Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

Species, causes, and outcomes of wildlife rehabilitation in New York State.

Wildlife rehabilitation is a publicly popular practice, though not without controversy. State wildlife agencies frequently debate the ecological impact of rehabilitation. By analyzing case records, we can clarify and quantify the causes for rehabilitation, species involved, and treatment outcomes. This data would aid regulatory agencies and rehabilitators in making informed decisions, as well as gaining insight into causes of species mortality. In New York State, the Department of Environmental Conservation (NYSDEC) has licensed rehabilitators since 1980 and annual reporting is required. In this study, we analyzed 58,185 individual wildlife cases that were attended by New York rehabilitators between 2012 and 2014. These encompassed 30,182 (51.9%) birds, 25,447 (43.7%) mammals, 2,421 (4.2%) reptiles, and 75 (0.1%) amphibians. We identified patterns among taxonomic representation, reasons for presentation to a rehabilitation center, and animal disposition. Major causes of presentation were trauma (n = 22,156; 38.1%) and orphaning (n = 21,679; 37.3%), with habitat loss (n = 3,937; 6.8%), infectious disease (n = 1,824; 3.1%), and poisoning or toxin exposure (n = 806; 1.4%) playing lesser roles. The overall release rate for animals receiving care was 50.2% while 45.3% died or were euthanized during the rehabilitation process. A relatively small number (0.3%) were permanently non-releasable and placed in captivity; 4.1% had unknown outcomes. A comparable evaluation in 1989 revealed that wildlife submissions have increased (annual mean 12,583 vs 19,395), and are accompanied by a significant improvement in release (50.2% in the study period vs 44.4% in 1989) (χ2(1) = 90.43, p < 0.0001). In this manuscript, we aim to describe the rehabilitator community in New York State, and present the causes and outcomes for rehabilitation over a three-year period.

How can we augment the few that remain? Using stable population dynamics to aid reintroduction planning of an iteroparous species.

Restoration of depleted populations is an important method in biological conservation. Reintroduction strategies frequently aim to restore stable, increasing, self-sustaining populations. Knowledge of asymptotic system dynamics may provide advantage in selecting reintroduction strategies. We introduce interactive software that is designed to identify strategies for release of females that are immediately aligned with stable population dynamics from species represented by 2-, 3-, 4-, and 5-stage life history strategies. The software allows managers to input a matrix of interest, the desired number of breeding females, and the desired management timeline, and calls upon stable population theory to give release strategies that are in concert with both stable population status and the management goals. We demonstrate how the software can aid in assessing various strategies ahead of a hypothetical restoration. For the purpose of demonstration of the tool only, we use published vital rates of an ungulate species, but remark that the selection of species for demonstration is not central to the use of this tool. Adaption of this tool to real-life restorations of any 2-, 3-, 4-, or 5-stage iteroparous species may aid in understanding how to minimize undesirable recovery complications that may naturally arise from transient population dynamics. The software is freely available at: https//cwhl.vet.cornell.edu/tools/stapopd.

A NOVEL ORTHOREOVIRUS ASSOCIATED WITH EPIZOOTIC NECROTIZING ENTERITIS AND SPLENIC NECROSIS IN AMERICAN CROWS (CORVUS BRACHYRHYNCHOS).

Epizootic mortalities in American Crows (Corvus brachyrhynchos) during the winter months, referred to as winter mortality of crows, have been recorded in North America for almost two decades. The most common postmortem findings include necrotizing enteritis, colitis, and fibrinous splenic necrosis. These findings are proposed to be due to infection with a Reovirus sp. Our objectives were to characterize the pathology and seasonality of the epizootics in New York State (NYS), confirm the causative role of an Orthoreovirus sp., and determine its phylogeny. On the basis of our proposed case definition for reovirosis, we examined case data collected by the NYS Wildlife Health Program for 16 yr. A total of 558 cases of reovirosis were recorded between 2001 and 2017. Reovirosis had a clear seasonal presentation: cases occurred almost exclusively in winter months (71% in December-January). Detailed data from a 2-yr period (2016-17) demonstrated that reovirosis caused up to 70% of all recorded crow deaths during epizootic months. Crows with positive orthoreovirus isolation from the spleen or intestine were 32 times more likely to die with characteristic histologic lesions of enteritis or enterocolitis and splenic necrosis than crows with negative isolation results. An in situ hybridization probe specific to virus isolated from NYS crow reovirosis cases demonstrated a direct association between viral presence and characteristic histologic lesions. Sigma C (capsid protein) sequences of isolates from NYS crows showed high homology with Tvärminne avian virus, recently proposed as a novel Corvus orthoreovirus clade, and only distantly related to the avian orthoreovirus clade. Our study indicated that a novel orthoreovirus was the cause of winter mortality (or reovirosis) of American Crows and placed the NYS isolates in the newly proposed genus of Corvid orthoreovirus.

Necrotizing Enteritis in Nesting Adult Eastern Bluebirds (Sialia sialis) in New York State, USA.

Increased mortalities of adult Eastern Bluebirds, Sialia sialis, breeding in artificial nesting boxes were recorded in New York State, US. A total of 46 dead bluebirds were reported from 23 sites between early April and mid-August 2017. The maximum distance between sites was over 600 km. A total of 27 carcasses were available for postmortem examination. The most common cause of death was necrotizing enteritis, found in 56% (9/16) of birds that could be examined histopathologically. Lesions consisted of foci of hypereosinophilic debris and inflammation rich in Gram-negative bacteria. Aerobic and anaerobic culture of intestines from 4/8 birds with necrotizing enteritis yielded no growth. Plagiorhynchus cylindraceus acanthocephalids were often (6/9, 67%) but not invariably present in affected birds. Occasional incidental lesions included foreign-body microgranulomas in the wall of the ventriculus and intravascular nematodiasis at the base of the heart. The cause of sporadic outbreaks of necrotizing enteritis in breeding bluebirds remains undetermined and warrants further investigation.

Genetic Characterization of Sarcoptes scabiei from Black Bears (Ursus americanus) and Other Hosts in the Eastern United States.

Since the early 1990s there has been an increase in the number of cases and geographic expansion of severe mange in the black bear (Ursus americanus) population in Pennsylvania. Although there are 3 species of mites associated with mange in bears, Sarcoptes scabiei has been identified as the etiologic agent in these Pennsylvania cases. Historically, S. scabiei-associated mange in bears has been uncommon and sporadic, although it is widespread and relatively common in canid populations. To better understand this recent emergence of sarcoptic mange in bears in Pennsylvania and nearby states, we genetically characterized S. scabiei samples from black bears in the eastern United States. These sequences were compared with newly acquired S. scabiei sequences from wild canids (red fox [Vulpes vulpes] and coyote [Canis latrans]) and a porcupine (Erethizon dorsatum) from Pennsylvania and Kentucky and also existing sequences in GenBank. The internal transcribed spacer (ITS)-2 region and cytochrome c oxidase subunit 1 (cox1) gene were amplified and sequenced. Twenty-four ITS-2 sequences were obtained from mites from bears (n = 16), red fox (n = 5), coyote (n = 2), and a porcupine. The sequences from bear samples were identical to each other or differed only at polymorphic bases, whereas S. scabiei from canids were more variable, but 2 were identical to S. scabiei sequences from bears. Eighteen cox1 sequences obtained from mites from bears represented 6 novel haplotypes. Phylogenetic analysis of cox1 sequences revealed 4 clades: 2 clades of mites of human origin from Panama or Australia, a clade of mites from rabbits from China, and a large unresolved clade that included the remaining S. scabiei sequences from various hosts and regions, including sequences from the bears from the current study. Although the cox1 gene was more variable than the ITS-2, phylogenetic analyses failed to detect any clustering of S. scabiei from eastern U.S. hosts. Rather, sequences from black bears grouped into a large clade that included S. scabiei from numerous hosts from Europe, Asia, Africa, and Australia. Collectively, these data suggest that the increasing number of S. scabiei cases in northeastern black bears is not due to the emergence and expansion of a single parasite strain.

The role of a PSP-producing Alexandrium bloom in an unprecedented diamondback terrapin (Malaclemys terrapin) mortality event in Flanders Bay, New York, USA.

Diamondback terrapins (Malaclemys terrapin) are a threatened or endangered species in much of their range along the U.S. Atlantic and Gulf coasts. Over an approximately three-week period from late April to mid-May 2015, hundreds of adult diamondback terrapins were found dead on the shores of Flanders Bay, Long Island, New York, USA. Concurrent with the mortality event, elevated densities of the paralytic shellfish toxin (PST)-producing dinoflagellate, Alexandrium fundyense (>104 cells L-1) and high levels of PST in bivalves (maximal levels = 540 µg STX eq. 100 g-1 shellfish tissue) were observed in the Flanders Bay region, resulting in shellfish bed closures in regional tributaries. Gross and histologic postmortem examinations of terrapins revealed no physical trauma to individuals or a common, underlying disease process to explain the deaths. PST compounds (0.2-12.5 µg STX eq. 100 g-1) were present in various M. terrapin tissues collected over the duration of the mortality event. High-throughput sequencing revealed that the ribbed mussel (Geukensia demissa, a PST vector) was present in the gastrointestinal tracks of all terrapin samples tested. While the potential of PST to cause mortality in chelonians has not been well-characterized, in the absence of other significant findings from necropsies and pathological analyses, we provide evidence that PST in shellfish was likely high enough to cause or contribute to the mortality in these small (<2.0 kg) animals.

Molecular Surveillance for Lymphoproliferative Disease Virus in Wild Turkeys (Meleagris gallopavo) from the Eastern United States.

Lymphoproliferative disease virus (LPDV) is a poorly understood, oncogenic avian retrovirus of domestic turkeys that has historically been restricted to Europe and Israel. However, a recent study reported LPDV in multiple wild turkey diagnostic cases from throughout the eastern United States of America (USA). To better understand the distribution of LPDV in the eastern USA, we surveyed 1,164 reportedly asymptomatic hunter-harvested wild turkeys from 17 states for the presence of LPDV proviral DNA by PCR. In total, 564/1,164 (47%) turkeys were positive for LPDV. Wild turkeys from each state had a relatively high prevalence of LPDV, although statewide prevalence varied from 26 to 83%. Phylogenetic analysis revealed two major clades of LPDV in the USA, although one was at a low frequency suggesting restricted transmission, as well as significant clustering by state of isolation. To determine the best tissue to target for diagnostic purposes, liver, spleen, and bone marrow were tested from a subset of 15 hunter-harvested wild turkeys and 20 wild turkey diagnostic cases. Overall, bone marrow provided the highest level of detection for both hunter-harvested turkeys and diagnostic cases. The sensitivity of LPDV detection between tissues was not significantly different for diagnostic cases, but was for hunter-harvested birds. These results indicate that LPDV infection is common and widespread in wild turkey populations throughout the eastern USA, even without overt signs of disease.

Prevalence of antibodies to Toxoplasma gondii in white-tailed deer (Odocoileus virginianus) in New York State, USA.

Sera collected from 299 white-tailed deer (Odocoileus virginianus) harvested in New York State by hunters in November 2010 were assayed for anti-Toxoplasma gondii IgG antibodies. White-tailed deer are a useful sentinel for risk of human and domestic animal exposure to Toxoplasma oocysts and pose a potential risk for infection to humans and other animals by ingestion of the meat. White-tailed deer share grazing space with domestic animals raised for meat and are likely to be exposed by horizontal transmission through oocyst consumption, similar to other grazing species of economic concern. Overall, 42.2% of samples were positive by enzyme-linked immunosorbent assay, indicating a true prevalence of 38.5%, with a significantly higher proportion of adult than immature deer antibody positive. No significant difference in prevalence was found between male and female deer nor was there a significant effect of local human population density on deer antibody prevalence. These results provide insight into the risk of environmental Toxoplasma exposure in New York State and support horizontal transmission through oocyst consumption as the most common mechanism of white-tailed deer infection.

Proliferative thyroid lesions and hyperthyroidism in captive fishers (Martes pennanti).

Diseases of the thyroid gland are common in many zoo species, but there are few descriptions of thyroid dysfunction in Mustelidae. A 7-yr-old, captive-bred female fisher (Martes pennanti) with progressive alopecia was diagnosed with clinical hyperthyroidism based on persistent elevation of both total and free serum thyroxine and triiodothyronine, ultrasound examination, and histologic evidence of adenomatous hyperplasia. Four additional geriatric adult fishers (two male and two female) were identified with thyroid adenomatous hyperplasia in a review of 23 postmortem records. Banked sera were available for thyroid hormone testing from three of the four necropsy cases. Total and free thyroxine were elevated in four of four animals tested, and triiodothyronine was elevated in two of three animals tested. Necropsy findings in four cases identified cardiac hypertrophy, congestive heart failure, and vascular lesions consistent with hypertension; complete tissues were not available from the remaining case. Clinical and subclinical hyperthyroidism may be a common but overlooked condition of captive fishers.

Evaluation of oral itraconazole administration in captive Humboldt penguins (Spheniscus humboldti).

Aspergillus spp. fungal infections are the most common cause of morbidity and mortality in captive penguins. Itraconazole has been the drug of choice for both therapeutic and prophylactic treatment; however, the pharmacokinetic and pharmacodynamic parameters can be highly variable in different species, and it has not been evaluated in penguins. In this study, four preliminary steady-state trials were performed to compare two oral formulations of itraconazole (commercial capsules compared with generic bulk compounded powder) at two different dosages (6 or 12 mg/kg once a day) administered in fish to small groups of captive Humboldt penguins (Spheniscus humboldti). Building on this data, a final steady-state trial was performed with the use of a 7 mg/kg oral dosage twice a day of commercial capsules given in fish to a group of 15 penguins. With sparse sampling, blood was drawn for testing from small subsets of each treatment group at 4-7 time points in the 24-hr period after the final dose of itraconazole on day 14. Steady-state plasma concentrations of itraconazole and hydroxyitraconazole, the major metabolite, were determined by reverse phase high-performance liquid chromatography with fluorescence detection. Treatment with the generic bulk compounded product resulted in plasma levels of itraconazole that were undetectable for 26 out of 30 blood samples, compared with seven out of 20 blood samples for the commercial product at the same dosage. On the basis of study results, an estimated oral dosage of either 8.5 mg/kg twice a day or 20 mg/kg once a day of the commercial itraconazole capsules given in fish would produce adequate steady-state therapeutic blood levels in Humboldt penguins.