PAIRED BIOCHEMICAL ANALYSIS OF PIGMENTED PLASMA SAMPLES FROM ZOO-KEPT AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER) USING A POINT-OF-CARE AND A STANDARD WET CHEMISTRY ANALYZER.

American flamingos (Phoenicopterus ruber) are commonly kept in zoological collections, making health monitoring essential. Use of point-of-care (POC) blood analyzers that require small volumes of whole blood samples produces prompt results allowing for rapid clinical decision-making. To evaluate and compare blood biochemistry analysis results analyzed by a POC biochemistry analyzer and a laboratory wet biochemistry analyzer, blood was collected from 17 apparently healthy zoo-kept American flamingos. Analyzer agreement was investigated using the Passing-Bablock regression analysis and Spearman correlation coefficients. Plasma samples from all birds were bright yellow in color. The results from the POC analyzer used in this study were found to be outside acceptance and clinical allowable error limits when compared with the laboratory analyzer for phosphorus (Phos), total protein (TP), albumin (Alb), glucose (Glu), creatine kinase (CK), and potassium (K). For aspartate aminotransferase (AST), results were within clinical allowable error but outside the acceptance limits, and for calcium (Ca) and sodium (Na), results were within both limits. The POC analyzer failed to measure the uric acid (UA) concentrations of all the samples, and reported all bile acids (BA) concentrations as below its minimal measurable limit. The use of analyzer-specific reference intervals is recommended for most analytes tested. The POC analyzer used in this study cannot be recommended for measuring UA concentrations in brightly colored samples from American flamingos.

SELECTED HEMATOLOGIC AND PLASMA BIOCHEMISTRY ANALYSIS OF FERRUGINOUS DUCKS (AYTHYA NYROCA) IN ISRAEL.

The ferruginous duck (Aythya nyroca) is a medium-sized chestnut-colored diving duck that inhabits wetlands of Europe and Asia. In recent years, this species has been declining throughout much of Europe--a decline that is attributed mainly to destruction of natural habitats, and to hunting and pollution. The ferruginous duck is listed as "near threatened" by the International Union for Conservation of Nature, and as a critically endangered nesting species in Israel. In 2009, a captive-breeding/reintroduction program was established in Israel, aiming to increase the species' population. The objective of this study was to collect data on normal hematology and plasma biochemistry analytes of ferruginous ducks in order to promote the species' conservation. Blood was collected from 49 birds, and 27 analytes were quantified. Compared to most other anseriformes studied, the ferruginous ducks in this study had lower white blood cell counts, which were dominated by heterophils rather than by lymphocytes.

SELECTED PLASMA BIOCHEMISTRY ANALYTES OF HEALTHY CAPTIVE SULCATA (AFRICAN SPURRED) TORTOISES (CENTROCHELYS SULCATA).

The sulcata or African spurred tortoise (Centrochelys sulcata) is a large tortoise species that is commonly kept in zoologic collections and as a pet. The objectives of this study were to establish reference intervals for selected biochemical analytes in clinically healthy captive sulcata tortoises and to evaluate the impact of blood sampling site and sex. Blood samples were collected from 60 tortoises from either the dorsal coccygeal (tail) vein or the subcarapacial venous plexus based on their body size. The packed cell volume and refractometric total solids (TS) were determined. The concentrations of selected plasma biochemical analytes were determined using the VetScan VS2 analyzer and included albumin, aspartate aminotransferase, bile acids, calcium, creatine kinase, globulins, glucose, potassium, sodium, phosphorous, total proteins (TP), and uric acid. The calcium-to-phosphorous ratio was calculated. Reference intervals were determined and evaluated for the potential effects of blood sampling site and sex. There were significant differences (P < 0.05) associated with the blood sampling site in TS, TP, phosphorus, and globulins, with higher values in samples from the tail versus the subcarapacial sampling site. No significant statistical differences were noted in the plasma biochemistry analytes between the sexes. Reading of the globulins by the analyzer failed in 36 of 60 of the samples and was largely associated with the subcarapacial plexus sampling site. The reference intervals defined by the VetScan analyzer in this study can be used for clinical medicine and conservation of this tortoise species. Sampling site was identified as a factor significantly affecting some blood analytes in this study; this factor should thus be taken into consideration when assessing sulcata tortoise health status and using this testing methodology.

Hematologic, plasma biochemistry, and acid-base analysis of adult Negev Desert tortoises (Testudo werneri) in Israel.

The Negev Desert tortoise (Testudo werneri) is one of the smallest tortoise species in the Mediterranean region. This is a critically endangered species (CITES I) in its native habitat, which includes the Saharo-Arabian sands of northern Egypt, Sinai, and the Negev Desert in Israel. Great efforts have been invested in captive breeding and reintroduction of this tortoise to the wild. The purpose of this study was to collect blood samples from healthy Negev Desert tortoises kept in well-managed zoologic collections in order to describe hematologic, plasma biochemistry, and acid-base analytes for this species. Data of 36 different blood analytes were collected using the Abaxis Vetscan bench-top analyzer and i-STAT handheld analyzer, and a significant difference was observed between males and females in 13 of the measured analytes. To the authors' knowledge, this is the first report of blood analytes for the Negev Desert tortoise; however, the data do not fully meet the strict ASVCP guidelines required for reference range determination and thus can only provide a rough estimate for evaluating the health status of Negev Desert tortoises using similar testing methodology.

Advanced diagnostic approaches and current management of proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a fatal inflammatory disease that affects mainly, but not exclusively, psittacine birds (Order: Psittaciformes). PDD has long been suspected to be a viral disease, but its causative agent, a novel Bornavirus, was only identified in 2008.

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (Nymphicus hollandicus). RESULTS: Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73-100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40-89% amino acid sequence identity with viruses in the Retroviridae and Astroviridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds. CONCLUSION: In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels.

Diabetes mellitus concurrent with hepatic haemosiderosis in two macaws (Ara severa, Ara militaris).

Diabetes mellitus was diagnosed in a 5-year-old male chestnut-fronted macaw (Ara severa) and an 8-year-old female Military macaw (Ara militaris) based on persistent hyperglycaemia and glucosuria. Hepatic biopsies showed marked hepatic haemosiderosis, while pancreatic biopsies showed no inflammatory lesions. Repeatable and titratable responses to bovine or porcine protamine zinc insulin were recorded in both patients, who were followed up for more than 2 years. In addition, iron-elimination therapy was initiated by chelation or phlebotomy, and the birds' diet was changed to low-iron content pellets. Both birds responded favourably to this therapy, showing a decreased demand for extrinsic insulin. Follow-up biopsies demonstrated marked reduction in hepatic haemosiderin. Plasma fructosamine and beta-hydroxybutyric acid levels were measured periodically in both birds and compared with euglycaemic psittacines. Both tests appeared useful for monitoring treatment success. The potential association between diabetes mellitus and excessive iron storage in birds should be further investigated.

Pathology and tissue distribution of West Nile virus in North American owls (family: Strigidae).

This study describes the macroscopic and microscopic lesions and the viral antigen distribution in 82 owls (Family: Strigidae) of 11 North American and one Eurasian species that died following natural West Nile virus infection. The range of lesions seen was greater than that previously reported for owls, and involved more organs. Two patterns of antigen distribution were identified: one that involved the blood and all major organs; and a second where antigen was sparse, localized, and absent from the blood. The first pattern was associated with species of northern natural breeding range, while the second was seen in owls of a more southern distribution and appeared to be associated with a more prolonged course of illness. Further differences in lesion and antigen distribution appeared to be either species related or individual. The findings underline the complexity and variability of West Nile virus pathology within birds of a relatively narrow taxonomic group.

Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.

West Nile virus outbreak in North American owls, Ontario, 2002.

From July to September 2002, an outbreak of West Nile virus (WNV) caused a high number of deaths in captive owls at the Owl Foundation, Vineland, Ontario, Canada. Peak death rates occurred in mid-August, and the epidemiologic curve resembled that of corvids in the surrounding Niagara region. The outbreak occurred in the midst of a louse fly (Icosta americana, family Hippoboscidae) infestation. Of the flies tested, 16 (88.9 %) of 18 contained WNV RNA. Species with northern native breeding range and birds >1 year of age were at significantly higher risk for WNV-related deaths. Species with northern native breeding range and of medium-to-large body size were at significantly higher risk for exposure to WNV. Taxonomic relations (at the subfamily level) did not significantly affect exposure to WNV or WNV-related deaths. Northern native breeding range and medium-to-large body size were associated with earlier death within the outbreak period. Of the survivors, 69 (75.8 %) of 91 were seropositive for WNV.

Detecting West Nile virus in owls and raptors by an antigen-capture assay.

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%-95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors.