Pathogenesis of Bohle iridovirus infection in Krefft's freshwater turtle hatchlings (Emydura macquarii krefftii).

Ranaviruses have been detected in over 12 families of reptiles including many genera of turtles, tortoises, and terrapins, but the pathogenesis of these infections is still poorly understood. Krefft's river turtle hatchlings (N = 36; Emydura macquarii krefftii) were inoculated intramuscularly with Bohle iridovirus (BIV, Ranavirus, isolate) or saline, and euthanized at 9 timepoints (3 infected and 1 control per timepoint) over a 24-day period. Samples of lung, liver, kidney, and spleen were collected for quantitative polymerase chain reaction (PCR); internal organs, skin, and oral cavity samples were fixed for histopathological examination. The earliest lesions, at 8 days postinoculation (dpi), were lymphocytic inflammation of the skin and fibrinoid necrosis of regional vessels at the site of inoculation, and mild ulcerative necrosis with lymphocytic and heterophilic inflammation in the oral, nasal, and tongue mucosae. Fibrinonecrotic foci with heterophilic inflammation were detected in spleen and gonads at 16 dpi. Multifocal hepatic necrosis, heterophilic inflammation, and occasional basophilic intracytoplasmic inclusion bodies were observed at 20 dpi, along with ulcerative lymphocytic and heterophilic tracheitis and bronchitis. Tracheitis, bronchitis, and rare bone marrow necrosis were present at 24 dpi. Of the viscera tested for ranaviral DNA by PCR, the liver and spleen had the highest viral loads throughout infection, and thus appeared to be major targets of viral replication. Testing of whole blood by qPCR was the most-effective ante-mortem method for detecting ranaviral infection compared with oral swabs. This study represents the first time-dependent pathogenesis study of a ranaviral infection in turtles.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

Pathogenesis of Bohle Iridovirus (Genus Ranavirus) in Experimentally Infected Juvenile Eastern Water Dragons ( Intellagama lesueurii lesueurii).

Juvenile eastern water dragons ( Intellagama lesueurii lesueurii) are highly susceptible to infection with Bohle iridovirus (BIV), a species of ranavirus first isolated from ornate burrowing frogs in Townsville, Australia. To investigate the progression of BIV infection in eastern water dragons, 11 captive-bred juveniles were orally inoculated with a dose of 104.33 TCID50 and euthanized at 3, 6, 8, 10, 12, and 14 days postinfection (dpi). Viral DNA was detected via polymerase chain reaction (PCR) in the liver, kidney, and cloacal swabs at 3 dpi. Mild lymphocytic infiltration was observed in the submucosa and mucosa of the tongue and liver at 3 dpi. Immunohistochemistry (IHC) first identified viral antigen in foci of splenic necrosis and in hepatocytes with intracytoplasmic inclusion or rare single-cell necrosis at 6 dpi. By 14 dpi, positive IHC labeling was found in association with lesions in multiple tissues. Selected tissues from an individual euthanized at 14 dpi were probed using in situ hybridization (ISH). The ISH labeling matched the location and pattern detected by IHC. The progression of BIV infection in eastern water dragons, based on lesion severity and virus detection, appears to start in the spleen, followed by the liver, then other organs such as the kidney, pancreas, oral mucosa, and skin. The early detection of ranaviral DNA in cloacal swabs and liver and kidney tissue samples suggests these to be a reliable source of diagnostic samples in the early stage of disease before the appearance of clinical signs, as well as throughout the infection.

Causes of mortality of harbor porpoises Phocoena phocoena along the Atlantic and Pacific coasts of Canada.

There is increasing public interest in the overall health of the marine environment. Harbor porpoises Phocoena phocoena have a coastal distribution, and stranded animals function as sentinels for population and ecosystem health. The goal of this retrospective study was to join datasets from the western Atlantic and eastern Pacific coasts of Canada to investigate causes of morbidity and mortality in this species. A total of 241 necropsy records were reviewed including 147 (61%) from the Pacific region and 94 (39%) from the Atlantic region from 1988 to 2011. A cause of death could be determined with confidence in 118 (49%) of these cases. Of these 118 cases, the leading cause of mortality for both regions, together and separately, was infectious disease. In the Pacific region, this was followed by traumatic and anthropogenic causes, whereas in the Atlantic region, it was followed by emaciation/starvation, mortality of dependent calves, and anthropogenic causes. Pathogens of potential zoonotic significance or indicative of environmental contamination, e.g. Salmonella sp. and Cryptococcus gattii, were identified. Numerous parasitic species were observed within the lungs, liver, stomach, middle ear, and subcutaneous tissues, although they were usually interpreted as incidental findings. Anthropogenic causes may be underrepresented as they are notoriously difficult to diagnose with certainty, thereby making up a proportion of the 'unknown causes of death' (51%) category. Improved standardization of data collection and documentation is required to better understand harbor porpoise and ecosystem health.

Poorly differentiated cutaneous carcinoma of non-sebaceous origin in a 3-year-old Mongolian gerbil (Meriones unguiculatus).

A 3-year-old female gerbil developed a non-healing skin wound due to a malignant neoplasm. Histology, immunohistochemistry (cytokeratin 19 positive; vimentin, estrogen, and progesterone receptor negative), and electron microscopy (no desmosomes or melanosomes) revealed an undifferentiated carcinoma with pulmonary metastasis. Unlike in previous reports, it did not arise from the abdominal pad's sebaceous gland.

Carcinome cutané d'origine non sébacée peu différencié chez une gerbille de Mongolie âgée de 3 ans(Meriones unguiculatus). Une gerbille femelle âgée de 3 ans a développé une plaie cutanée qui ne guérissait pas en raison d'un néoplasme malin. Des examens histologiques, par immunohistochimie (positif pour la cytokératine 19; négatif pour les récepteurs de vimentine, d'œstrogène et de progestérone) et par microscopie électronique (pas de desmosomes ni de mélanosomes) ont révélé un carcinome indifférencié avec métastase pulmonaire. Contrairement aux rapports antérieurs, il n'était pas causé par la glande sébacée du coussinet abdominal.(Traduit par Isabelle Vallières).

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae.

Amphibian populations suffer massive mortalities from infection with frog virus 3 FV3, genus Ranavirus, family Iridoviridae, a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica Lithobates sylvaticus, was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host response. Oral dosing was successful LD50 was 10(2.93 2.423.44) p.f.u. for frogs averaging 35mm in length. Onset of clinical signs occurred 614days post-infection p.i. median 11 days p.i. and time to death was 1014 days p.i. median 12 days p.i.. Each tenfold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in faeces 710 days p.i. 34.5days before death and skin sheds 10 days p.i. 01.5days before death of some frogs dead from infection. Most common lesions were dermal erosion and haemorrhages haematopoietic necrosis in bone marrow, kidney, spleen and liver and necrosis in renal glomeruli, tongue, gastrointestinal tract and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies probably viral were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a ranaviruswood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.

Molecular characterization of Trichomonas gallinae isolates recovered from the Canadian Maritime provinces' wild avifauna reveals the presence of the genotype responsible for the European finch trichomonosis epidemic and additional strains.

Finch trichomonosis, caused by Trichomonas gallinae, emerged in the Canadian Maritime provinces in 2007 and has since caused ongoing mortality in regional purple finch (Carpodacus purpureus) and American goldfinch (Carduelis tristis) populations. Trichomonas gallinae was isolated from (1) finches and rock pigeons (Columbia livia) submitted for post-mortem or live-captured at bird feeding sites experiencing trichomonosis mortality; (2) bird seed at these same sites; and (3) rock pigeons live-captured at known roosts or humanely killed. Isolates were characterized using internal transcribed spacer (ITS) region and iron hydrogenase (Fe-hyd) gene sequences. Two distinct ITS types were found. Type A was identical to the UK finch epidemic strain and was isolated from finches and a rock pigeon with trichomonosis; apparently healthy rock pigeons and finches; and bird seed at an outbreak site. Type B was obtained from apparently healthy rock pigeons. Fe-hyd sequencing revealed six distinct subtypes. The predominant subtype in both finches and the rock pigeon with trichomonosis was identical to the UK finch epidemic strain A1. Single nucleotide polymorphisms in Fe-hyd sequences suggest there is fine-scale variation amongst isolates and that finch trichomonosis emergence in this region may not have been caused by a single spill-over event.

Detection and characterization of a Trichomonas isolate from a rehabilitated bald eagle (Haliaeetus leucocephalus).

A hatching-year bald eagle (Haliaeetus leucocephalus) was presented for clinical examination after being found unable to fly. Upon admission, routine wet-mount microscopy detected no trichomonads. Five months later, oral cavity inspection found no abnormalities, but the eagle was swabbed for research on trichomonosis in maritime birds. The swab was used to inoculate an InPouch TF culture and trichomonads were visible within 24 hr. Genotyping (ITS) revealed a Trichomonas isolate that was 100% identical to an isolate from a bearded vulture (Gypaetus barbatus) from the Czech Republic. The eagle was treated with metronidazole (50 mg/kg q 12h PO for 5 consecutive days). Following treatment, the eagle was swabbed and the inoculated InPouch TF culture was monitored daily for 1 wk. No trichomonads were observed. Rehabilitation centers interested in surveillance should consider combining the InPouch TF technique with clinical inspection of live birds to confirm trichomonosis and for future research.

Taenia crassiceps Cysticercosis in a Wild Muskrat and a Domestic Dog in the Northeastern United States.

Taenia crassiceps is a parasite of wild canids and dogs that serve as definite hosts, harboring the adult cestode, whereas rodents are the intermediate hosts in which the metacestode/cysticercus/larval stage occurs. Fecal-oral transmission ensures the parasite's lifecycle. At times, dogs and humans act as accidental intermediate hosts. Despite the public health concern this parasite warrants, its epidemiology remains unclear. In this report, we document the occurrence of metacestodes of T. crassiceps in a muskrat (Ondatra zibethicus) and a domestic dog from the northeastern United States, a development that necessitates increased awareness and surveillance to tackle this disease of "one health" significance. Taenia crassiceps cysticercosis was confirmed in an adult male muskrat in February 2018 and in a 4-year-old female spayed Staffordshire Bull Terrier in December 2020. Parasitological and histopathologic examination of both cases revealed cysticerci with the characteristic rostellar hook morphology that aided in Taenia species identification. In the muskrat case specifically, partial sequencing of the mitochondrial cytochrome oxidase gene confirmed the species identity as T. crassiceps. We report T. crassiceps occurrence in a muskrat in New York State for the first time and document a case presentation in a domestic dog from New Jersey that was infected with metacestode stages of this parasite. Given the detection of this parasite in the northeastern United States, T. crassiceps infection, which otherwise is considered a rare disease, should be on the radar of veterinary, medical and wildlife biologists for timely diagnosis and interventions.

Valvular endocarditis and septic thrombosis associated with a radial fracture in a red-tailed hawk (Buteo jamaicensis).

A free-ranging adult female red-tailed hawk died suddenly after 3 weeks in rehabilitation for a radial fracture. Cause of death was septic thrombosis from a chronic bacterial valvular endocarditis, probably associated with injury at the fracture site. The challenge of clinical diagnosis of sepsis in wild birds is emphasized.

THE EFFECT OF DEXAMETHASONE ON HEMATOLOGIC PROFILES, HEMOSPORIDIAN INFECTION, AND SPLENIC HISTOLOGY IN HOUSE FINCHES (HAEMORHOUS MEXICANUS).

Research on host response to infectious disease often involves pharmacological induction of immunosuppression, frequently through administration of dexamethasone. Reports on the effect of dexamethasone in birds are largely restricted to poultry and pigeons. This study describes changes in white blood cell (WBC) differentials, hemoparasite counts, splenic histology, and splenic CD3 immunoreactivity in House Finches (Haemorhous mexicanus). Experimental group birds (n=9) were treated with a daily intramuscular injection of 25 µg of dexamethasone for 8 d; a control group (n=9) received daily saline solution. Smears were made with blood collected immediately before the first dose (day 0) and on d 4, 8, and 9, and stained with modified Wright. The WBC differential counts were performed by three blinded observers, parasite counts by two blinded observers, and histology by one blinded observer. Dexamethasone-treated birds experienced relative heterophilia and lymphopenia on d 4 (P=0.008); heterophilia was also present at d 8 (P=0.018). Hemosporidian counts were significantly increased in dexamethasone-treated birds on d 4 and 8 (P=0.048 and P=0.031, respectively). In contrast with control birds, all dexamethasone-treated birds lacked histologically apparent splenic lymphoid follicles (P<0.001). No significant difference was observed in splenic CD3 immunoreactivity between groups. Our results indicate that dexamethasone has an effect on the hematologic profile of House Finches and suggest that it may be a useful method to induce immunosuppression in this species.

Trichomoniasis in finches from the Canadian Maritime provinces--An emerging disease.

Trichomoniasis was diagnosed in multiple incidents of mortality in wild purple finch (Carpodacus purpureus) and American goldfinch (Carduelis tristis) in the Canadian Maritimes. Birds exhibited regurgitation, emaciation, and hyperplastic oropharyngitis, ingluvitis, and esophagitis. Trichomonas gallinae was identified by histopathology and polymerase chain reaction (PCR). Trichomoniasis (trichomonosis) is an emerging disease in wild finches of eastern Canada.

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.

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.

Common midwife toad ranaviruses replicate first in the oral cavity of smooth newts (Lissotriton vulgaris) and show distinct strain-associated pathogenicity.

Ranavirus is the second most common infectious cause of amphibian mortality. These viruses affect caudates, an order in which information regarding Ranavirus pathogenesis is scarce. In the Netherlands, two strains (CMTV-NL I and III) were suspected to possess distinct pathogenicity based on field data. To investigate susceptibility and disease progression in urodeles and determine differences in pathogenicity between strains, 45 adult smooth newts (Lissotriton vulgaris) were challenged via bath exposure with these ranaviruses and their detection in organs and feces followed over time by PCR, immunohistochemistry and in situ hybridization. Ranavirus was first detected at 3 days post infection (p.i.) in the oral cavity and upper respiratory mucosa. At 6 days p.i, virus was found in connective tissues and vasculature of the gastrointestinal tract. Finally, from 9 days p.i onwards there was widespread Ranavirus disease in various organs including skin, kidneys and gonads. Higher pathogenicity of the CMTV-NL I strain was confirmed by higher correlation coefficient of experimental group and mortality of challenged animals. Ranavirus-exposed smooth newts shed virus in feces intermittently and infection was seen in the absence of lesions or clinical signs, indicating that this species can harbor subclinical infections and potentially serve as disease reservoirs.

Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control.

The introduction of a new group of dendrobatid frogs to an established captive amphibian collection was followed by several acute mortalities in both resident and introduced frog populations. Chytridiomycosis, caused by Batrachochytrium dendrobatidis, was diagnosed by histology in two of the dead frogs. Following the diagnosis, all amphibians were moved to a specially made quarantine room with strict handling protocols and treated with itraconazole. Frogs, being terrestrial amphibians, were treated with itraconazole (Sporanox, 10 mg/ml) at 0.01% in 0.6% saline in a 5-min bath for 11 consecutive days. Axolotls (Ambystoma mexicanum) and Kaup's caecilians (Potymotyphlus kaupii), being aquatic amphibians, were treated with itraconazole administered directly in their primary tank water to achieve a concentration of 0.01% for 30 min every 5 days for four treatments. Itraconazole was removed from the tank water after 30 min by high-rate-of-flow activated charcoal filters. The treatment and quarantine procedures were successful in eradicating the disease. The few amphibian mortalities that occurred in the 18 mo after the start of the treatment have been histologically negative for the presence of chytrid fungi. The collection is now considered free of chytridiomycosis.

Clinical pathology of amphibians: a review.

Amphibian declines and extinctions have worsened in the last 2 decades. Partly because one of the main causes of the declines is infectious disease, veterinary professionals have increasingly become involved in amphibian research, captive husbandry, and management. Health evaluation of amphibians, free-living or captive, can benefit from employing the tools of clinical pathology, something that is commonly used in veterinary medicine of other vertebrates. The present review compiles what is known of amphibian clinical pathology emphasizing knowledge that may assist with the interpretation of laboratory results, provides diagnostic recommendations for common amphibian diseases, and includes RIs for a few amphibian species estimated based on peer-reviewed studies. We hope to encourage the incorporation of clinical pathology in amphibian practice and research, and to highlight the importance of applying veterinary medicine principles in furthering our knowledge of amphibian pathophysiology.

Persistence of Trichomonas gallinae in Birdseed.

Trichomonas gallinae has emerged worldwide as a cause of mortality in songbirds (passerines). The congregation of numerous birds, including the reservoir hosts, pigeons and doves (columbids), at backyard feeding and watering sources has been suggested as a potential driver for the outbreaks. Evidence supporting a role for water in transmission has been established, but the role of birdseed in the transmission of trichomoniasis remained to be investigated. We assessed the survival of T. gallinae in three commercial birdseeds (mixed seed, black-oil sunflower seed, and niger seed) routinely used to attract passerine birds to local properties. Trichomonad suspensions were inoculated (low dose: 1 × 103; high dose: 1 × 105) into each of the three seed types in petri dishes, using both dry and moist (water-soaked) conditions, in triplicate. Petri dishes were incubated at 37 C and monitored for T. gallinae survival for 48 hr by wet-mount microscopy and by InPouch™ TF medium culture for 10 days. Surviving trichomonads were not detected in any of the dry birdseed treatments. In moist conditions, however, trichomonads were found to survive ≤24 hr in all three seed types and ≤48 hr in the mixed seed that contained organic debris. We demonstrate that T. gallinae has the ability to survive in moist birdseed, which suggests that public bird-feeding sites may play a significant role in the transmission of trichomoniasis.

Hematologic reference intervals for Xenopus tropicalis with partial use of automatic counting methods and reliability of long-term stored samples.

BACKGROUND: The African frog, Xenopus tropicalis, is widely used in biomedical and toxicologic research. Reference intervals (RI) for hematologic variables, valuable to research and health status assessment, have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RI of X tropicalis, and establish whether automated cell counting can facilitate routine hematologic assessment in frogs. METHODS: Blood from 41 adult healthy X tropicalis was collected via cardiac puncture, and diluted in Natt-Herrick solution. Complete WBC, RBC, and thrombocyte counts (hemocytometry), differential WBC counts (Wright-Giemsa-stained smears), PCV (centrifugation), total protein (refractometry), and automated total cell counts (WBC + RBC + thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients calculated the agreement between total cell counts obtained by hemocytometry and automated particle counting, and between total cell counts at collection and after 2 years of storage. RESULTS: Leukocyte morphology was similar to other amphibians and mammals. PCV was similar to other frogs; RBC counts were higher, and MCV was lower than in other frog species. Neutrophils were the most numerous WBC. Agreement was good between hemocytometry and automated cell counts. Subtracting the hemocytometer WBC and thrombocyte counts from the automated total cell count reliably yielded the RBC count. Cellular integrity evaluated 2 years post collection was good, and automated counts were not clinically different from counts at collection. CONCLUSION: We provide hematologic RI for X tropicalis, suggest how automated cell counts may facilitate hematologic assessments of frogs, and establish that blood in Natt-Herrick solution is stable 2 years post collection.

Hematologic reference intervals for Rana sylvatica (Lithobates sylvaticus) and effect of infection with Frog Virus 3 (Ranavirus sp., Iridoviridae).

BACKGROUND: Although the Wood Frog, Rana sylvatica, is used in research on infectious diseases of amphibians, hematologic RIs or response to infection have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RIs for adult Wood Frogs and alterations associated with infection with Frog Virus 3 (FV3, Ranavirus sp.). METHODS: Blood was collected from 40 wild-caught adult Wood Frogs that had been in captivity for 6 months. Complete (Natt-Herrick solution hemocytometry) and differential (Wright-Giemsa-stained smears) WBC, RBC, and thrombocyte cell counts, PCV, and automated total cell counts (WBC+RBC+thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients determined agreement between hemocytometric and automated total cell counts. Thirteen frogs were orally infected with a lethal dose of 10(4.43) plaque-forming units of FV3 and terminally sampled 4, 9, or 14 days postinfection (dpi). Pre- and postinfection variables for each frog were compared. RESULTS: Leukocyte morphology was similar to that of other amphibians and mammals. Lymphocytes were the most numerous WBC. PCV and RBC counts were similar to other frogs in the same family. Agreement was good between hemocytometry and automated total cell counts. Infection with FV3 caused neutrophilia, increase in undifferentiated blast-like cells, and reduction in the percentage of basophils. Lymphocytes decreased at 4 and 9 dpi but increased at 14 dpi. From 9 dpi onwards, nuclear deterioration and mild toxic change were present in neutrophils; viral cytoplasmic inclusion bodies were observed in lymphocytes, monocytes, neutrophils, and eosinophils. CONCLUSION: We provide hematology RIs for Rana sylvatica, and report hematologic changes associated with a lethal FV3 infection.