Given that some DNA viruses have been found to exhibit virus-host co-evolution and establish lifelong infection, mammals with unique evolutionary histories in island ecosystems likely host exceptionally diverse viruses. Madagascar is inhabited by endemic non-human primate and rodent lineages interacting with expansive populations of introduced non-native rodents across the island. Using a viral metagenomic workflow on 189 oral swabs of lemurs and rodents in southeastern Madagascar, we characterized genomic sequences of DNA viruses in the families Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, Parvoviridae and Polyomaviridae and assessed their phylogenetic relationships to known viruses. Endemic lemurs and tufted-tailed rats displayed particularly novel DNA viral diversity mirroring the geographic isolation and subsequently rich evolutionary history of their hosts. Notably, we provide the first coding-complete sequences in lemurs of herpesviruses, polyomaviruses, adeno-associated viruses and circoviruses. In contrast, the DNA viral communities of black rats in Madagascar were similar to those found in globally distributed black and brown rat populations, given their broad geographic spread and relatively recent introduction to the island. Given the scarcity of viral research in natural populations of lemurs and rodents in Madagascar despite the island's exceptional biodiversity and escalating anthropogenic pressures, this study provides a genomic and phylogenetic foundation for DNA viruses infecting Malagasy lemurs and rodents.

Strategies to improve survival of wildlife impacted by emerging infectious disease are needed for species of conservation concern. We investigated how prior infection by the skin fungal pathogen, Batrachochytrium dendrobatidis (Bd), affected subsequent disease outcomes in a highly susceptible and critically endangered toad, Atelopus glyphus. Our experimental design consisted of: (1) Bd infection followed by itraconazole clearance and Bd re-infection (Bd-Bd n = 20), (2) mock infection followed by itraconazole treatment and Bd infection (mock-Bd n = 20), (3) Bd infection followed by itraconazole clearance and mock infection (Bd-mock n = 10), (4) mock infection followed by itraconazole treatment and mock infection (mock-mock n = 10), (5) untreated animals (untreated n = 10). Prior infection shifted the skin bacterial and fungal communities to a new state enriched in putatively anti-Bd bacteria. However, prior infection worsened disease outcomes, accelerating Bd infection and mortality in prior infected animals compared to naïve animals. Prior infection animals not re-infected (Bd-mock) showed immune gene downregulation compared to re-infected Bd-Bd animals suggesting that even after Bd clearance, Bd caused sustained immunosuppression in genes that respond to Bd; this likely led to lowered immune efficacies when re-infected. Upon re-infection, as Bd load increased, Bd became the primary driver of both immune expression and bacterial composition changes regardless of infection history. Observed bacterial community differences were closely tied to host immune gene expression. The fungal community did not shift in response to re-infection, suggesting that the initial shift was due to itraconazole treatment. Prior infected Bd-Bd animals did not show any novel shifts in immune gene expression, rather they had similar expression patterns that were just more pronounced compared to naïve mock-Bd animal. This suggests that the infection-clearance-infection treatment was ineffective in improving animal survival because there was limited long-lasting effects on immune genes once the pathogen was re-encountered. Unlike previous studies demonstrating protective effects following Bd clearance in other species, this highly susceptible amphibian showed no evidence of acquired resistance. Our results underscore the species-specific nature of disease outcomes and the value of integrative approaches for examining strategies to reduce pathogen susceptibility in endangered wildlife.

Non-native rodents serve as bridges between anthropogenic and natural landscapes. They have expanded across the planet alongside humans while bringing competition, predation, and pathogens, such as poxviruses, to naïve ecosystems. Although rodents serve as reservoirs for multiple zoonotic poxviruses, limited research has focused on rodents for identification of unknown poxviruses. Here, we characterized a divergent metagenome-assembled poxvirus, madamurpox virus, from the oral swab of a black rat in southeastern Madagascar. While madamurpox virus shares a phylogenetic relationship with human-infecting molluscum contagiosum virus and bat-associated Rousettus poxvirus, madamurpox virus presents extensive genetic variation and represents a putative new species and genus in the Chordopoxvirinae subfamily. Further, although madamurpox virus has a similar genome organization to molluscum contagiosum virus and Rousettus poxvirus, madamurpox virus lacks key immune modulators seen in molluscum contagiosum virus. Our findings highlight that substantial unexplored poxvirus diversity likely exists in rodents, with globally distributed, non-native rodent populations of increased interest.

A 27-yr-old female pygmy hippopotamus (Choeropsis liberiensis) had two consecutive stillbirths with no overt signs of labor, suggestive of uterine inertia. After a third pregnancy was confirmed, an induction protocol was developed. Cloprostenol and betamethasone were administered on d 200 of gestation (time 0 h). Additional doses of cloprostenol were administered at 24 and 48 h and oxytocin at 30, 31, and 48 h. Each injection resulted in preparturient behavior without overt evidence of contractions. Fetal membranes presented at the vulva at 54.5 h after initial cloprostenol and betamethasone administration with no progression of labor. Transvaginal palpation and manual delivery of a live calf followed. Despite confirmed nursing, the serum glutaraldehyde coagulation test was negative. Failure of passive transfer may have been secondary to the induction protocol. The calf was treated with broad-spectrum antimicrobial agents due to diarrhea, and clinical signs resolved. This clinical brief details the first known induction of parturition in a pygmy hippopotamus, which can serve as the basis for further development of the technique.

The endangered Limosa harlequin frog Atelopus limosus has experienced significant chytridiomycosis-related declines, but has been successfully bred in captivity as part of the Panama Amphibian Rescue and Conservation Project. We conducted the first Atelopus release trial using 83 captive-bred A. limosus , and monitored individuals as they transitioned from captivity back into the wild at a site within their historic distribution with no extant population. We acclimated 23 animals to the environment prior to release by holding them in mesocosms for 30 days (soft release) and released a further 60 animals without prior acclimation (hard release). We radio-tracked a subsample of animals in each treatment group. We used a Bayesian mark–recapture analysis to integrate known fate data from radio-tracked individuals with data from resighted individuals that had unknown fates. For this analysis, we allowed survival to differ between release treatments (hard vs . soft) and assumed detection probability would differ between tracking methods. Per-survey detection probability was about 0.02 for individuals without radio transmitters vs . 0.88 for radio-tracked frogs. We observed that hard-released animals initially dispersed further than soft-released animals, and, despite the fact that 25% of the soft-released animals were Batrachochytrium dendrobatidis (Bd)-positive on release day, their estimated 30-day survival probability was 0.46 ( vs . 0.31 for the hard-released animals). The insights from this release trial can be used in an adaptive management framework to improve and refine release methods that will inform the nascent field of amphibian reintroduction ecology.

During the early stages of the COVID-19 pandemic in 2020, the first author, then a fourth-year student at Harvard Medical School, was enrolled in a One Health clinical experience at Zoo New England where he was introduced to a transdisciplinary approach to integrate human, animal, and ecosystem health. Seeing the vast impact of the pandemic and knowing its roots as a zoonotic disease, he realized this approach was critical to his medical education and for preparation against future novel infectious diseases. Zoonotic diseases have been emerging into human populations with increasing frequency, leading to public health emergencies such as Ebola, avian influenza, and SARS. The SARS-CoV-2 narrative, starting in bats and then mutating through an intermediate host into humans, is another striking example of the interconnectedness between human, animal, and ecosystem health that underlies these infections. Preventing future pandemics will require a transdisciplinary One Health approach, and physicians should be prepared to participate in these discussions while advocating for One Health initiatives for the benefit of their current and future patients. Integration of One Health education into medical school curricula will also prepare future physicians for other complex and urgently important health issues such as climate change, antimicrobial resistance, and the impact of biodiversity loss. As the consequences of the COVID-19 pandemic persist, education in One Health must become a priority; it is essential to break down the conventional disciplinary silos of human medicine, veterinary medicine, environmental health, public health, and the social sciences, so that future health crises can be prevented and mitigated collaboratively.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearingAtelopus variustadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

There is an urgent need to integrate One Health education into medical school curricula. One Health is the concept that human health is intrinsically connected to animal health and ecosystem health. The current COVID-19 pandemic highlights the need for physicians and medical students to understand how deforestation, climate change and the loss of biodiversity impact human health and often perpetuate racial inequities. We describe a One Health elective that we developed at Harvard Medical School where students rotate through the Franklin Park Zoo while studying a curriculum focused on disease ecology, climate change and the importance of biodiversity which is taught by the zoo’s head veterinarian. Living through the SARS-CoV-2 pandemic should be a call for our heightened understanding that our health is intimately linked to the health of our planet and its other living creatures in a shared ecosystem. Only through education and training will our future physicians begin to understand these connections and become advocates for climate protections and the preservation of biodiversity.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several free-ranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations >250 ng/ml (voriconazole) and >1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations >250 ng/ml for 12-24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1-17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02-1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanox®) was evaluated in seven apparently healthy free-ranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species.

Serum 25-hydroxyvitamin D concentrations were assessed in subadult to adult captive lowland gorillas ( Gorilla gorilla gorilla) (n = 26) at two institutions with different husbandry and management practices. Serum 25-hydroxyvitamin D (25[OH]D) concentrations for gorillas managed predominantly indoors was low (14.2 ± 5.9 ng/ml), despite consuming commercial biscuits fortified with vitamin D3. Concentrations of 25(OH)D in gorillas with near daily outdoor access were significantly higher than gorillas managed indoors, although many individuals still had serum values below concentrations recommended for adult humans. Consideration should be given to assessing 25(OH)D concentrations in all captive gorillas and providing specific supplementation, particularly to juveniles without access to direct sunlight.

A 20-yr-old female Matschie's tree kangaroo (Dendrolagus matschiei) was diagnosed with hypoaldosteronism, a rare condition in which the body fails to produce normal amounts of the mineralocorticoid aldosterone. Aldosterone plays a key role in body salt homeostasis, increasing sodium reabsorption and promoting excretion of potassium. Hypoaldosteronism resulted in decreased appetite, lethargy, and weight loss in conjunction with hyponatremia, hyperkalemia, and hypercalcemia in this tree kangaroo. The animal was successfully managed with mineralocorticoid replacement using desoxycorticosterone pivalate. To the authors' knowledge this is the first report of hypoaldosteronism in a tree kangaroo and one of the few reports in the veterinary literature in any species.

An 18-year-old female Bali mynah (Leucopsar rothschildi) was presented for polyphagia, weight loss, and incoordination. Diabetes mellitus was diagnosed based on the history and clinical findings, including persistent hyperglycemia with concurrent hypoinsulinemia and glucosuria. A treatment protocol was developed that led to improvement of clinical signs and management of hyperglycemia over several months. Because of the advanced age of the animal, difficulty in maintaining euglycemia, and the stress of handling and treatment, euthanasia was elected 167 days after initial presentation. At postmortem examination, no pancreatic lesions were detected histologically that would account for the diabetes mellitus. To our knowledge this is the first reported case of diabetes mellitus and clinical management of this condition in a passerine species.

An increase in mortality in a captive flock of budgerigars (Melopsittacus undulatus) coincided with the isolation of attaching and effacing Escherichia coli from postmortem samples. Common histologic lesions included hepatitis, enteritis, and in one case attaching and effacing lesions along the intestinal tract. Retrospective review of necropsy records and increased sampling led to the identification of several cases of E. coli with the attaching and effacing (eae) virulence gene. Factors such as environment, nutrition, and concomitant pathogens were thought to contribute to mortality in the flock. Although it is not clear whether E. coli was a primary pathogen during the period of increased mortality, the presence of the eae gene combined with associated histologic lesions supports the conclusion that this organism was a significant contributor to mortality. Manipulation of diet, environment, and the addition of probiotic supplementation resulted in a decline in mortality rate and decreased shedding of E. coli based on negative follow-up cultures of intestines, liver, and feces.