Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Correction: Detection of novel coronaviruses in bats in Myanmar.

[This corrects the article DOI: 10.1371/journal.pone.0230802.].

Estimating individual exposure to predation risk in group-living baboons, Papio anubis.

In environments with multiple predators, vulnerabilities associated with the spatial positions of group-living prey are non-uniform and depend on the hunting styles of the predators. Theoretically, coursing predators follow their prey over long distances and attack open areas, exposing individuals at the edge of the group to predation risk more than those at the center (marginal predation). In contrast, ambush predators lurk unnoticed by their prey and appear randomly anywhere in the group; therefore, isolated individuals in the group would be more vulnerable to predators. These positions of vulnerability to predation are expected to be taken by larger-bodied males. Moreover, dominant males presumably occupy the center of the safe group. However, identifying individuals at higher predation risk requires both simultaneous recording of predator location and direct observation of predation events; empirical observations leave ambiguity as to who is at risk. Instead, several theoretical methods (predation risk proxies) have been proposed to assess predation risk: (1) the size of the individual 'unlimited domain of danger' based on Voronoi tessellation, (2) the size of the 'limited domain of danger' based on predator detection distance, (3) peripheral/center position in the group (minimum convex polygon), (4) the number and direction of others in the vicinity (surroundedness), and (5) dyadic distances. We explored the age-sex distribution of individuals in at-risk positions within a wild baboon group facing predation risk from leopards, lions, and hyenas, using Global Positioning System collars. Our analysis of the location data from 26 baboons revealed that adult males were consistently isolated at the edge of the group in all predation risk proxies. Empirical evidence from previous studies indicates that adult male baboons are the most frequently preyed upon, and our results highlights the importance of spatial positioning in this.

Assessing Behavioral Risk Factors Driving Zoonotic Spillover Among High-risk Populations in Myanmar.

The increasing global emergence of zoonoses warrants improved awareness of activities that predispose vulnerable communities to greater risk of disease. Zoonotic disease outbreaks regularly occur within Myanmar and at its borders partly due to insufficient knowledge of behavioral risks, hindering participatory surveillance and reporting. This study employed a behavioral surveillance strategy among high-risk populations to understand the behavioral risks for zoonotic disease transmission in an effort to identify risk factors for pathogen spillover. To explore behavioral mechanisms of spillover in Myanmar, we aimed to: (1) evaluate the details around animal contact and types of interaction, (2) assess the association between self-reported unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) and animal contact activities and (3) identify the potential risk factors including behavioral practices of self-reported illness. Participants were enrolled at two community sites: Hpa-An and Hmawbi in Southern Myanmar. A behavioral questionnaire was administered to understand participants' animal exposures, behaviors and self-reported illnesses. From these responses, associations between (1) animal contact activities and self-reported unusual illnesses, and (2) potential risk factors and self-reported unusual illness were tested. Contact with poultry seemed to be very frequent (91.1%) and many participants reported raising, handling and having poultry in their houses as well as slaughtering or being scratched/bitten by them, followed by contact with rodents (57.8%) and swine (17.9%). Compared to participants who did not have any unusual symptoms, participants who had unusual symptoms in the past year were more likely to have sold dead animals (OR = 13.6, 95% CI 6.8-27.2), slaughtered (OR = 2.4, 95% CI 1.7-3.3), raised (OR = 3.4, 95% CI 2.3-5.0) or handled animals (OR = 2.1, 95% CI 1.2-3.6), and had eaten sick (OR = 4.4, 95% CI 3.0-6.4) and/or dead animals (OR = 6.0, 95% CI 4.1-8.8) in the same year. Odds of having reported unusual symptoms was higher among those involved in animal production business (OR = 3.4, 95% CI 1.9-6.2) and animal-involved livelihoods (OR = 3.3, 95% CI 1.5-7.2) compared to other livelihoods. The results suggest that there is a high level of interaction between humans, livestock and wild animals in communities we investigated in Myanmar. The study highlights the specific high-risk behaviors as they relate to animal contact and demographic risk factors for zoonotic spillover. Our findings contribute to human behavioral data needed to develop targeted interventions to prevent zoonotic disease transmission at human-animal interfaces.

Non-invasive assessment of ovarian activity in free-ranging eastern black rhinoceros (Diceros bicornis michaeli) in Kenya.

Eastern black rhinos (Diceros bicornis michaeli) are a critically endangered species living in diverse habitats across Africa. In Kenya, once threatened with extinction due to massive poaching pressures, increased protection has resulted in losses being less than 1% annually today. Still, some populations have failed to achieve desired population growth targets. At Ol Jogi Wildlife Conservancy, some individuals are experiencing sub-optimal reproduction based on historical calving records and long inter-calving intervals (>3 years). Hormones drive the reproductive process, so non-invasive assessments of endocrine patterns can be useful indicators of individual reproductive health. In this study, we analysed longitudinal fecal progestagen metabolite (fPM) concentrations in all breeding female eastern black rhinos at Ol Jogi (n = 17) and compared the prevalence of irregular estrous cycles (longer or shorter than 20-40 days) and anestrous periods (interluteal period more than twice the length of a normal follicular phase, i.e. > 10 days) between optimal (inter-calving interval < 3 years) and sub-optimal (>3 years) reproducing individuals. Ten rhinos were pregnant during at least part of the study period. A total of 12 complete cycles were observed in seven females with an average length of 36 ± 3 days and equal numbers of regular and irregular cycles. Single anestrous periods averaging 67 ± 13 days were observed in five females. Surprisingly, a majority of cycles in optimal reproducing individuals were categorized as irregular based on fPM profiles. Overall, results suggest that irregular ovarian activity and isolated bouts of anestrus do not have negative impacts on reproductive performance in this subpopulation at Ol Jogi. A high priority is to continue using noninvasive hormone monitoring to evaluate how ecological or other variables influence reproductive success in this and other eastern black rhino subpopulations in Kenya.

Exposure to diverse sarbecoviruses indicates frequent zoonotic spillover in human communities interacting with wildlife.

BACKGROUND: Sarbecoviruses are a subgenus of Coronaviridae that mostly infect bats with known potential to infect humans (SARS-CoV and SARS-CoV-2). Populations in Southeast Asia, where these viruses are most likely to emerge, have been undersurveyed to date. METHODS: We surveyed communities engaged in extractive industries and bat guano harvesting from rural areas in Myanmar. Participants were screened for exposure to sarbecoviruses, and their interactions with wildlife were evaluated to determine the factors associated with exposure to sarbecoviruses. RESULTS: Of 693 people screened between July 2017 and February 2020, 12.1% were seropositive for sarbecoviruses. Individuals were significantly more likely to have been exposed to sarbecoviruses if their main livelihood involved working in extractive industries (logging, hunting, or harvesting of forest products; odds ratio [OR] = 2.71, P = 0.019) or had been hunting/slaughtering bats (OR = 6.09, P = 0.020). Exposure to a range of bat and pangolin sarbecoviruses was identified. CONCLUSION: Exposure to diverse sarbecoviruses among high-risk human communities provides epidemiologic and immunologic evidence that zoonotic spillover is occurring. These findings inform risk mitigation efforts needed to decrease disease transmission at the bat-human interface, as well as future surveillance efforts warranted to monitor isolated populations for viruses with pandemic potential.

Surgical placement of implantable cardiac loop recorders in great apes.

Cardiovascular disease is the leading cause of morbidity and mortality in zoologically managed adult great apes, accounting for 29%-77% of adult deaths in the North American population depending on the species. In an effort to better understand the underlying causes of heart disease, implantable loop recorders (ILRs) have been used in some cases to monitor great apes with suspected or known cases of arrhythmia. This is a 10-year review of the Great Ape Heart Project's experience of implanting 21 ILRs in 7 gorillas (Gorilla gorilla gorilla; 9 total ILR devices), 5 chimpanzees (Pan troglodytes, 11 total ILR devices), and 1 orangutan (Pongo abelii, 1 ILR device) in an effort to develop effective methods for surgical implantation and remote collection of the data for analysis.

Intralesional application of ribavirin in two American flamingos (Phoenicopterus ruber) with poxvirus infection.

We report the successful treatment of poxvirus lesions in two juvenile American flamingos (Phoenicopterus ruber) with experimental low-dose intralesional ribavirin injection. In the first flamingo, the size and location of a beak verrucosity interfered with feeding, and after multiple surgical interventions, an experimental therapy of low-dose intralesional ribavirin was implemented with close blood parameter monitoring to minimize any potential side effects due to systemic antiviral administration. The second flamingo had a poxvirus lesion on the tibiotarsus, which recurred after unsuccessful conservative medical treatment and surgical intervention and a course of intralesional ribavirin therapy was implemented. Regression of the lesions in both flamingos commenced within 3 days of ribavirin treatment resulting in complete resolution within 6 weeks of onset of ribavirin treatment.

Metagenomic Investigation of Ticks From Kenyan Wildlife Reveals Diverse Microbial Pathogens and New Country Pathogen Records.

Focusing on the utility of ticks as xenosurveillance sentinels to expose circulating pathogens in Kenyan drylands, host-feeding ticks collected from wild ungulates [buffaloes, elephants, giraffes, hartebeest, impala, rhinoceros (black and white), zebras (Grévy's and plains)], carnivores (leopards, lions, spotted hyenas, wild dogs), as well as regular domestic and Boran cattle were screened for pathogens using metagenomics. A total of 75 host-feeding ticks [Rhipicephalus (97.3%) and Amblyomma (2.7%)] collected from 15 vertebrate taxa were sequenced in 46 pools. Fifty-six pathogenic bacterial species were detected in 35 pools analyzed for pathogens and relative abundances of major phyla. The most frequently observed species was Escherichia coli (62.8%), followed by Proteus mirabilis (48.5%) and Coxiella burnetii (45.7%). Francisella tularemia and Jingmen tick virus (JMTV) were detected in 14.2 and 13% of the pools, respectively, in ticks collected from wild animals and cattle. This is one of the first reports of JMTV in Kenya, and phylogenetic reconstruction revealed significant divergence from previously known isolates and related viruses. Eight fungal species with human pathogenicity were detected in 5 pools (10.8%). The vector-borne filarial pathogens (Brugia malayi, Dirofilaria immitis, Loa loa), protozoa (Plasmodium spp., Trypanosoma cruzi), and environmental and water-/food-borne pathogens (Entamoeba histolytica, Encephalitozoon intestinalis, Naegleria fowleri, Schistosoma spp., Toxoplasma gondii, and Trichinella spiralis) were detected. Documented viruses included human mastadenovirus C, Epstein-Barr virus and bovine herpesvirus 5, Trinbago virus, and Guarapuava tymovirus-like virus 1. Our findings confirmed that host-feeding ticks are an efficient sentinel for xenosurveillance and demonstrate clear potential for wildlife-livestock-human pathogen transfer in the Kenyan landscape.

Strengthening global health security by improving disease surveillance in remote rural areas of low-income and middle-income countries.

The COVID-19 pandemic has underscored the need to strengthen national surveillance systems to protect a globally connected world. In low-income and middle-income countries, zoonotic disease surveillance has advanced considerably in the past two decades. However, surveillance efforts often prioritise urban and adjacent rural communities. Communities in remote rural areas have had far less support despite having routine exposure to zoonotic diseases due to frequent contact with domestic and wild animals, and restricted access to health care. Limited disease surveillance in remote rural areas is a crucial gap in global health security. Although this point has been made in the past, practical solutions on how to implement surveillance efficiently in these resource-limited and logistically challenging settings have yet to be discussed. We highlight why investing in disease surveillance in remote rural areas of low-income and middle-income countries will benefit the global community and review current approaches. Using semi-arid regions in Kenya as a case study, we provide a practical approach by which surveillance in remote rural areas can be strengthened and integrated into existing systems. This Viewpoint represents a transition from simply highlighting the need for a more holistic approach to disease surveillance to a solid plan for how this outcome might be achieved.

PERINATAL CENTRILOBULAR HEPATIC NECROSIS IN GIANT PANDAS (AILUROPODA MELANOLEUCA): A RETROSPECTIVE STUDY.

Between 1983 and 2012, six giant panda cubs (Ailuropoda melanoleuca) born at a zoological institution were stillborn or died between the ages of 3 and 200 h. Two of the six cubs had panhepatic centrilobular hepatic necrosis (CHN), granulocytic extramedullary hematopoiesis (GEM), positive liver culture for Staphylococcus species, and terminal liver failure. Another low-weight cub was administered oxygen therapy immediately after birth and developed hyaline membranes in air spaces and hepatic necrosis restricted to the hilar region. A retrospective analysis of liver and lung lesions, pulmonary microanatomy, blood-gas barrier ultrastructure, and hepatic myofibroblast proliferation was conducted on the six cubs. Neonates with CHN had concurrent severe periportal GEM accompanied by severe myofibroblast proliferation. The pulmonary blood-gas barrier was markedly increased in one cub with CHN. Developmentally, the lungs of all but one cub were at the late saccular stage, and the lowest-weight cub was in early saccular stage, consistent with immaturity, and had pneumonia comparable to neonatal respiratory distress syndrome (RDS). Stage of lung development was eliminated as the primary factor leading to CHN. The pathogenesis of CHN in these neonates is proposed to be transformation of hepatic stellate cells to myofibroblasts initiating blockage and microvascular constriction of hepatic sinusoids, resulting in insufficient perfusion and cellular hypoxia of hepatocytes surrounding central veins in acinar zone 3.

Movements of Indian Flying Fox in Myanmar as a Guide to Human-Bat Interface Sites.

Frugivorous bats play a vital role in tropical ecosystems as pollinators and seed dispersers but are also important vectors of zoonotic diseases. Myanmar sits at the intersection of numerous bioregions and contains habitats that are important for many endangered and endemic species. This rapidly developing country also forms a connection between hotspots of emerging human diseases. We deployed Global Positioning System collars to track the movements of 10 Indian flying fox (Pteropus giganteus) in the agricultural landscapes of central Myanmar. We used clustering analysis to identify foraging sites and high-utilization areas. As part of a larger viral surveillance study in bats of Myanmar, we also collected oral and rectal swab samples from 29 bats to test for key emerging viral diseases in this colony. There were no positive results detected for our chosen viruses. We analyzed their foraging movement behavior and evaluated selected foraging sites for their potential as human-wildlife interface sites.

Ranking the risk of animal-to-human spillover for newly discovered viruses.

The death toll and economic loss resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are stark reminders that we are vulnerable to zoonotic viral threats. Strategies are needed to identify and characterize animal viruses that pose the greatest risk of spillover and spread in humans and inform public health interventions. Using expert opinion and scientific evidence, we identified host, viral, and environmental risk factors contributing to zoonotic virus spillover and spread in humans. We then developed a risk ranking framework and interactive web tool, SpillOver, that estimates a risk score for wildlife-origin viruses, creating a comparative risk assessment of viruses with uncharacterized zoonotic spillover potential alongside those already known to be zoonotic. Using data from testing 509,721 samples from 74,635 animals as part of a virus discovery project and public records of virus detections around the world, we ranked the spillover potential of 887 wildlife viruses. Validating the risk assessment, the top 12 were known zoonotic viruses, including SARS-CoV-2. Several newly detected wildlife viruses ranked higher than known zoonotic viruses. Using a scientifically informed process, we capitalized on the recent wealth of virus discovery data to systematically identify and prioritize targets for investigation. The publicly accessible SpillOver platform can be used by policy makers and health scientists to inform research and public health interventions for prevention and rapid control of disease outbreaks. SpillOver is a living, interactive database that can be refined over time to continue to improve the quality and public availability of information on viral threats to human health.

Cefovecin pharmacokinetics after single-dose intramuscular administration in cheetahs (Acinonyx jubatus).

Cefovecin is a third-generation cephalosporin with potential value for use in exotic felids due to its long duration of action. A sparse sampling protocol was implemented with 18 zoo-housed cheetahs (Acinonyx jubatus) to evaluate the pharmacokinetics of cefovecin (Convenia® ) after a single 8 mg/kg intramuscular injection. Blood was collected serially for 15 days following administration, and plasma cefovecin concentrations were determined using high-pressure liquid chromatography with ultraviolet detection. Pharmacokinetic parameters were estimated using population pharmacokinetic methods and non-linear mixed effects modeling (NLME). Cefovecin was well tolerated by all cats, with no adverse effects observed. Peak plasma cefovecin concentration was 84.75 µg/ml, with a mean residence time of 207.9 h and an elimination half-life of 144.1 h (6.00 days). Plasma concentrations of cefovecin were maintained >7 µg/ml in all individuals for the entire study duration (15 days). These concentrations are lower, and the half-life slightly shorter, than the values reported for domestic cats. Cefovecin was highly protein-bound (approximately 99.9%) in cheetah plasma, which is nearly identical to domestic cats. These results indicate that cefovecin is potentially useful as a long-acting antibiotic in cheetahs.

Home range, sleeping site use, and band fissioning in hamadryas baboons: Improved estimates using GPS collars.

Variation in spatial and temporal distribution of resources drives animal movement patterns. Links between ecology and behavior are particularly salient for the multilevel society of hamadryas baboons, in which social units cleave and coalesce over time in response to ecological factors. Here, we used data from GPS collars to estimate home range size and assess temporal patterns of sleeping site use in a band of hamadryas baboons in Awash National Park, Ethiopia. We used GPS data derived from 2 to 3 collared baboons over three 8-12-month collaring intervals to estimate annual and monthly home ranges using kernel density estimators (KDEs) and minimum convex polygons (MCPs). The 95% KDE home range was 64.11 km2 for Collaring Interval I (July 2015-March 2016), 85.52 km2 for Collaring Interval II (October 2016-October 2017), 76.43 km2 for Collaring Interval III (July 2018-May 2019), and 75.25 km2 across all three collaring intervals. MCP home ranges were 103.46 km2 for Collaring Interval I, 97.90 km2 for Collaring Interval II, 105.22 km2 for Collaring Interval III, and 129.33 km2 overall. Ninety-five percent KDE home range sizes did not differ across months, nor correlate with temperature or precipitation, but monthly MCP home ranges increased with monthly precipitation. Our data also revealed a southward home range shift over time and seven previously unknown sleeping sites, three of which were used more often during the wet season. Band cohesion was highest during dry months and lowest during wet months, with fissioning occurring more frequently at higher temperatures. One pair of collared individuals from Collaring Interval III spent 95% of nights together, suggesting they were members of the same clan. Our results both suggest that previous studies have underestimated the home range size of hamadryas baboons and highlight the benefits of remote data collection.

Use of a Portable Analyzer for Venous Blood Gas and Biochemistry Analysis in Free-Ranging Indian Flying Foxes (Pteropus giganteus) in Myanmar.

We determined venous blood gas, acid-base, and biochemical parameters for thirteen free-ranging Indian flying foxes (Pteropus giganteus) in Myanmar, using a handheld i-STAT analyzer with CG8+ and CHEM8 cartridges. For field-based projects, portable blood analyzers enable identification and management of electrolyte and acid-base imbalances and collection of physiologic data, but present logistical challenges.

The intersection of land use and human behavior as risk factors for zoonotic pathogen exposure in Laikipia County, Kenya.

A majority of emerging infectious diseases (EIDs) are zoonotic, mainly caused through spillover events linked to human-animal interactions. We conducted a survey-based human behavioral study in Laikipia County, Kenya, which is characterized by a dynamic human-wildlife-livestock interface. Questionnaires that assessed human-animal interactions, sanitation, and illnesses experienced within the past year were distributed to 327 participants among five communities in Laikipia. This study aimed to 1) describe variation in reported high-risk behaviors by community type and 2) assess the relationship between specific behaviors and self-reported illnesses. Behavioral trends were assessed in R via Fisher's exact tests. A generalized linear mixed model with Lasso penalization (GLMMLasso) was used to assess correlations between behaviors and participants' self-reported illness within the past year, with reported behaviors as independent variables and reported priority symptoms as the outcome. Reported behaviors varied significantly among the study communities. Participants from one community (Pastoralist-1) were significantly more likely to report eating a sick animal in the past year (p< 0.001), collecting an animal found dead to sell in the past year (p<0.0001), and not having a designated location for human waste (p<0.0001) when compared to participants from other communities. The GLMMLasso revealed that reports of an ill person in the household in the past year was significantly associated with self-reported illness. Sixty-eight percent of participants reported that bushmeat is available within the communities. Our study demonstrates community-level variation in behaviors that may influence zoonotic pathogen exposure. We further recommend development of targeted studies that explore behavioral variations among land use systems in animal production contexts.

Physiological impacts of temperature variability and climate warming in hellbenders (Cryptobranchus alleganiensis).

Cold-adapted hellbender salamanders that inhabit cool mountain streams are expected to fare poorly under warmer projected climate scenarios. This study investigated the physiological consequences of long-term, naturalistic temperature variation on juvenile hellbenders under simulated current and warmer (+1.6 C) climates vs. controlled steady temperatures. Mean temperature and temperature variability were both important predictors of growth as indicated by monthly body mass change (%), stress as indicated by neutrophil:lymphocyte (N:L) ratio and bacteria-killing ability of blood. Cold exposure in hellbenders was associated with weight loss, increased N:L ratios and reduced Escherichia coli killing ability of blood, and these effects were less pronounced under a warmer climate scenario. These observations suggest that cold periods may be more stressful for hellbenders than previously understood. Growth rates peaked in late spring and late fall around 14-17°C. Hellbenders experiencing warmer simulated climates retained body condition better in winter, but this was counter-balanced by a prolonged lack of growth in the 3-month summer period leading up to the fall breeding season where warmer simulated conditions resulted in an average loss of -0.6% body mass/month, compared to a gain +1.5% body mass/month under current climate scenario. Hellbenders can physiologically tolerate projected warmer temperatures and temperature fluctuations, but warmer summers may cause animals to enter the fall breeding season with a caloric deficit that may have population-level consequences.

Effects of Lupron and surgical castration on fecal androgen metabolite concentrations and intermale aggression in capybaras (Hydrochoerus hydrochaeris).

To curb agonistic interactions in a bachelor group of three male capybaras (Hydrochoerus hydrochaeris), a single dose of leuprolide acetate (Lupron®) was used in an attempt to chemically sterilize the males. Concurrently, fecal androgen metabolite (FAM) concentrations were quantified via enzyme immunoassay to monitor changes in testosterone production after injection of the gonadotropin-releasing hormone agonist. When Lupron proved ineffective in suppressing intraspecific aggression, surgical castration was performed on two males, with continued noninvasive endocrine monitoring. In all three capybaras, FAM concentrations increased initially as a result of the luteinizing hormone surge, but then decreased significantly following chemical sterilization. Surgical castration resulted in further, persistent declines in FAM concentrations in two males, while the third, intact male demonstrated a rise in FAM to pre-Lupron concentrations at 8.5 and 9.5-month postadministration. Despite successful suppression of sperm and testosterone production, intermale aggression continued, ultimately necessitating separation of the animals and transfer to other holding institutions. Under this set of conditions, a single Lupron dose was inadequate for suppressing intraspecific aggression in a group of three males with a pre-established history of aggression.