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.

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.

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.

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.

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.

Blood collection from the facial (maxillary)/musculo-cutaneous vein in true frogs (family Ranidae).

Collection of blood from amphibians, as in other classes of vertebrate animals, is essential to evaluate parameters of health, diagnose hemoparasitism, identify viral and bacterial pathogens, and measure antibodies. Various methods of blood collection have been described for amphibians. Most can be cumbersome (venipucture of femoral vein, ventral abdominal vein or lingual venus plexus) or result in pain or deleterious health consequences (cardiac puncture and toe-clipping). We describe an easy and practical technique to collect blood from frogs and toads that can be used in multiple species and is minimally invasive. The technique consists of puncturing either the facial or, less commonly, the musculo-cutaneous vein and collecting the blood with a capillary tube. These veins run dorsal and parallel to the maxillary bone and can be accessed by quick insertion and withdrawal of a needle through the skin between the upper jawline and the rostral or caudal side of the tympanum. The needle should be of 27 or 30 gauge for anurans weighing more or less than 25 g, respectively. Although the technique has been used by some amphibian researchers for years, it is little known by others and has never been fully described in a peer-reviewed publication.