated differences in antibiotic resistance of Escherichia coli isolates from human and the endangered black rhinoceros in Lambwe Valley, Kenya. We used standard microbiological techniques to carry out susceptibility assays using eight antibiotics of clinical and veterinary importance. Standard PCR method was used to characterize antibiotic resistance genes. There was no difference in resistance between E. coli isolates from human and those from rhinoceros (U = 25, p = 0.462). However, higher resistance in isolates from humans was noted for cotrimoxazole (p = 0.000, OR = 0.101), ceftriaxone (p = 0.005, OR = 0.113) and amoxicillin/clavulanic acid (p = 0.017, OR = 0.258), whereas isolates from rhinoceros showed higher gentamicin resistance (p = 0.001, OR = 10.154). Multi-drug resistance phenotype was 69.0% in humans and 43.3% in rhinoceros. Isolates from both species contained blaTEM, tetA, tetB, dfrA1 and sul1 genes. Resistance profiles in the two species suggest potential for cross-transfer of resistance genes or exposure to comparable selective pressure and call for a multi-sectorial action plan on surveillance of antibiotic resistance at the human-wildlife interface. Genome-wide studies are needed to explicate the direction of transfer of genes that confer antibiotic resistance at the human-wildlife interface.

Risks from disease caused by Mycobacterium orygis as a consequence of Greater one-horned Rhinoceros (Rhinoceros unicornis) translocation in Nepal.

The greater one-horned rhinoceros (Rhinoceros unicornis) is listed as vulnerable by the IUCN Red List. Mycobacterium orygis-associated disease was identified in a single greater one-horned rhino in Chitwan National Park in February 2015 prior to a planned translocation of five greater one-horned rhinoceros from Chitwan National Park to Bardia National Park for conservation purposes. This paper describes a qualitative disease risk analysis conducted retrospectively post-translocation for Mycobacterium orygis and this translocation, with the aim to improve the understanding of disease threats to the conservation of greater one-horned rhino. The disease risk analysis method used was devised by Sainsbury & Vaughan-Higgins (Conservation Biology, 26, 2017, 442) with modifications by Bobadilla Suarez et al (EcoHealth, 14, 2017, 1) and Rideout et al (EcoHealth, 14, 2017, 42) and included the use of a scenario tree and an analysis of uncertainty as recommended by Murray et al. (Handbook on import risk analysis for animals and animal products. Volume 1. Introduction and qualitative risk analysis, 2004), and the first time this combination of methods has been used to assess the risk from disease in a conservation translocation. The scenario tree and analysis of uncertainty increased the clarity and transparency of the analysis. Rideout et al.'s (EcoHealth, 14, 2017, 42) criteria were used to assess the source hazard and may be useful in comparative assessment of source hazards for future conservation translocations. The likelihood of release into the destination site of Mycobacterium orygis as a source hazard was estimated as of low risk, the risk of exposure of populations at the destination was of high risk and the likelihood of biological and environmental consequences was low. Overall, the risk from disease associated with Mycobacterium orygis as a result of this translocation was found to be low. Recommendations on disease risk management strategies could be improved with a better understanding of the epidemiology including the presence/absence of Mycobacterium orygis in greater one-horned rhino to develop effective disease risk management strategies.

An interferon-gamma release assay for the diagnosis of the Mycobacterium bovis infection in white rhinoceros (Ceratotherium simum).

Mycobacterium bovis (M. bovis), the cause of bovine tuberculosis, is endemic in Kruger National Park (KNP), South Africa. The risk of spread of M. bovis infection currently prevents translocation of white rhinoceros (Ceratotherium simum) from this population. Therefore, accurate assays are necessary for screening this threatened species. Interferon gamma (IFN-γ) release assays (IGRA) are commonly used for tuberculosis diagnosis in humans and other wildlife species. Hence, the aim of this study was to develop an IGRA for M. bovis detection in white rhinoceros. Heparinized whole blood was collected from immobilized white rhinoceros in KNP (n = 131) and incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes, after which the plasma was harvested following centrifugation. Tissue samples for mycobacterial culture were available from a subset of 21 rhinoceros. The concentration of IFN-γ in plasma samples was measured using the Mabtech equine IFN-γ ELISAPRO kit. An IGRA result was calculated as the difference in IFN-γ concentrations in the QFT Nil and TB antigen tubes. Using test results for the white rhinoceros with known infection status, a diagnostic cut-off value was calculated as 21 pg/ml. Additionally, cut-off values for IFN-γ concentrations for plasma from QFT Nil and QFT Mitogen tubes were calculated to increase confidence in IGRA result interpretation. The combination of the QFT stimulation platform and Mabtech equine IFN-γ ELISA is a promising diagnostic test to distinguish between of M. bovis-infected and -uninfected white rhinoceros.

Gut microbiome differences between wild and captive black rhinoceros - implications for rhino health.

A number of recent studies have shown the importance of the mammalian gut microbiome in host health. In the context of endangered species, a few studies have examined the relationship between the gut microbiome in wild versus captive populations due to digestive and other health issues. Unfortunately, the results seem to vary across taxa in terms of captive animals having higher, lower, or equivalent microbiome diversity relative to their wild counterparts. Here, we focus on the black rhinoceros as captive animals suffer from a number of potentially dietary related health effects. We compared gut microbiomes of wild and captive black rhinos to test for differences in taxonomic diversity (alpha and beta) and in functional diversity of the microbiome. We incorporated a more powerful metagenomic shotgun sequencing approach rather than a targeted amplification of the 16S gene for taxonomic assignment of the microbiome. Our results showed no significant differences in the alpha diversity levels between wild and captive black rhinos, but significant differences in beta diversity. We found that bacterial taxa traditionally associated with ruminant guts of domesticated animals had higher relative abundances in captive rhinos. Our metagenomic sequencing results suggest that unknown gut microbes of wild rhinos are being replaced by those found in conventional human-domesticated livestock. Wild rhinos have significantly different functional bacterial communities compared to their captive counterparts. Functional profiling results showed greater abundance of glycolysis and amino acid synthesis pathways in captive rhino microbiomes, representing an animal receiving sub-optimal nutrition with a readily available source of glucose but possibly an imbalance of necessary macro and micronutrients. Given the differences observed between wild and captive rhino gut microbiomes, we make a number of recommendations for potentially modifying captive gut microbiome to better reflect their wild counterparts and thereby hopefully improve overall rhino health in captivity.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species-the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota's transformation of dietary phytoestrogens in captive SWR females.IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

Rare gut microbiota associated with breeding success, hormone metabolites and ovarian cycle phase in the critically endangered eastern black rhino.

BACKGROUND: Host microbiomes play a role in hormone production and subsequent fertility in humans, but this is less well understood in non-model organisms. This is of particular relevance to species in zoo-based conservation breeding programmes, as relationships between host microbiome composition and reproductive output may allow for the development of microbial augmentation strategies to improve success. Here, we characterise faecal bacterial communities of breeding and non-breeding eastern black rhino (Diceros bicornis michaeli) using 16S rRNA gene amplicon sequencing and quantify progestagen and glucocorticoid metabolite concentrations through enzyme immunoassays to identify such relationships. RESULTS: We identified significant differences in black rhino gut microbiome composition according to ID, institution, breeding success and ovarian cycle phase. In particular, the gut microbiome during pregnancy and post-parturition was significantly altered. Around a third of bacterial genera showed more than ± 10% correlation with either progestagen and/or glucocorticoid concentration, and in general, microbial genera correlated with both hormones in the same direction. Through a combination of analyses, we identified four genera (Aerococcaceae, Atopostipes, Carnobacteriaceae and Solobacterium) that were significantly associated with breeding success, pregnancy and/or post-parturition, and higher faecal progestagen metabolite concentrations. These genera had a lower-than-average relative abundance in the gut microbiome. CONCLUSION: Our results indicate that many members of the gut microbiome of black rhino are associated with hormone production and breeding success, and some members of the rare microbiota appear to be particularly important. Although the directionality of the relationship is unclear, the variation in gut microbiome communities represents a potential biomarker of reproductive health. We identified four genera that were associated with multiple indicators of reproductive output; these could be candidate probiotics to improve the breeding success of black rhino in zoo-based conservation breeding programmes. Further work is required to understand the efficacy and feasibility of this, either directly through microbial augmentation (e.g. probiotics) or indirectly via dietary manipulation or prebiotics.

Pansusceptible Escherichia coli isolates obtained from faeces of free-ranging Baird's tapirs (Tapirus bairdii) suggests a low selective pressure for resistance determinants in the northwestern region of the Talamanca Mountain Range, Costa Rica.

OBJECTIVES: The main objective of this study was to analyse the antimicrobial susceptibility profile of Escherichia coli isolates obtained from faecal samples of free-ranging Baird's tapirs (Tapirus bairdii) in the northwestern region of the Talamanca Mountain Range, Costa Rica. METHODS: Faecal samples were collected by opportunistic search of the study area from February-September 2017 during seven field expeditions. Escherichia coli isolates were recovered using selective and differential MacConkey agar medium and were subjected to biochemical identification and antimicrobial susceptibility testing using a VITEK®2 Compact automated system and the AST-N279 card. RESULTS: A total of 60 E. coli isolates were obtained from 63 faecal samples. Following evaluation of nine different antimicrobial classes, 98% (59/60) of the isolates were characterised as pansusceptible; only 1 isolate presented resistance to nalidixic acid. CONCLUSION: We propose that the commensal intestinal microbiota of free-ranging Baird's tapirs in this area remains isolated from antibiotic selective pressure, probably because seven different protected areas converge, thus giving a possible low anthropogenic activity to the region.