A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

STRECK CELL PRESERVATIVE STABILIZES KOALA ( PHASCOLARCTOS CINEREUS) WHOLE BLOOD FOR COMPLETE BLOOD COUNTS.

Wildlife health assessments at remote sites may lead to delayed testing of whole blood for complete blood counts (CBC) resulting in artifacts affecting clinical interpretation. Streck Cell Preservative (SCP) is a proprietary liquid stabilization reagent designed to preserve human leukocytes and may be applicable to wildlife health assessments when immediate processing of blood is not possible. The purpose of this study was to determine if SCP adequately preserved EDTA-anticoagulated whole blood from koalas ( Phascolarctos cinereus) for up to 14 days. Blood from 12 captive adult koalas was collected and combined with SCP in a 1 : 1 ratio and refrigerated. Paired samples of SCP treated and untreated blood had CBCs performed at five time-points over 14 days. Streck Cell Preservative extended koala EDTA-anticoagulated whole blood viability to 14 days by decreasing cellular lysis. Species- and method-specific reference intervals for SCP should be generated to avoid clinical misinterpretation, especially when evaluating anemia.

Reprint of: Assay Validation of the Cardiac Isoform of Troponin I in Double Crested Cormorant (Phalacrocorax auritus) Plasma for Diagnosis of Cardiac Damage.

Cardiac abnormalities, initially found in Deepwater Horizon weathered MC252 crude oil exposed Double Crested Cormorants (DCCOs) upon gross necropsy, were further investigated using echocardiography. Clinical and statistically significant changes including decreased ventricular myocardial contractility and arrhythmia were elucidated by echocardiography and interpreted by boarded cardiologists as potentially life threatening. The objective of this investigation was to initiate development of an antemortem, sensitive blood screening test for cardiac damage due to oil exposure of avian species. An assay for the cardiac isoform of troponin I (cTnI) which is known to be highly cross-reactive across mammalian species was chosen and analytically validated in DCCO. This is the first time this test has been analytically validated in avian species. All plasma samples from birds assessed as healthy had trace concentrations (<0.016ng/ml). The assays was precise and accurate revealing a coefficient of variation <3% and an R2>0.99. Diagnostic investigation revealed that the test appears to have diagnostic potential for the diagnosis of cardiomyocyte damage. Diagnostic sensitivity and specificity were 91% and 73% in this laboratory population. Due to an equivocal sample population in which health could not be proven, further investigation is needed to diagnostically validate troponin I in the assessment of oil exposure in DCCO.

Assay validation of the cardiac isoform of troponin I in double crested cormorant (Phalacrocorax auritus) plasma for diagnosis of cardiac damage.

Cardiac abnormalities, initially found in Deepwater Horizon weathered MC252 crude oil exposed Double Crested Cormorants (DCCOs) upon gross necropsy, were further investigated using echocardiography. Clinical and statistically significant changes including decreased ventricular myocardial contractility and arrhythmia were elucidated by echocardiography and interpreted by boarded cardiologists as potentially life threatening. The objective of this investigation was to initiate development of an antemortem, sensitive blood screening test for cardiac damage due to oil exposure of avian species. An assay for the cardiac isoform of troponin I (cTnI) which is known to be highly cross-reactive across mammalian species was chosen and analytically validated in DCCO. This is the first time this test has been analytically validated in avian species. All plasma samples from birds assessed as healthy had trace concentrations (<0.016ng/ml). The assays was precise and accurate revealing a coefficient of variation <3% and an R2>0.99. Diagnostic investigation revealed that the test appears to have diagnostic potential for the diagnosis of cardiomyocyte damage. Diagnostic sensitivity and specificity were 91% and 73% in this laboratory population. Due to an equivocal sample population in which health could not be proven, further investigation is needed to diagnostically validate troponin I in the assessment of oil exposure in DCCO.

Effect of oral exposure to artificially weathered Deepwater Horizon crude oil on blood chemistries, hepatic antioxidant enzyme activities, organ weights and histopathology in western sandpipers (Calidris mauri).

Shorebirds were among birds exposed to Mississippi Canyon 252 (MC252) crude oil during the 2010 Deep Water Horizon (DWH) oil spill in the Gulf of Mexico. The western sandpiper (Calidris mauri) was chosen as one of four species for initial oral dosing studies conducted under Phase 2 of the avian toxicity studies for the DWH Natural Resource Damage Assessment (NRDA). Thirty western sandpipers were assigned to one of three treatment groups, 10 birds per group. The control group was sham gavaged and the treatment groups were gavaged with 1 or 5mL oil kg bw-1 daily for 20 days. Periodic blood samples for hemoglobin measurements were collected during the trial. A final blood sample used to determine hemoglobin concentration in addition to complete blood counts, plasma clinical chemistries, haptoglobin concentration and plasma electrophoresis was collected when birds were euthanized and necropsied on day 21. Tissues were removed, weighed and processed for subsequent histopathological evaluation. There were numerical decreases in hemoglobin concentrations in oil-dosed birds over the 21-day trial, but values were not significantly different compared to controls on day 21. There were no significant differences between controls and oiled birds in complete blood counts, plasma chemistries, haptoglobin concentration, and plasma electrophoresis endpoints. Of the hepatic oxidative stress endpoints assessed, the total antioxidant capacity assessment (Trolox equivalents) for the control group was lower compared to the 1mL oil kg bw-1 group. Absolute liver weights in the 5mL oil kg bw-1 group were significantly greater compared to controls. While not conclusive, the numerical decrease in hemoglobin concentration and significant increase in absolute liver weight are consistent with exposure to oil. Histological changes in the adrenal gland could be considered a non-specific indicator of stress resulting from exposure to oil. It is possible that the quantity of oil absorbed was not sufficient to induce clearly evident hemolytic anemia or that the western sandpiper is relatively insensitive to ingested oil.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Organ weights and histopathology of double-crested cormorants (Phalacrocorax auritus) dosed orally or dermally with artificially weathered Mississippi Canyon 252 crude oil.

A series of toxicity tests were conducted to assess the effects of low to moderate exposure to artificially weathered Deepwater Horizon Mississippi Canyon 252 crude oil on representative avian species as part of the Natural Resource Damage Assessment. The present report summarizes effects of oral exposure (n=26) of double-crested cormorants (DCCO; Phalacrocorax auritus) to 5 or 10ml oil kg-1 day-1 for up to 21 days or dermal application (n=25) of 13ml oil to breast and back feathers every three days totaling 6 applications in 21 days on organ weights and histopathology. Absolute and relative kidney and liver weights were increased in birds exposed to oil. Additionally, gross and/or histopathologic lesions occurred in the kidney, heart, pancreas and thyroid. Clinically significant renal lesions in the orally dosed birds included squamous metaplasia and increased epithelial hypertrophy of the collecting ducts and renal tubules and mineralization in comparison to controls. Gross cardiac lesions including thin walls and flaccid musculature were documented in both orally and dermally dosed birds and myocardial fibrosis was found in low numbers of dermally dosed birds only. Cytoplasmic vacuolation of the exocrine pancreas was noted in orally dosed birds only. Thyroid follicular hyperplasia was increased in dermally dosed birds only possibly due to increased metabolism required to compensate damaged feather integrity and thermoregulate. Gastrointestinal ulceration was found in orally dosed birds only. There were no significant hepatic histopathologic lesions induced by either exposure route. Therefore, hepatic histopathology is likely not a good representation of oil-induced damage. Taken together, the results suggest that oral or dermal exposure of DCCOs to artificially weathered MC252 crude oil induced organ damage that could potentially affect survivability.

Toxicological and thermoregulatory effects of feather contamination with artificially weathered MC 252 oil in western sandpipers (Calidris mauri).

The external contamination of bird feathers with crude oil might have effects on feather structure and thus on thermoregulation. We tested the thermoregulatory ability of western sandpipers (Calidris mauri) in a respirometry chamber with oil applied either immediately prior, or three days before the experiment. The birds were then exposed to a sliding cold temperature challenge between 27°C and -3°C to calculate thermal conductance. After the experiment, a large blood sample was taken and the liver extracted to measure a range of parameters linked to toxicology and oxidative stress. No differences in thermal conductance were observed among groups, but birds exposed to oil for three days had reduced body temperatures and lost more body mass during that period. At necropsy, oiled birds showed a decrease in plasma albumin and sodium, and an increase in urea. This is reflective of dysfunction in the kidney at the loop of Henle. Birds, especially when exposed to the oil for three days, showed signs of oxidative stress and oxidative damage. These results show that the ingestion of externally applied oil through preening or drinking can cause toxic effects even in low doses, while we did not detect a direct effect of the external oil on thermoregulation over the temperature range tested.

Changes in white cell estimates and plasma chemistry measurements following oral or external dosing of double-crested cormorants, Phalacocorax auritus, with artificially weathered MC252 oil.

Scoping studies were designed whereby double-crested cormorants (Phalacocorax auritus) were dosed with artificially weathered Deepwater Horizon (DWH) oil either daily through oil injected feeder fish, or by application of oil directly to feathers every three days. Preening results in oil ingestion, and may be an effective means of orally dosing birds with toxicant to improve our understanding of the full range of physiological effects of oral oil ingestion on birds. Blood samples collected every 5-6 days were analyzed for a number of clinical endpoints including white blood cell (WBC) estimates and differential cell counts. Plasma biochemical evaluations were performed for changes associated with oil toxicity. Oral dosing and application of oil to feathers resulted in clinical signs and statistically significant changes in a number of biochemical endpoints consistent with petroleum exposure. In orally dosed birds there were statistically significant decreases in aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) activities, calcium, chloride, cholesterol, glucose, and total protein concentrations, and increases in plasma urea, uric acid, and phosphorus concentrations. Plasma electrophoresis endpoints (pre-albumin, albumin, alpha-2 globulin, beta globulin, and gamma globulin concentrations and albumin: globulin ratios) were decreased in orally dosed birds. Birds with external oil had increases in urea, creatinine, uric acid, creatine kinase (CK), glutamate dehydrogenase (GLDH), phosphorus, calcium, chloride, potassium, albumin, alpha-1 globulin and alpha-2 globulin. Decreases were observed in AST, beta globulin and glucose. WBC also differed between treatments; however, this was in part driven by monocytosis present in the externally oiled birds prior to oil treatment.

Assessment of toxicity and coagulopathy of brodifacoum in Japanese quail and testing in wild owls.

Based on detection of hepatic residues, scavenging and predatory non-target raptors are widely exposed to second generation anticoagulant rodenticides (SGARs). A small proportion, generally <10%, of tested birds are diagnosed as acutely poisoned. Little is known, however, of sub-lethal effects of SGARs, such as interaction of clotting capacity with traumatic injury. Assessment of coagulation function of birds submitted live to wildlife rehabilitators or veterinarians may provide a means of establishing the proportion of animals suffering sub-lethal coagulopathies, as well as identifying individuals requiring treatment. As a first step in exploring the potential of this approach, we dosed Japanese quail (Coturnix japonica) with the SGAR, brodifacoum, at 0, 0.8, 1.4, 1.9, and 2.5 mg/kg and sampled birds at 1, 3, 5 and 7 days post-dosing. Prothrombin time (PT), which measures the extrinsic coagulation pathway, was significantly prolonged in 98% of brodifacoum-exposed quail in a dose- and time-dependent manner. 50-fold prolongation of PT occurred at higher brodifacoum dosages and correlated to hemorrhage found at necropsy. Activated clotting time (ACT), a measure of the intrinsic pathway also increased with dose and time. Hemoglobin (Hb) and hematocrit (Hct) decreased dose- and time-dependently at doses ≥1.4 mg/kg with no significant change at 0.8 mg/kg. Reference intervals for PT (10.0-16.2 s), ACT (30-180 s), Hb (9.6-18.4 g/dl), and Hct (34-55%) were established in Japanese quail. Species-specific reference intervals are required as barn owl PT (17-29 s) and quail PT were different. The proportion of brodifacoum-exposed quail with hemorrhage was not correlated with liver residues, but was correlated with PT, suggesting that this assay is a useful indicator of avian anticoagulant rodenticide exposure. PTs measured in free-living barn owls sampled between April 2009 and August 2010 in the lower Fraser Valley of BC do not suggest significant exposure to SGARs.

Blood mineral concentrations in manatees (Trichechus manatus latirostris and Trichechus manatus manatus).

Limited information is available regarding the role of minerals and heavy metals in the morbidity and mortality of manatees. Whole-blood and serum mineral concentrations were evaluated in apparently healthy, free-ranging Florida (Trichechus manatus latirostris, n = 31) and Belize (Trichechus manatus manatus, n = 14) manatees. Toxicologic statuses of the animals and of their environment had not been previously determined. Mean mineral whole-blood (WB) and serum values in Florida (FL) and Belize (BZ) manatees were determined, and evaluated for differences with respect to geographic location, relative age, and sex. Mean WB and serum silver, boron, cobalt, magnesium, molybdenum, and WB cadmium concentrations were significantly higher in BZ versus FL manatees (P < 0.05). Mean WB aluminum, calcium, manganese, sodium, phosphorus, vanadium, and serum zinc concentrations were significantly lower in BZ versus FL manatees. Adult manatees had significant and higher mean WB aluminum, manganese, sodium, antimony, vanadium, and serum manganese and zinc concentrations compared to juvenile animals. Significant and lower mean WB and serum silver, boron, cobalt, and serum copper and strontium concentrations were present in adults compared to juveniles (P < or = 0.05). Females had significant and higher mean WB nickel and serum barium compared to males (P < or = 0.05). Mean WB arsenic and zinc, and mean serum iron, magnesium, and zinc concentrations fell within toxic ranges reported for domestic species. Results reveal manatee blood mineral concentrations differ with location, age, and sex. Influence from diet, sediment, water, and anthropogenic sources on manatee mineral concentration warrant further investigation.

Analytical validation of a novel point-of-care immunoassay for canine N-terminal pro-brain natriuretic peptide analysis.

BACKGROUND: N-terminal pro-brain natriuretic peptide (NT-proBNP) is a widely used point-of-care (POC) cardiac biomarker in human medicine. Canine NT-proBNP is used less in veterinary medicine, possibly due to the lack of a POC canine NT-proBNP assay resulting in temporal delay, increased degradation in transport, and high reported variability in the available assay. A new quantitative POC analyzer allows fast, onsite measurement of NT-proBNP, minimizing preanalytical error and reducing variability. OBJECTIVE: We aimed to analytically validate an NT-proBNP assay (Vcheck) according to American Society of Veterinary Clinical Pathology (ASVCP) and Clinical Laboratory Improvement Amendments (CLIA) specifications. METHODS: Archived and prospective plasma and serum samples were collected from male and female, client-owned dogs of various breeds with cardiac abnormalities (n = 81) and a healthy control population (n = 225). Precision, accuracy, analytical sensitivity, and specificity, and other statistical analyses were performed. RESULTS: Imprecision was considered acceptable with a coefficient of variation ranging from 9% at 4000 pmol/L to 20% at 600 pmol/L. The lower limit of quantitation was 650 pmol/L based on repetitive measures evaluation. Comparison of the Vcheck assay with the Cardiopet NT-proBNP assay revealed an excellent correlation with minimal bias when preanalytical factors were controlled. Significant degradation of NT-proBNP occurred when current methods were used at refrigerated and room temperatures, which could change diagnostic and prognostic decision-making. Age-partitioned reference intervals have high reference values of 750 pmol/L and 1280 pmol/L for juvenile and adult dogs, respectively. CONCLUSIONS: The Vcheck NT-ProBNP assay provides analytically acceptable results. Onsite testing can minimize variability related to preanalytical error and provide clinically useful contemporaneous results. Samples should be centrifuged immediately and analyzed within 2 hours of collection.

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris).

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate.

Hematology of healthy Florida manatees (Trichechus manatus).

BACKGROUND: Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. OBJECTIVES: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. METHODS: Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. RESULTS: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. CONCLUSIONS: Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

Debromoaplysiatoxin in Lyngbya-dominated mats on manatees (Trichechus manatus latirostris) in the Florida King's Bay ecosystem.

Proliferation of the potentially toxic cyanobacterium, Lyngbya, in Florida lakes and rivers has raised concerns about ecosystem and human health. Debromoaplysiatoxin (DAT) was measured in concentrations up to 6.31 microg/g wet weight lyngbyatoxin A equivalents (WWLAE) in Lyngbya-dominated mats collected from natural substrates. DAT was also detected (up to 1.19 microg/g WWLAE) in Lyngbya-dominated mats collected from manatee dorsa. Ulcerative dermatitis found on manatees is associated with, but has not been proven to be caused by DAT.

Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae.

The Afrotheria represent an early branching of placental mammals. Only two herpesviruses from Afrotheria have been previously identified, and the genus Proboscivirus in the subfamily Betaherpesvirinae has been proposed for them. Six novel gammaherpesviruses were identified in four species in the superorder Afrotheria by detection and analysis of their DNA polymerase genes. Elephantid herpesvirus 3 (ElHV3) and Elephantid herpesvirus 4 (ElHV4) were identified from conjunctival swabs from Asian elephants (Elephas maximus). ElHV3 was also found in a vaginal swab from one elephant with vaginitis. Elephantid herpesvirus 5 (ElHV5) was identified from vaginal swabs of two Asian elephants with vaginal plaques. Elephantid herpesvirus 6 was discovered in a conjunctival swab from an African elephant (Loxodonta africana). Procavid herpesvirus 1 (PrHV1) was found in spleen and conjunctival swabs of rock hyrax (Procavia capensis). Trichechid herpesvirus 1 (TrHV1) was identified from skin and buffy coats of Florida manatees (Trichechus manatus latirostris). ElHV3 and ElHV4 form a distinct cluster, and ElHV5, ElHV6, TrHV1, and PrHV1 form a second cluster. These viruses may have codiverged with their host species. Phylogenetic analysis of these novel herpesviruses suggests that two separate groups of gammaherpesviruses may have codiverged with the Afrotheria.

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.

Sample Collection.

Results may be changed with suboptimal sample collection and transport, which then result in incorrect diagnoses. Quality management of samples must start in the patient, extend through sampling itself, include appropriate short transport, and then be correctly accessioned at the referral laboratory or in-house station to ensure accurate diagnosis of disease. A quality assurance plan at the laboratory chosen is mandatory under American Society for Veterinary Clinical Pathology guidelines.

Validation of an immunocytochemical assay for immunophenotyping of lymphoma in guinea pigs (Cavia porcellus).

BACKGROUND: Lymphoma is an important disease of pet guinea pigs, although validation of immunophenotyping techniques based on cytologic or hematologic samples has not been reported. OBJECTIVE: To describe an immunocytochemical method for immunophenotyping of lymphoma (as either T- or B-cell) in guinea pigs, and to validate antibodies for this purpose. METHODS: Blood and tissues were obtained at the time of necropsy from laboratory guinea pigs and a privately owned dog (control) euthanized for reasons unrelated to lymphoproliferative disease. Fine-needle aspirates of enlarged peripheral lymph nodes were obtained from a case of spontaneous lymphoma in a pet guinea pig. Anti-CD3 and anti-Pax5 antibodies were validated by a combination of western blotting performed on splenic lysates of both the dog and guinea pigs, immunohistochemical studies on normal guinea pig tissues, and immunocytochemistry on normal guinea pig peripheral blood and splenic impression smears. RESULTS: The antibodies bound to antigens of an appropriate size in both the dog and guinea pig splenic lysates by Western blot analysis. Immunohistochemistry and immunocytochemistry demonstrated the expected distribution of putative T- and B-lymphocytes in normal tissues, peripheral blood, and splenic impression smears. As a proof-of-principle for its clinical utility, this immunocytochemical assay was used to diagnose a B-cell phenotype in a spontaneous lymphoma case in a pet guinea pig. CONCLUSIONS: Here, we validated an immunocytochemical method for immunophenotyping of lymphoma in guinea pigs as either a T- or B-cell phenotype. This enables future research into the clinical attributes of these subtypes and may ultimately improve both prognostication and therapy of lymphoma in guinea pigs.

ASVCP guidelines: Allowable total error hematology.

The purpose of this document is to provide total allowable error (TEa ) recommendations for commonly analyzed hematology measurands for veterinary personnel. These guidelines define relevant terminology and highlight considerations specific to hematology measurands. They also provide reasons and guidelines for using TEa in instrument performance evaluation, including recommendations for when the total observed error exceeds the recommended TEa . Biological variation-based quality specifications are briefly discussed. The appendix describes the derivation of the hematology TEa recommendations and provides resources for external quality assurance/proficiency testing programs and a worksheet for implementation of the guidelines.