GENOTYPE IDENTIFICATION OF TOXOPLASMA GONDII IN MACROPODS FROM A ZOOLOGICAL PARK IN FLORIDA, USA.

There are limited reports of the genetic characterization of Toxoplasma gondii infecting captive macropods in North America. A novel genotype, ToxoDB PCR-RFLP genotype 263, was reported from six wallabies at a zoological facility in Virginia, USA, prompting an investigation into the genotypes from T. gondii strains infecting macropods at a zoological park in Florida, USA. Cardiac muscle and/or lung samples from an agile wallaby (Macropus agilis, n = 1), red kangaroos (Macropus rufus, n = 8), red-necked wallaby (Macropus rufogriseus, n = 1), and a tammar wallaby (Macropus eugenii, n = 1) that died between 2014 and 2018 were collected. All 11 cases were confirmed to have died from systemic toxoplasmosis by histopathology and immunohistochemical staining. Multilocus PCR-RFLP genotyping of T. gondii was performed directly on tissue samples or on parasites isolated from myocardium by mouse bioassay. Two cases of toxoplasmosis were identified as the reported novel genotype, ToxoDB PCR-RFLP genotype 263, but no common source of exposure could be identified. Five cases were identified as genotype 2 (type III strain, haplogroup 3), and four cases were identified as genotype 216, which has been previously reported in North American wildlife. There were no overt differences in lesion severity or distribution related to genotype. These results suggest that the premise was contaminated with at least three genotypes of T. gondii causing systemic toxoplasmosis in macropods. The largest cluster of fatal toxoplasmosis in macropods in the study period occurred following severe rainfall flooding of the exhibit, suggesting the transmission of T. gondii by water and pointing out the importance of this transmission mechanism. In summary, our study revealed three T. gondii outbreaks that caused significant loss of macropods within 5 yr in a zoological facility in Florida. More studies are needed to understand transmission and prevention of toxoplasmosis in sensitive zoo animals.

SEROSURVEY, HEMATOLOGY, AND CAUSES OF MORTALITY OF FREE-RANGING AMERICAN MARTENS ( MARTES AMERICANA) IN MICHIGAN.

To better understand the clinical pathology, diseases, and causes of mortality of reintroduced American martens ( Martes americana) in Michigan, a study was conducted from 2011 to 2015 in the Upper and Lower Peninsulas of Michigan. Samples obtained from live trapping ( n = 58) or harvested carcasses ( n = 34) were serologically tested for select pathogens. Antibodies against Toxoplasma gondii and canine distemper virus were detected in 58 and 3.4% of samples, respectively. All samples were seronegative for Leptospira spp. and negative for Dirofilaria immitis antigen. Urine samples tested for Leptospira spp. via immunofluorescent antibody assay ( n = 7), polymerase chain reaction ( n = 6) , or both ( n = 3) were all negative. Parvovirus DNA was detected in 9.1% of small intestine samples ( n = 22) collected from carcasses and in 3.7% of fecal samples ( n = 27) collected during live trapping. Complete blood counts ( n = 64) and serum biochemistries ( n = 63) were obtained from 49 live-trapped martens. Biochemical parameters found to be significantly different ( P < 0.05) between genders were calcium, creatinine, glucose, and phosphorus. There was no significant difference between genders for any hematologic parameter. Significant differences ( P < 0.05) between summer and winter seasons were found in total estimated white blood cell count, neutrophils, lymphocytes, monocytes, alkaline phosphatase, bicarbonate, calcium, creatinine, globulin, glucose, phosphorus, potassium, sodium, and total protein. There was no significant difference in blood cell count or serum biochemistry values between radio-collared ( n = 17) and noncollared ( n = 47) martens. Animals seropositive for T. gondii were found to have significantly higher ( P < 0.05) eosinophil and globulin levels than seronegative animals. The primary natural cause for mortality of radio-collared American martens was predation. Histologic examinations revealed a high percentage (60%) of martens with verminous or granulomatous pneumonia.

Gastrolithiasis in prehensile-tailed porcupines (Coendou prehensilis): nine cases and pathogenesis of stone formation.

Gastrolithiasis was diagnosed in nine prehensile-tailed (PT) porcupines (Coendou prehensilis) housed at six zoologic institutions in the United States and Canada. Affected animals were either asymptomatic or had clinical signs, including weight loss, diarrhea, and depression. Abdominal palpation was adequate for diagnosis in all six antemortem cases, and radiographs confirmed a soft tissue density mass effect produced by the concretion. These gastroliths were all successfully surgically removed. Recurrence of gastrolith formation was common and occurred in four of the cases. Three cases were diagnosed postmortem, with the gastrolith causing gastric perforation in one case. Gastroliths from four cases were identified by mass spectrometry as bile acid precipitates consisting of the insoluble acid form of endogenous glycine-conjugated bile acids.

Point-of-care viscoelastic coagulation monitor parameters in Amazon parrots (Amazona spp.).

OBJECTIVE: To evaluate the feasibility of the point-of-care viscoelastic coagulation monitor (VCM) in Amazon parrots (Amazona spp.) and describe the parameters with fresh whole-blood samples in healthy Amazon parrots. DESIGN: A total of 18 Amazon parrots were enrolled. Physical examinations, a CBC, and a biochemistry profile, including bile acids, were performed on all parrots. VCM tracings were obtained at the time of venipuncture for baseline laboratory work. MEASUREMENTS AND MAIN RESULTS: The median clot time was 2102 seconds (range: 38.6-3599 s), median clot formation time was 929 seconds (range: 21.4-1711 s), median alpha angle was 20 (range: 6-67), and the median maximum clot formation was 8.5 (range: 0-36). The median lysis index at 30 minutes (LI30) was 100 (range: 98-100), and the median lysis index at 45 minutes (LI45) was 100 (range: 90-100). Of 18 samples, alpha angles were not reported in 7 samples, LI30 was not reported in 10 samples, and LI45 was not reported in 12 samples. Of the qualitative curves, 6 reflected normal mammalian curves, and the remainder were consistent with a hypocoaguable state. CONCLUSIONS: The results were markedly variable, with the majority of VCM tracings being hypocoagulable in comparison with reference intervals established for dogs and cats. Using these protocols, the VCM is not reliable in Amazon parrots. Future areas of investigation include altering the temperature during sample analysis, the use of activators, or an exchange of clotting reagents for an extrinsic pathway activator, which may contribute to the success of this device in avian species.

Isolation of Toxoplasma gondii in cell culture: an alternative to bioassay.

Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii.

Pan-primate studies of age and sex.

Age and sex have a profound effect on cytosine methylation levels in humans and many other species. Here we analyzed DNA methylation profiles of 2400 tissues derived from 37 primate species including 11 haplorhine species (baboons, marmosets, vervets, rhesus macaque, chimpanzees, gorillas, orangutan, humans) and 26 strepsirrhine species (suborders Lemuriformes and Lorisiformes). From these we present here, pan-primate epigenetic clocks which are highly accurate for all primates including humans (age correlation R = 0.98). We also carried out in-depth analysis of baboon DNA methylation profiles and generated five epigenetic clocks for baboons (Olive-yellow baboon hybrid), one of which, the pan-tissue epigenetic clock, was trained on seven tissue types (fetal cerebral cortex, adult cerebral cortex, cerebellum, adipose, heart, liver, and skeletal muscle) with ages ranging from late fetal life to 22.8 years of age. Using the primate data, we characterize the effect of age and sex on individual cytosines in highly conserved regions. We identify 11 sex-related CpGs on autosomes near genes (POU3F2, CDYL, MYCL, FBXL4, ZC3H10, ZXDC, RRAS, FAM217A, RBM39, GRIA2, UHRF2). Low overlap can be observed between age- and sex-related CpGs. Overall, this study advances our understanding of conserved age- and sex-related epigenetic changes in primates, and provides biomarkers of aging for all primates.

Treatment of vasculitis and dermatitis in a 59-yr-old Nile hippopotamus (Hippopotamus amphibius).

A 59-yr-old female Nile hippopotamus (Hippopotamus amphibius) was diagnosed and treated for severe dermatitis. Lesions included large areas of depigmentation, erosions, and ulcerations on glabrous skin areas, limbs, and perineal region. Histopathologic lesions included a markedly edematous, focally eroded, ulcerative to necrotic epidermis; foci of keratinocyte apoptosis; and a mixed suppurative dermatitis. Most of the dermal vessels had variable hyalinized walls with plump endothelial cells and frequent intramural neutrophils, and some vessels had vascular thrombi consistent with vasculitis. Culture of the lesions yielded beta-hemolytic Streptococcus, Morganella morgannii, and Enterococcus sp. The hippopotamus was successfully treated with sulfamethoxazole and trimethoprim, amoxicillin, and pentoxifylline for more than 2 mo, and the condition did not recur over the subsequent 16 mo.

Toxoplasma gondii prevalence in carnivorous wild birds in the eastern United States.

Toxoplasma gondii is an important zoonotic protozoan parasite that can infect all warm-blooded animals including mammals and birds. Raptors can be intermediate hosts for T. gondii and the infection may be dependent on their feeding habits. In this study, we investigated the seroprevalence of T. gondii in ten raptor species from Florida, Pennsylvania, and Tennessee followed by a parasite bioassay on select seropositive samples. From a total of 155 raptors, we detected T. gondii antibodies using a modified agglutination test (cutoff 1:25) in 32 (20.6%) birds. The T. gondii seroprevalence was 44.8% in Falconiformes (13/29), 75% in Strigiformes (15/20), and 3.8% in Ciconiiformes (4/106). All Ciconiiformes samples (hearts and sera) were collected from Pennsylvania during nuisance wildlife removal projects and all birds were apparently healthy. Falconiform and Strigiform samples were collected from an exotics clinic in Tennessee and a rehabilitation center in Florida. All sampled birds were dead or euthanatized due to failure of rehabilitation or treatment. There was no statistically significant difference in T. gondii seroprevalence between Tennessee and Florida in the tested raptors. There was also no statistically significant difference in T. gondii exposure between males and females or adults and subadults. Mice bioassay attempts using fresh brain and/or heart tissue were performed on four seropositive birds. We isolated viable T. gondii tachyzoites from one red-shouldered hawk (Buteo lineatus) and genotyped the isolate using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ten genetic markers. The isolated strain was designated as TgHawkFL1, which is ToxoDB PCR-RFLP genotype #28. Further research is needed to investigate the prevalence of T. gondii in raptors in the United States to obtain a better understanding of the life cycle, wildlife population impacts, and transmission dynamics of the parasite.

Detection of Trichomonas gallinae in Wild Birds Admitted to a Rehabilitation Center, Florida, USA.

We screened raptors (n=188) and columbids (n=2) presenting to a Tampa, Florida, US rehabilitation center from June 2016 to April 2017 for Trichomonas gallinae. One juvenile Cooper's Hawk (Accipiter cooperii) was culture- and PCR-positive and sequencing identified as genotype E1. A subset of culture-negative (10%, n=19) samples were negative via PCR.

BIOACCUMULATION OF MERCURY IN A TERRESTRIAL CARNIVORE, AMERICAN MARTEN (MARTES AMERICANA).

Studies that assess mercury bioaccumulation in small carnivores in terrestrial habitats are limited. We quantified total mercury (THg) in American marten (Martes americana) that were harvested for fur in Michigan, US, during 2013 and 2014. We quantified THg (dry weight) in hair, kidney, and liver samples and further analyzed hair for potential demographic and ecological factors that influence THg bioaccumulation. We found THg concentrations to be the highest in hair (1.228±0.475 µg/g, n=40), followed by kidney (0.922±0.651 µg/g, n=29) and liver (0.344±0.219 µg/g, n=26). Total mercury distributed predictably and significantly between tissue types, and hair was moderately predictive at modeling THg in kidney (R2=0.50, P<0.001, n=29) and weakly predictive in liver (R2=0.35, P<0.001, n=26), suggesting that hair, which is easily obtained, could be a useful sample type for future biomonitoring programs. The concentrations of THg in hair were higher in adults relative to juveniles, and adult female martens had the highest levels of THg (1.980±0.188 µg/g), as compared to juveniles and adult males. Results of generalized linear modeling suggested that THg hair concentrations were positively associated with marten age and trophic position (stable isotope ratio, δ15N). An interaction between δ15N and the year marten carcasses were collected showed that δ15N alone could be highly predictive of THg in some years but not in others. Annual changes in diet could lead to differing rates of mercury bioaccumulation and alter the usefulness of δ15N to predict THg in marten tissues. Further research should explore the connections between changes in prey availability, types of prey consumed, and the influence on bioaccumulation rates of mercury in terrestrial system mesocarnivores.

Intervertebral disc extrusion and spinal decompression in a binturong (Arctictis binturong).

A 10-yr-old binturong (Arctictis binturong) developed an acute onset of hind limb paralysis. Neurological examination revealed sensorimotor paraplegia. Myelography and computed tomography demonstrated a ventrolateral extradural compression of the spinal cord centered over the L3-L4 intervertebral disc space. Spinal decompression was performed via hemilaminectomy and excision of degenerate nucleus pulposus, confirmed by histopathologic examination. The binturong regained slight motor function by day 8 postoperatively but succumbed to pancreatitis 19 days postoperatively.

EFFECTS OF LIVE-TRAPPING AND ISOFLURANE ANESTHESIA ON FREE-RANGING AMERICAN MARTENS (MARTES AMERICANA).

Seventy-two free-ranging American martens ( Martes americana ) in Michigan, US were immobilized using isoflurane from 2011 to 2015. In total, 129 anesthetic procedures were performed with no mortalities. Hypothermia and hyperthermia were the most common anesthetic complications, and the mean rectal temperatures were significantly higher during summer than in winter. Dental abnormalities were common; the majority of abnormal findings were broken or discolored teeth attributed to previous dental trauma and were not trap-induced. Blood (n=72) was analyzed from 53 martens for venous blood gas, lactate, hematocrit, and/or selected serum biochemistry analytes. Lactate concentration was measured by two different devices (VetScan i-STAT 1 and Lactate Plus) and compared for clinical agreement for 26 samples. Both methods for lactate measurement provided statistically similar results. Using domestic feline reference ranges, the acid-base status and relative arterial oxygen saturation of anesthetized martens in this study were normal as determined by blood pH and pulse oximetry, respectively. Serum biochemistry parameters, multiple environmental parameters, and marten-specific attributes were evaluated for their influence on lactate in American martens using linear regression and an information-theoretic approach with model averaging. Blood urea nitrogen was in all of the top models and was positively related to lactate (ß=0.02, 95% confidence interval: 0.00-0.04). Initial body temperature, ambient temperature, and time from trap discovery until immobilization of martens were informative predictors for lactate level. Recommendations for the live-trapping and isoflurane anesthesia of free-ranging martens include using caution during warmer summer months, minimizing disturbance prior to induction, monitoring lactate in addition to vital rates, and being prepared to prevent or treat both hypothermia and hyperthermia during any time of year.

Endoparasites of American marten (Martes americana): Review of the literature and parasite survey of reintroduced American marten in Michigan.

The American marten (Martes americana) was reintroduced to both the Upper (UP) and northern Lower Peninsula (NLP) of Michigan during the 20th century. This is the first report of endoparasites of American marten from the NLP. Faeces from live-trapped American marten were examined for the presence of parasitic ova, and blood samples were obtained for haematocrit evaluation. The most prevalent parasites were Capillaria and Alaria species. Helminth parasites reported in American marten for the first time include Eucoleus boehmi, hookworm, and Hymenolepis and Strongyloides species. This is the first report of shedding of Sarcocystis species sporocysts in an American marten and identification of 2 coccidian parasites, Cystoisospora and Eimeria species. The pathologic and zoonotic potential of each parasite species is discussed, and previous reports of endoparasites of the American marten in North America are reviewed.