Rh: review of mountain chicken frog necropsy findings a retrospective review of post-metamorphic mountain chicken frog (leptodactylus fallax) necropsy findings from european

The mountain chicken frog (Leptodactylus fallax) is the largest endemic amphibian species in the Western Hemisphere. Since 1998, this Critically Endangered species has been maintained as a European Endangered Species Programme (EEP) but low breeding success and a 25 high mortality rate threatens the sustainability of the captive frog population. In the current study, we analyzed gross and histopathological post-mortem information from 212 mountain chicken frogs which died in European zoological collections, 1998 2018. Thin body condition was the most commonly reported finding across all submissions, observed in 125 frogs. The gastrointestinal and urinary systems were reported to have the highest prevalence of pathological 30 findings on gross and histopathological examination. Inflammatory disease was the most frequent diagnosis following histopathological examination of relevant tissues with intestinal inflammatory disease (n=76) followed by tubulointerstitial nephritis (n=26) most commonly reported. Neoplasia was reported in 42/212 (19.8%) frogs, all of which were adults. A defined cause of death, or reason for euthanasia, was proposed for 164/212 (77.4%) frogs with 35 inflammatory diseases processes (74/212; 34.9%) most commonly implicated. Intestinal adenocarcinoma, seemingly restricted to the colon, caused the deaths of 31 adult frogs. Further investigations to determine factors contributing to the high incidence of inflammatory disease processes and neoplasia are advocated in order to help improve the health and sustainability of the captive mountain chicken frog population. 40 INTRODUCTION The mountain chicken frog (Leptodactylus fallax) is the largest endemic amphibian in the 45 Western Hemisphere but its population has been decimated by habitat loss, hunting, introduced predators and, most notably, the infectious fungal agent, Batrachochytrium dendrobatidis (Bd). In recent times, mountain chicken frogs (MCFs) have been restricted to just two Caribbean islands, Dominica and Montserrat. Between 2002-2004, outbreaks of chytridiomycosis caused an 85% reduction in the MCF population on Dominica and, since the first detection of Bd on 50 Montserrat in 2009, the island’s MCF population has experienced a catastrophic decline with the species now thought to be extinct on the island. In 1998, wild MCFs were first brought to Europe with the aim of founding a sustainable captive breeding population. In 2009, during the chytrid outbreak on Montserrat, a further 50 wild frogs were brought to Europe from this island to form the core of a captive breeding 55 population, maintained under strict biosecurity conditions, with the ultimate aim of future reintroduction to the wild. The long-term viability of the captive MCF population is uncertain due to low breeding success and high rates of mortality; however, the precise causes and potential underlying pathologies have not been determined. Mortality reviews relating to other wild and captive 60 anuran species have been been published; however, these are limited to non-Leptodactylid species and are unlikely to accurately reflect the situation facing the MCF. Whilst case reports exist which describe the diagnosis of neoplasia and metabolic bone disease in individual captive MCFs, the importance of these, or other, disease processes at a population level has not previously been determined. 65 This study is the first comprehensive review of post-mortem data relating to captive MCFs from multiple European institutions. The results will help to identify possible disease risk factors and guide future areas of investigation with the aim of improving the health and sustainability of the captive MCF population. 70 MATERIALS AND METHODS Necropsy reports All European Endangered Species Programme (EEP) establishments holding MCFs were asked to supply necropsy reports relating to MCFs which died whilst housed at these institutions between 1 January 1998 and 31 August 2018. Information pertaining to each animal’s age and 75 sex was recorded. If known, the frog’s birth status (wild born versus captive bred) and biosecure versus non-biosecure status was also recorded. Animals were categorized as juvenile (zero days to two years post-metamorphosis) or adult (>2 years). All wild born frogs were considered adults at the time of capture so, for the purposes of this study, the age at death for these individuals was estimated by adding two years to the time period they were held in captivity. Tadpoles were 80 excluded from the study because L. fallax are not officially recorded in the EEP until they have metamorphosed and reliable data is not available. Details relating to environmental parameters and husbandry practices at different holding institutions were not requested. Each necropsy report was individually reviewed and assessed for quality of information provided and the state of carcass preservation (no autolysis; mild autolysis; severe autolysis) at 85 the time of the necropsy. Records which did not describe post-mortem examination findings and reports stating severe carcass autolysis were excluded from the study. Body tissues were considered abnormal on gross examination if unusual macroscopic observations relating to shape, size, color or texture were reported; however, histopathology was required to confirm and characterize disease processes further. All pathological findings were 90 recorded. Based on relevant gross and histopathological information provided by pathologists within submitted reports, a primary cause of death or reason for euthanasia (hereafter “cause of death”) was assigned to each frog. Pathological findings and cause of death were categorized by main body system affected (body as a whole; cardiopulmonary; celomic cavity; gastrointestinal (GI); 95 integumentary; hepatobiliary; musculoskeletal; reproductive; special senses; urinary; multisystemic; not determined) and primary disease process (inflammatory; neoplasia; otherdetermined; not determined). Statistical analysis 100 For any particular pathological finding, prevalence was calculated using the number of animals for which that particular organ, tissue or specific finding was reported as screened for, rather than using the total number of post-mortem reports. This was done to prevent bias caused by variation in the breadth and depth of information contained within submitted necropsy reports. All further analyses were performed using a statistics program (SPSS Statistics Version 24; 105 IBM. Armonk, NY 10504, USA). Statistical significance was set at P < 0.05. Analyses were performed to determine whether significant differences existed between pathologic findings and sex, age, wild born versus captive bred status and biosecure versus non-biosecure status. Pearson’s chi-square test was performed to examine relationships between mutually exclusive categorical data. The Cramer’s V strength test was used to test the data when a significant Chi110 square result had been obtained. If the assumptions of the adequate expected cell count of 5 or more had been violated, the Fisher’s Exact test was used. Two sample t-tests were used to analyze differences between the occurrence of pathologic alterations and age. Information relating to post-mortem microbiological testing was limited and not standardized and was therefore not considered suitable for statistical analysis. 115 RESULTS Study Population Reports were received for 343 post-metamorphic MCF from eleven different European institutions. Due to poor carcass preservation and or lack of information relating to post-mortem 120 examination findings, 131 reports were excluded from the study. Of the 212 records included in the statistical analysis, 53 post-mortem reports contained only gross necropsy findings whilst 149 reports contained information from both gross and histopathological examinations. A further ten reports contained information relating to histopathological examination only. The breadth and depth of information provided varied 125 between reports. One-hundred-and-fifty-eight reports were for captive bred frogs and 54 were from wild born animals. Sixty-one frogs (28.8%) were from the biosecure captive population whilst 136 frogs (64.1%) were held under non-biosecure conditions. The status of 15 frogs was not reported. Adult frogs were most represented (148/212; 70.1%) with 62 juveniles and two frogs of unknown 130 age also included. Wild born frogs lived, on average, significantly longer (7y2m) than captivebred frogs which had reached adult status (3y7m) (t=6.08, p<0.001). Sex was confirmed as female in 86 frogs (40.6%) and male in 78 animals (36.8%) whilst the sex of 48 frogs was not recorded, 44 (91.2%) of which were juveniles. 135 Post-mortem findings The most prevalent findings observed on gross and histopathological examination are shown in Table 1. Body condition was reported in 200 out of 212 submitted reports (94.3%) with thin body condition, often to the point of suspect emaciation, commonly described (125/200; 62.5%). This 140 finding was significantly more prevalent in adult (Chi=31.9, p<0.001) and captive bred frogs (Chi=14.1, p<0.001). An underlying disease process was reported in 98 of these individuals. The gastrointestinal tract was the organ system with the highest frequency of lesions reported. Gross examination of the gastrointestinal tract was performed in 202 animals and lesions were observed in 82 (40.6%) of these animals, 77 of which were adult frogs. Adhesions between the 145 colon and the urinary bladder was the most commonly reported lesion involving this body system (45/202; 22.3%) with concurrent fistula formation seen in 11 cases. These adhesions were more commonly reported in adult frogs (F, p<0.001). Soft tissue intestinal masses (31/202; 15.3%) (Figure 1), intestinal distension (29/202; 14.1%) and intestinal impaction (18/202; 8.9%) were also commonly reported. 150 Histopathological examination was performed on GI tissues from