Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring.

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng µL-1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80-120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

APPLICATION OF A NOVEL ASPERGILLUS LATERAL-FLOW DEVICE IN THE DIAGNOSIS OF ASPERGILLOSIS IN CAPTIVE GENTOO PENGUINS (PYGOSCELIS PAPUA PAPUA).

Aspergillosis is the primary fungal disease affecting captive penguins globally. Its diagnosis remains challenging, and currently no tests are both sensitive and specific for the detection of early infection. The present study evaluated a recently developed Aspergillus lateral-flow device (AspLFD) for the detection of Aspergillus spp. antigen in plasma and glottis mucus from captive penguins. In a pilot retrospective study, banked frozen plasma samples from captive penguins were reviewed: samples from 11 gentoo penguins (Pygoscelis papua papua) and 4 king penguins (Aptenodytes patagonicus) fulfilled the inclusion criteria and were used in the analysis. Positive plasma AspLFD test results were found in 80% (four of five) of the aspergillosis-positive cases tested. All of the aspergillosis-negative cases tested negative (10 of 10) on the AspLFD test. In a cohort prospective study, paired plasma and glottis swab samples were opportunistically and nonrandomly collected from captive gentoo penguins. In total, 26 penguins were tested. In the negative control group, AspLFD test was negative on plasma and swab in 100% of birds (14 of 14). In the aspergillosis-positive group, AspLFD test was positive on plasma samples from 33% (4 of 12) of birds, on swab samples from 50% (6 of 12) of birds, and on either plasma or swab samples from 75% (9 of 12) of birds. The AspLFD is currently used for the diagnosis of aspergillosis in humans and also shows promise for use in penguins. Larger prospective studies are recommended.

USE OF CLINDAMYCIN IN PALLAS' CATS [OTOCOLOBUS (FELIS) MANUL] TO REDUCE JUVENILE TOXOPLASMOSIS-ASSOCIATED MORTALITY RATES.

Pallas' cat [Otocolobus (Felis) manul] experiences a high mortality rate from toxoplasmosis. During the period 2006-2016, the overall mortality rate for this species from all causes during the first year of life was 71.59% in European Association of Zoos and Aquaria institutions, with the most significant infectious cause from systemic toxoplasmosis (20.6%) as confirmed by postmortem examination and histopathology. Clindamycin was used starting in 2014 in two collections that had previously experienced 100% mortality rates by toxoplasmosis in kittens less than one year of age, covering key Toxoplasma gondii exposure periods for kittens (n = 17) as a prophylactic measure. This protocol resulted in a 67.03% (95% confidence interval 41.76-78.61%) reduction in the first year mortality rate over a two-year period to 5.88% in those animals treated.

HYPERVITAMINOSIS D IN A GIANT ANTEATER (MYRMECOPHAGA TRIDACTYLA) AND A LARGE HAIRY ARMADILLO (CHAETOPHRACTUS VILLOSUS) RECEIVING A COMMERCIAL INSECTIVORE DIET.

Hypervitaminosis D was diagnosed in a giant anteater (Myromecophaga tridactyla) and a large hairy armadillo (Chaetophractus villosus) being fed a commercial insectivore diet. Clinical findings included weight loss, reduced appetite, vomiting, and suspected abdominal discomfort. Hypercalcemia (3.68 and 2.04 mmol/L total and ionized calcium, respectively) was detected in the anteater, and plasma 25(OH)D levels were measured and found to be 808.7 and 379.4 nmol/L for the anteater and armadillo, respectively. Dietary change resulted in a reduction of 25(OH)D levels in both animals and resolution of hypercalcemia in the giant anteater. Dietary analysis of the commercial insectivore food revealed levels of vitamin D3 higher than the data-sheet values. This case report demonstrates that hypervitaminosis D in Xenarthra can be associated with significant clinical signs.

Evidence of Leptospira species and their significance during reintroduction of Eurasian beavers (Castorfiber) to Great Britain.

The Scottish Beaver Trial (SBT) reintroduced the Eurasian beaver (Castor fiber) in 2009 using wild-caught Norwegian beavers. This included a six-month prerelease quarantine in Devon, England. The International Union for Conservation of Nature (IUCN) and government guidelines for health screening were followed, including testing for Leptospira species. Unlicensed beavers, from Germany, were also identified in Scotland (Tayside) and Devon (later forming the River Otter Beaver Trial (ROBT)) and were health-screened under licence. Due to positive Leptospira species results and lack of prerelease screening in ROBT and Tayside, beavers from Germany and Norway (range sources) were screened. One hundred and fifty-six samples from 151 beavers were analysed by Leptospira species quantitative PCR (qPCR) (n=73 kidney (postmortem)/urine samples (antemortem)) or microscopic agglutination test (MAT, Leptospira pools 1-6) (n=83 serum samples). No beavers from Norway (95 per cent confidence interval (CI) 0-5.6 per cent, n=52), Tayside or SBT postrelease (95 per cent CI 0-4.6 per cent, n=63) tested positive. Seven beavers from Germany and Devon were positive. This gives an overall 9.3 per cent (95 per cent CI 5.2-15.1 per cent) exposure level, of which 4.6 per cent (95 per cent CI 1.9-9.3 per cent) suggested infection on a positive qPCR (n=1) or MAT titre of at least 1/400 (n=6), although none had abnormal physical, biochemical or haematological changes. This study suggests that Leptospira species infection in wild Eurasian beavers occurs at a low level, has no sex bias and does not appear to cause significant morbidity or mortality.

CLINICAL SIGNS, ANTEMORTEM DIAGNOSTICS, AND PATHOLOGICAL FINDINGS ASSOCIATED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS INFECTION IN MISHMI TAKIN ( BUDORCAS TAXICOLOR TAXICOLOR).

Mycobacterium avium subspecies paratuberculosis (MAP) is a cause of contagious and typically fatal enteric disease, primarily affecting ruminant and pseudoruminant species. During a MAP outbreak in a captive collection, six of nine adult Mishmi takin ( Budorcas taxicolor taxicolor) showed marked weight loss over 1-3 mo, followed by an acute deterioration. Fecal culture and microscopy failed to identify MAP shedding. Necropsy findings included grossly normal intestines and marked enlargement of mesenteric lymph nodes. Histological findings included multibacillary granulomatous enteritis, mesenteric lymphadenitis, and periportal hepatitis. MAP was confirmed by culture of intestinal and lymph node tissues from the index case. Results of antemortem serological testing using an indirect ELISA (ID SCREEN® Paratuberculosis Indirect) were corroborated by findings at necropsy or survival of the outbreak. Mishmi takin appear to show high MAP susceptibility and a rapid disease course compared with domestic ruminant species.

SUSPECTED HYPOVITAMINOSIS A-ASSOCIATED SALT GLAND ADENITIS IN NORTHERN ROCKHOPPER PENGUINS ( EUDYPTES MOSELEYI).

Supraorbital salt-excreting glands are present in at least 10 avian orders and are largest in marine species, including penguins. Diseases of the avian salt gland have been described infrequently. From September 2015, five captive northern rockhopper penguins ( Eudyptes moseleyi) were presented over a 6-wk period for unilateral or bilateral supraorbital swellings. In September 2016, two cases recurred and two additional cases were identified. Histopathology demonstrated salt gland adenitis with extensive squamous metaplasia. Blood plasma testing demonstrated marked vitamin A and E deficiencies within the colony. Prolonged frozen storage of feed-fish was implicated as a cause of vitamin depletion; reducing storage times and addition of dietary supplementation prevented recurrence.

MANDIBULAR SQUAMOUS CELL CARCINOMA IN A ROCK HYRAX ( PROCAVIA CAPENSIS).

A 9-yr-old, entire female captive rock hyrax ( Procavia capensis) was presented with a 1-wk history of sialorrhea. On clinical examination, a mass was identified, encompassing the rostral mandible and intermandibular area, with associated mucosal ulceration, marked gingival recession, and loosening or loss of adjacent teeth. Skull radiography and cytology of fine-needle aspirates of the mass were suggestive of squamous cell carcinoma. Based on a suspected poor prognosis, the animal was humanely euthanized. Postmortem histological examination of samples confirmed a diagnosis of oral squamous cell carcinoma with invasion of mandibular bone. Neoplasia is uncommonly reported in hyraxes, which has led to the assumption that they may share mechanisms of cancer resistance with elephants, their closest extant relatives. This is the first report of squamous cell carcinoma in this species.

Plasma protein electrophoresis as a prognostic indicator in Aspergillus species-infected Gentoo penguins (Pygoscelis papua papua).

BACKGROUND: Avian aspergillosis presents a significant threat to captive penguin populations. Currently, a lack of objective prognostic factors limits disease staging, objective reassessment throughout treatment, comparative evaluation of treatment regimes, and appropriate timing of euthanasia. OBJECTIVE: The study objective was to investigate absolute and relative plasma protein fractions by agarose gel electrophoresis (EPH) as predictors of survival in Gentoo penguins (Pygoscelis papua papua) under treatment for aspergillosis. METHODS: One hundred and eighty-three EPH profiles from individual clinical cases were examined retrospectively. Animal survival to 90 days post sampling was established from clinical records; birds either survived (n = 146) or died within 90 days (n = 37), and time to death was recorded. RESULTS: Fourteen variables showed statistically significant differences (P < .05) between surviving and dying birds. Receiver operating characteristic curve analysis identified total albumin concentration (albumin + prealbumin) and albumin-to-globulin (A:G) ratio as having strongest discriminatory values (95% CI) at 0.788 (0.710-0.866) and 0.784 (0.696-0.871), respectively. Albumin (concentration and percentage of total protein) displayed moderate discriminatory value but additionally a weak positive correlation with time to death (95% CI); r = .353 (0.033-0.608) and .424 (0.116-0.658), respectively. CONCLUSIONS: Optimized test cutoffs for total albumins, albumin (concentration and percentage of total protein), and A:G ratio achieved moderate sensitivity and specificity, strong negative predictive values, but weak positive predictive values due to a low prevalence of death. Selection of appropriate test cutoff values may provide valuable adjunctive prognostic tools for clinical decision-making when the prognosis is difficult to assess clinically.

Haematology and Serum Biochemistry Parameters and Variations in the Eurasian Beaver (Castor fiber).

Haematology parameters (N = 24) and serum biochemistry parameters (N = 35) were determined for wild Eurasian beavers (Castor fiber), between 6 months - 12 years old. Of the population tested in this study, N = 18 Eurasian beavers were from Norway and N = 17 originating from Bavaria but now living extensively in a reserve in England. All blood samples were collected from beavers via the ventral tail vein. All beavers were chemically restrained using inhalant isoflurane in 100% oxygen prior to blood sampling. Results were determined for haematological and serum biochemical parameters for the species and were compared between the two different populations with differences in means estimated and significant differences being noted. Standard blood parameters for the Eurasian beaver were determined and their ranges characterised using percentiles. Whilst the majority of blood parameters between the two populations showed no significant variation, haemoglobin, packed cell volume, mean cell haemoglobin and white blood cell counts showed significantly greater values (p<0.01) in the Bavarian origin population than the Norwegian; neutrophil counts, alpha 2 globulins, cholesterol, sodium: potassium ratios and phosphorus levels showed significantly (p<0.05) greater values in Bavarian versus Norwegian; and potassium, bile acids, gamma globulins, urea, creatinine and total calcium values levels showed significantly (p<0.05) greater values in Norwegian versus Bavarian relict populations. No significant differences were noted between male and female beavers or between sexually immature (<3 years old) and sexually mature (≥3 years old) beavers in the animals sampled. With Eurasian beaver reintroduction encouraged by legislation throughout Europe, knowledge of baseline blood values for the species and any variations therein is essential when assessing their health and welfare and the success or failure of any reintroduction program. This is the first study to produce base-line blood values and their variations for the Eurasian beaver.