Hepatic lipid accumulation is associated with multiple metabolic pathway alterations but not dyslipidemia and insulin resistance in central bearded dragons (Pogona vitticeps).

OBJECTIVE: To investigate associations between hepatic fat accumulation, fibrosis, and plasma values of primary metabolites, biochemical measurands, insulin, and lipoproteins in bearded dragons. ANIMALS: 48 adult central bearded dragons (Pogona vitticeps). METHODS: Dragons were sedated with alfaxalone, and a blood sample was collected. Plasma was submitted for untargeted primary metabolomics using gas chromatography time-of-flight mass spectrometry, a biochemistry panel, and a lipoprotein panel determined by PAGE. Hepatic lipid content was quantified by liver attenuation measurements from CT images and digital image analysis of standardized histologic sections of the liver. Fibrosis was quantified by digital image analysis on Masson's trichrome-stained histologic sections. Severity was determined from pathologic review of liver sections according to a standardized grading system. Statistical associations were investigated using serial linear models adjusted for false discovery rate and multivariate statistics. RESULTS: Both hepatic fat and fibrosis had a significant effect on CT liver attenuation values. Several oligosaccharides (maltotriose, maltose, ribose, trehalose) and alkaline phosphatase were significantly and linearly increased with hepatic lipid content (all q < .05). On partial least square-discriminant analysis, ß-hydroxybutyric acid was the most important discriminatory variable between fatty liver severity grades on histology. No significant associations were found with insulin, lipoproteins, and succinic acid. CLINICAL RELEVANCE: Bearded dragons with hepatic lipid accumulation experienced multiple metabolic pathway disruptions, some being compatible with mitochondrial dysfunction. No evidence of insulin resistance or dyslipidemia was found. Hepatic biopsy and histopathology remain recommended for reliably diagnosing and staging fatty liver disease in bearded dragons.

Lipoprotein characterization in Quaker parrots (Myiopsitta monachus) using gel-permeation high-performance liquid chromatography.

BACKGROUND: Lipid accumulation disorders, such as atherosclerosis and hepatic lipidosis, are common in psittacine birds and associated with various dyslipidemias. Gel-permeation high-performance liquid chromatography (GP-HPLC) is a reference method for advanced lipoprotein profiling based on particle size separation, followed by an analysis of lipid contents. OBJECTIVES: The objectives were to (a) characterize Quaker parrot lipoproteins using a commercial GP-HPLC method (Liposearch panel), and (b) obtain preliminary information on the reliability of the Friedewald formula for low-density lipoprotein-cholesterol (LDL-C) measurements. METHODS: Plasma samples were collected from 12 fasted healthy Quaker parrots. Cholesterol concentrations, triglyceride concentrations, particle sizes, and particle numbers were determined by GP-HPLC for four classes and 20 sub-fractions of lipoproteins. The LDL-C concentrations obtained using the Friedewald formula and direct measurements were compared with Bland-Altman plots. Alternate formulas were determined using multiple linear regression. RESULTS: High-density lipoprotein (HDL) was the predominant lipoprotein in Quaker parrots, and most particles were of medium-to-small sizes belonging to two sub-fractions (average size, 10.6 nm). LDL was the second most common lipoprotein and included large-to-small particles belonging to three sub-fractions (average size, 24.9 nm). Very-low-density lipoproteins (VLDL) and portomicrons were present in low concentrations. The Friedewald formula underestimated LDL-C concentrations with a significant bias of 0.44 mmol/L. An alternate formula was proposed: LDL-C = 0.75*Non-HDL-C. CONCLUSIONS: GP-HPLC allowed unprecedented characterization of plasma lipoproteins in Quaker parrots. Characterizing psittacine lipoprotein is useful for validation and interpretation of routine clinical tests as well as for use in epidemiologic and experimental research on psittacine lipid accumulation disorders.

Morphologic and quantitative evaluation of bone marrow aspirates from Hispaniolan Amazon parrots (Amazona ventralis).

BACKGROUND: Bone marrow aspirate assessments provide valuable information about hematopoietic status and hematologic disease. Hematopoietic cell differentials and morphologies have been anecdotally described in psittacines, but quantitative studies are lacking. OBJECTIVES: We aimed to determine differential cell counts and calculate granulocyte:erythroid (G:E) ratios in bone marrow aspirates from Hispaniolan Amazon parrots and report representative morphologies of the hematopoietic cells. METHODS: Bone marrow aspirates were collected from 32 clinically healthy, captive, parrots. Peripheral blood was obtained for CBCs. Bone marrow differential cell counts (%) were determined by counting 500 cells on modified Wright's-stained smears. G:E ratios were calculated. Representative images of hematopoietic cells at all stages of development were taken. RESULTS: Of the 32 parrots sampled, 17 bone marrow samples were of sufficient cellularity and quality for evaluation. Erythroid cells comprised 68.9 ± 8.6% (total ± SD) of the hematopoietic cells and consisted primarily of early- and late-stage polychromatophilic rubricytes (43.6 ± 2.1% of total erythroid cells). Granulocytic cells comprised 28.1 ± 3.8% of the hematopoietic cells and consisted primarily of mature and band heterophils (11.9 ± 5.2% and 6.5 ± 3.4%, respectively, of total granulocytic cells). A unique morphologic finding in avian progranulocytes was the presence of multiple different granules. The G:E ratio was 0.4 ± 0.2 (median 0.4, range 0.1-0.9). Thrombocyte lineage cells could not be reliably identified and were not counted. CBC results were largely within expected limits. CONCLUSIONS: The low G:E ratios observed could be normal in this species; however, these ratios could be affected by factors related to sampling and cell identification. These findings will be a valuable resource for the diagnostic evaluation of clinical bone marrow samples from Hispaniolan Amazon parrots and could serve as a general reference for psittacine bone marrow evaluation.

EFFECT OF ROUTINE HANDLING AND TRANSPORTATION ON BLOOD LEUKOCYTE CONCENTRATIONS AND PLASMA CORTICOSTERONE IN CAPTIVE HISPANIOLAN AMAZON PARROTS ( AMAZONA VENTRALIS).

Increased glucocorticoids cause a characteristic stress leukogram in mammalian taxa. It is assumed that avians exhibit a similar response, but to date, there have been no controlled studies to correlate serial endogenous corticosterone levels to hematologic values. An established flock of 18 Hispaniolan Amazon parrots ( Amazona ventralis) was used as a model in a crossover study. The treatment group was subjected to the stress of transport, restraint, and common clinical procedures with serial blood samples collected at 20-min intervals for hematology and corticosterone levels; the control group was sampled at the same intervals. Longitudinal data analysis was performed with linear mixed modeling. For all hematologic analytes, the baseline value had a significant positive effect on subsequent values (all P < 0.001). The white blood cell, heterophil, and eosinophil counts and heterophil to lymphocyte ratio increased over time in the treatment group, whereas it remained stable in the control group ( P = 0.016, P < 0.001, P < 0.001, P = 0.02, respectively, for the time*treatment effect). Lymphocyte absolute counts decreased over time, although not significantly; the decrease was significant for the relative lymphocyte count in the treatment group. Monocytes and basophils were not significantly altered. The treatment group had a higher mean corticosterone level overall than the control group by approximately 60% ( P = 0.008). The mean corticosterone level also increased over time in both groups by three- to fourfold ( P < 0.001) by 20 min then plateaued. These results demonstrate that some significant hematologic changes may arise with routine handling and transportation of birds and should be accounted for in hematologic interpretation of cell counts.

Venous blood gases, plasma biochemistry, and hematology of wild-caught common chameleons (Chamaeleo chamaeleon).

The purpose of this study was to determine a wide range of selected hematologic, venous blood gases, and plasma biochemistry analytes in common chameleons (Chamaeleo chamaeleon). Blood samples were collected from the ventral tail vein of 41 common chameleons to determine reference intervals for 30 different blood analytes. The calcium-to-phosphorus ratio, packed cell volume (PCV), refractometric total solids (TS), blood cell counts, and differentials were also determined. The microscopic evaluation of blood smears revealed inclusion bodies in monocytes in 7 of the samples. Females showed significantly higher values of plasma proteins and calcium and cholesterol concentrations and males showed significantly higher values of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) plasma concentrations. Significant differences were found between similar analytes determined by different testing methodologies in the PCV/hematocrit, electrolytes (sodium, potassium), and plasma proteins [TS, total protein (TP) and albumin]. Blood analytes determined in this study can provide baseline data that may be useful when evaluating the health status of common chameleons, taking into consideration the potential effects of gender and the type of analyzer used.

Des prélèvements sanguins ont été obtenus de la veine caudale ventrale sur 41 caméléons communs (Chamaeleo chamaeleon) dans l'optique d'établir des intervalles de référence pour 30 analytes sanguins. Le ratio calcium/phosphore, le PCV, les solides totaux par réfractométrie (TS), la numération-formule sanguine ont aussi été déterminés. L'évaluation microscopique des frottis sanguins ont révélé la présence de corps d'inclusions ressemblant aux corps d'inclusions a Chlamydia dans les monocytes sur sept échantillons. Les femelles avaient des valeurs significativement plus élevées pour l'AST et la GGT. Des différences significatives entre les mêmes analytes mais d'après différentes techniques analytiques ont été trouvées pour le PCV/hématocrite, des électrolytes (sodium, potassium) et les protéines plasmatiques (TS, TP et albumine). Les analytes sanguins rapportés dans cette étude pourront fournir des données de base utiles pour l'évaluation de l'état de sante des caméléons communs, tout en considérant les effets potentiels du sexe et de l'analyseur utilisé.(Traduit par les auteurs).

Avian Oncology: Diseases, Diagnostics, and Therapeutics.

Companion birds are increasingly living longer due to improved husbandry, nutrition, and veterinary care. As a consequence, a growing number of geriatric disease conditions are diagnosed and managed by veterinarians. Awareness of bird owners of diagnostic and treatment options for neoplastic diseases in humans and domestic animals has led to increasing demand to provide advanced diagnostic and treatment modalities for companion birds diagnosed with neoplasia. Treatment remains challenging in many companion birds due to the lack of information regarding prognosis and efficacy of antineoplastic treatments in these species. There is no established standard of care for most tumors in companion birds.

Evaluation of peripheral blood and abdominal fluid variables as predictors of intestinal surgical site failure in dogs with septic peritonitis following celiotomy and the placement of closed-suction abdominal drains.

OBJECTIVE To evaluate peripheral blood and abdominal fluid variables as predictors of intestinal surgical site failure in dogs with septic peritonitis following celiotomy and closed-suction abdominal drain (CSAD) placement. DESIGN Prospective study. ANIMALS 26 dogs with septic peritonitis that underwent celiotomy and CSAD placement. PROCEDURES Abdominal fluid and blood samples were collected prior to surgery and daily thereafter until CSAD removal. Abdominal fluid was collected through the CSAD. Analysis of all samples included pH, PCO2, PO2, PCV, WBC count, and total solids, glucose, lactate, and electrolyte concentrations. Abdominal fluid samples also underwent cytologic evaluation and bacterial culture, and the volume of fluid removed through the drain was recorded daily. The blood-to-fluid glucose and lactate differences, fluid-to-blood lactate ratio and blood-to-fluid WBC and neutrophil ratios were determined daily. Dogs were categorized into 2 groups on the basis of whether they had an uneventful recovery (UR) or developed postoperative septic peritonitis (POSP). RESULTS 23 dogs had a UR and 3 developed POSP. On the third day after surgery, the abdominal fluid WBC count was significantly lower and the blood-to-fluid WBC and neutrophil ratios were significantly higher for dogs in the POSP group, compared with those for dogs in the UR group. None of the other blood and abdominal fluid variables assessed differed significantly between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE Results failed to identify any objective predictive indicators for POSP in dogs with CSADs. Use of blood-to-fluid WBC and neutrophil ratios as predictive indicators for POSP requires further investigation.

Laboratory blood analysis in Strigiformes-Part I: hematologic reference intervals and agreement between manual blood cell counting techniques.

BACKGROUND: While hematologic reference intervals (RI) are available for multiple raptorial species of the order Accipitriformes and Falconiformes, there is a lack of valuable hematologic information in Strigiformes that can be used for diagnostic and health monitoring purposes. OBJECTIVES: The objective was to report RI in Strigiformes for hematologic variables and to assess agreement between manual cell counting techniques. METHODS: A multi-center prospective study was designed to assess hematologic RI and blood cell morphology in owl species. Samples were collected from individuals representing 13 Strigiformes species, including Great Horned Owl, Snowy Owl, Eurasian Eagle Owl, Barred Owl, Great Gray Owl, Ural Owl, Northern Saw-Whet Owls, Northern Hawk Owl, Spectacled Owl, Barn Owl, Eastern Screech Owl, Long-Eared Owl, and Short-Eared Owl. Red blood cell count was determined manually using a hemocytometer. White blood cell count was determined using 3 manual counting techniques: (1) phloxine B technique, (2) Natt and Herrick technique, and (3) estimation from the smear. Differential counts and blood cell morphology were determined on smears. Reference intervals were determined and agreement between methods was calculated. RESULTS: Important species-specific differences were observed in blood cell counts and granulocyte morphology. Differences in WBC count between species did not appear to be predictable based on phylogenetic relationships. Overall, most boreal owl species exhibited a lower WBC count than other species. Important disagreements were found between different manual WBC counting techniques. CONCLUSIONS: Disagreements observed between manual counting techniques suggest that technique-specific RI should be used in Strigiformes.

Laboratory blood analysis in Strigiformes-Part II: plasma biochemistry reference intervals and agreement between the Abaxis Vetscan V2 and the Roche Cobas c501.

BACKGROUND: Limited plasma biochemical information is available in Strigiformes. Only one study investigated the agreement between a point-of-care with a reference laboratory analyzer for biochemistry variables in birds. OBJECTIVES: The objective was to report reference intervals (RI) for plasma biochemistry variables in Strigiformes, and to assess agreement between the Abaxis Vetscan V2 and Roche Cobas c501. METHODS: A prospective study was designed to assess plasma biochemistry RI for concentration of calcium, phosphorus, total protein, albumin, globulin, glucose, bilirubin, uric acid, bile acids, sodium, potassium, and chloride, and activities of AST, GGT, CK, amylase, lipase, LDH, and GLDH. In addition, the agreement between the Vetscan and the Cobas in owl species was assessed. RESULTS: A total of 190 individuals were sampled belonging to 12 Strigiformes species including Barn Owls, Barred Owls, Great Horned Owls, Eurasian Eagle Owls, Spectacled Owls, Eastern Screech Owls, Long-Eared Owls, Short-Eared Owls, Great Gray Owls, Snowy Owls, Northern Saw-Whet Owls, and Northern Hawk-Owls. Order-, species-, and method-specific RI were determined on both analyzers. Although Vetscan data were not equivalent to the Cobas, 4 analytes (glucose, AST, CK, and total protein, with correction for bias) were within acceptable agreement, 3 analytes (uric acid, calcium, and phosphorus) were within close agreement, and the remaining analytes were in strong disagreement. Species-specific differences were observed notably for the concentration of glucose in Barn Owls and electrolytes in Northern Saw-Whet Owls. CONCLUSIONS: Overall, this study suggests that the Vetscan has acceptable clinical performance in Strigiformes for some analytes and highlights discrepancies for several analytes.

Cluster of atherosclerosis in a captive population of black kites (Milvus migrans subsp.) in France and effect of nutrition on the plasma lipid profile.

From January 2010 to March 2013, a captive colony of 83 black kites (Milvus migrans subsp.) in France experienced increased mortality related to atherosclerosis with an incidence of 4.4% per year. On histopathology, all kites had advanced atherosclerotic lesions, with several birds presenting abdominal hemorrhage and aortic rupture. In January 2012, a dietary change was instituted and consisted of introducing fish into the kites' diet. During the following 15 mo, the plasma lipid profile was monitored as well as body weight, food offered, and flight activity. Total and low-density lipoprotein cholesterol initially increased, but in December 2012 and March 2013, an overall decrease from initial values was observed. High-density lipoprotein cholesterol also increased during this period. Despite positive plasma lipid changes induced by dietary modifications, there was no decrease in mortality from atherosclerosis, which was probably associated with the severity of the atherosclerotic lesions at time of dietary management. However, owing to the long and progressive development of atherosclerotic lesions, long-term beneficial effects are probable. This report suggests that black kites are particularly susceptible to atherosclerosis and aortic dissection in captivity. To prevent degenerative diseases associated with captivity in birds of prey, species-specific lifestyle and dietary requirements and susceptibility to these diseases should be considered.

Association of plasma lipid levels with atherosclerosis prevalence in psittaciformes.

The prevalence of atherosclerosis is high in the captive psittacine population and increases with age and female sex. The genera Psittacus, Amazona, and Nymphicus are predisposed to atherosclerosis, whereas the genera Cacatua and Ara are less susceptible. Plasma cholesterol and lipoprotein abnormalities have been suggested as risk factors in the development of atherosclerosis as observed in mammals. To investigate whether the psittacine genera susceptibility to atherosclerosis and the known risk factors of age and sex could be associated with differences in the lipid profile, a retrospective analysis was conducted on blood lipid values from 5625 birds. Prevalence values were obtained from a previously published, large, case-control study and were compared with identified trends in plasma lipid profiles. Genus-specific differences were identified in plasma total cholesterol values that corresponded to observed trends in the prevalence of clinically important atherosclerotic lesions, which were also highly correlated. The effect of age was significant but was mild and may not account for the dramatic increase in atherosclerosis prevalence observed with age. In addition, Quaker parrots ( Myiopsitta monachus ), which were used as experimental models for psittacine atherosclerosis and dyslipidemia, were found to have the highest values in all lipid profile parameters. The results of this study suggest that the differences observed in prevalence among species of the psittacine genera may partly be explained by differences in plasma total cholesterol levels. Results also support the use of Quaker parrots as models for studying atherosclerosis and dyslipidemia.

Complete blood cell count in psittaciformes by using high-throughput image cytometry: a pilot study.

The avian hemogram is usually performed in veterinary diagnostic laboratories by using manual cell counting techniques and differential counts determined by light microscopy. There is no standard automated technique for avian blood cell count and differentiation to date. These shortcomings in birds are primarily because erythrocytes and thrombocytes are nucleated, which precludes the use of automated analyzers programmed to perform mammal complete blood cell counts. In addition, there is no standard avian antibody panel, which would allow cell differentiation by immunophenotyping across all commonly seen bird species. We report an alternative hematologic approach for quantification and differentiation of avian blood cells by using high-throughput image cytometry on blood smears in psittacine bird species. A pilot study was designed with 70 blood smears of different psittacine bird species stained with a Wright-Giemsa stain. The slides were scanned at 0.23 microm/pixel. The open-source softwares CellProfiler and CellProfiler Analyst were used for analyzing and sorting each cell by image cytometry. A "pipeline" was constructed in the CellProfiler by using different modules to identify and export hundreds of measures per cell for shape, intensity, and texture. Rules for classifying the different blood cell phenotypes were then determined based on these measurements by iterative feedback and machine learning by using CellProfiler Analyst. Although this approach shows promises, avian Leukopet results could not be duplicated when using this technique as is. Further studies and more standardized prospective investigations may be needed to refine the "pipeline" strategy and the machine learning algorithm.

Prevalence of and risk factors associated with atherosclerosis in psittacine birds.

OBJECTIVE: To estimate the prevalence of clinically relevant atherosclerotic lesions in birds and identify epidemiological variables and illness types associated with development of atherosclerosis. DESIGN: Retrospective case-control study. SAMPLE: Records of 7683 psittacine birds, including 525 with advanced atherosclerosis. PROCEDURES: 5 pathology centers provided databases and access to histopathology slides. Age and sex were collected for all birds of the Amazona, Ara, Cacatua, Nymphicus, and Psittacus genera. Databases were searched for atherosclerosis cases, and slides were reviewed for the presence of type IV through VI atherosclerotic lesions. Results were used to build several multiple logistic models to define the association between advanced atherosclerosis and age, sex, genus, illness type, and specific lesions. Prevalence was reported as a function of age, sex, and genus. RESULTS: In the first model including 7683 birds, age, female sex, and the genera Psittacus, Amazona, and Nymphicus were significantly associated with clinically relevant atherosclerosis detected via necropsy. Subsequent models of 1,050 cases revealed further associations with reproductive disease, hepatic disease, and myocardial fibrosis, controlling for age, sex, and genus. CONCLUSIONS AND CLINICAL RELEVANCE: Age, female sex, and 3 genera appeared to be positively associated with the presence of advanced atherosclerotic lesions in psittacine birds. This information may be useful in clinical assessment of the cardiovascular system and patient management. Reproductive diseases were the only potentially modifiable risk factor identified and could be a target for prevention in captive psittacine birds.

Preliminary evaluation of a quantitative polymerase chain reaction assay for diagnosis of feline immunodeficiency virus infection.

Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and specificity for detection of feline immunodeficiency virus (FIV) antibodies, but does not distinguish between infection and vaccination. Real-time quantitative PCR (qPCR) assays may distinguish infected from vaccinated cats. Performance of a commercial qPCR assay was assessed with blood samples from 29 FIV-infected non-vaccinated, 26 FIV-uninfected vaccinated and 35 FIV-uninfected non-vaccinated cats. FIV infection status of cats was assigned based on a combination of vaccination and medical history, prevention of contact with potentially infected cats and two FIV antibody ELISA results. Test sensitivity and specificity were determined against this gold standard. The qPCR test yielded positive results in samples from 23/29 FIV-infected non-vaccinated, 2/26 FIV-uninfected vaccinated and 0/35 FIV-uninfected non-vaccinated cats. It was concluded that the qPCR test was moderately sensitive and highly specific for the diagnosis of FIV infection, and that it may be suitable for ruling out FIV infection in cats with a positive antibody ELISA result and unknown vaccination history.

Successful treatment of a radioulnar synostosis in a Mississippi kite (Ictinia mississippiensis).

A Mississippi kite (Ictinia mississippiensis) was presented to the Wildlife Hospital of Louisiana, School of Veterinary Medicine, Louisiana State University, for an inability to fly and was subsequently diagnosed with a minimally displaced, closed, simple, short oblique, diaphyseal fracture of the distal third of the right ulna. A figure-8 bandage was initially applied to the wing to stabilize the fracture. Over a 5-week period, the kite developed progressive reduction in wing extension, and serial radiographs revealed a bridging callus at the ulnar fracture site, as well as development of a radioulnar synostosis. To restore wing function and extension, surgical excision of the bony union between the radius and the ulna was performed, after which a polypropylene mesh implant was interposed between the 2 bones. Within 2 weeks after the surgical procedure, the kite had recovered normal wing extension and was able to fly without noticeable impairment. The bird was released 2 weeks after surgery. This case illustrates a common complication of external coaptation used as sole means of managing fractures of the avian ulna, radius, or both, which results from lack of interosseous soft tissue in the antebrachial area. On the basis of the successful outcome in this case, surgical excision of the interdiaphyseal callus and interposition of a polypropylene mesh could be a viable option for management of posttraumatic radioulnar synostosis in birds of prey.

Methods for assessing feline immunodeficiency virus infection, infectivity and purification.

Infection of cats with the feline immunodeficiency virus (FIV) recapitulates many aspects of infection of humans with HIV, including highly activated but ineffectual immune responses. Infected hosts remain seropositive for life, and detection of antibodies is the mainstay of diagnosis. However, to quantify virus for research or prognosis, viral proteins, nucleic acids or enzymes, are typically measured by ELISA, PCR or activity, respectively. While such assays are in wide use, they do not distinguish whole, infectious viral particles from defective or disrupted viruses. Titers of infectious viral particles may be estimated from tissue culture infectious doses or by enumerating cell-associated viral proteins, viral transcriptional activity or formation of syncytia. To analyze the viral proteome and the incorporation of host components into viral envelopes, pure lentiviral preparations are required. Methods for purifying lentiviruses include ultracentrifugation to separate particles by size, mass and/or density; chromatography to separate particles by charge, affinity or size; and additional removal of extraviral proteins and exosomes through subtilisin digestion or immunoaffinity. This article reviews advantages and disadvantages of different approaches to purification of lentiviruses with special reference to suitability for FIV, and highlights effects of purification on immune responses and immune assays.