Non-specific markers of inflammation in bare-nosed wombats (Vombatus ursinus) with sarcoptic mange.

Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei, negatively impacts the health, welfare, and local abundance of bare-nosed wombats (Vombatus ursinus) in Australia. Improved understanding of the host immune response to disease and its contribution to pathophysiology could be used to inform management actions for this species in and ex situ. To evaluate the immune response of bare-nosed wombats to sarcoptic mange, we validated three assays (haptoglobin, agarose gel electrophoresis, and micro-erythrocyte sedimentation rate) measuring non-specific markers of inflammation using serum samples from free-living wombats from Tasmania (n = 33). We then analysed correlations between the assay results for each non-specific marker of inflammation and wombat's sarcoptic mange scores, and performed histopathological examinations to investigate association of the acute phase response with systemic amyloidosis. We present evidence that haptoglobin and erythrocyte sedimentation rate increased, and albumin decreased, in association with sarcoptic mange scores. This research demonstrates links between the acute phase response and sarcoptic mange severity in bare-nosed wombats, highlighting the utility of non-specific markers of inflammation for aiding assessment of the systemic effects of mange. Showing the value of agarose gel electrophoresis, we also identified specific acute phase proteins warranting future evaluation and found evidence of an immunoglobulin response in mange-affected wombats, revealed by increasing γ-globulins in association with apparent disease severity. Meanwhile, owing to its relatively low resource requirements and rapidity, the erythrocyte sedimentation rate assay may be useful as a point-of-care test to support therapeutic decisions in the field. Our methods and findings are likely to be applicable to a range of other clinical and population health scenarios in captive and free-living wombats, and species impacted by sarcoptic mange globally.

An Update on the Biologic Effects of Fenbendazole.

Fenbendazole remains the drug of choice to treat pinworm infection in laboratory rodents. When fenbendazole was last reviewed (15 y ago), the literature supported the drug's lack of toxic effects at therapeutic levels, yet various demonstrated physiologic effects have the potential to alter research outcomes. Although more recent reports continue to reflect an overall discordancy of results, several studies support the premise that fenbendazole affects the bone marrow and the immune system. No effects on reproduction were reported in an extensive study that assessed common treatment protocols in mice, and food intake was unchanged in rats. Behavioral studies are sparse, with only a single report of a subtle change in a rotarod performance in mice. Notably, unexpected results in tumor models during facility treatment with fenbendazole have prompted preclinical and clinical studies of the potential roles of benzimidazoles in cancer.

Development of methodology and reference intervals for the analysis of the free-ranging Atlantic horseshoe crab Limulus polyphemus hemolymph.

BACKGROUND: The Atlantic horseshoe crab, Limulus polyphemus, is valuable to fishing and biomedical industries, for education in public aquaria, and of ecological importance because the eggs provide a critical food source for migratory shorebirds. While hematologic and biochemical analyses are fundamental tools for health assessment, reports are limited for the four extant species of horseshoe crabs; L polyphemus (East Coast North America), Carcinoscorpius rotundicauda (mangrove horseshoe crab Southeast Asia), Tachypleus gigas, and Tachypleus tridentatus (Southeast and East Asia). OBJECTIVES: This prospective study measured hemolymph values to determine RIs from free-ranging horseshoe crabs during the annual breeding aggregation in Delaware Bay, USA. METHODS: Near-shore animals were briefly manually restrained for hemolymph collection from the cardiac sinus. Hematologic samples (25 males, 25 females) were collected for hemocytometer counts using the Natt and Herrick method. A second set of 50 animals (25 males, 25 females) was sampled for biochemistry measurands, including copper concentration by spectrophotometer, osmolality by vapor pressure osmometer, and protein electrophoresis. RESULTS: Total hemocyte counts varied widely (RI 6600-44 300/µL). Electrolytes were consistent with osmoconformers living in seawater. There was a marked difference in sexes, with females showing much lower protein, glucose, and copper levels. Eleven females had multiple outliers, and all data were excluded from RI calculations. CONCLUSIONS: Further studies of this species outside of spawning season are needed. Expanding our knowledge of horseshoe crab health is important to decreasing morbidity and mortality in research applications and maximizing longevity and overall health in display settings.

SERUM ACUTE-PHASE PROTEINS IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) AND CORRELATION WITH COMMONLY UTILIZED INFLAMMATORY INDICES.

Acute-phase proteins (APP) are the foundation to the innate immune response and valuable biomarkers that increase with inflammation, infection, neoplasia, stress, and trauma.2,4,16 Little is known about the acute-phase response in cetaceans and if these proteins can be used for health monitoring in individuals and free-ranging populations. The purpose of this study was to characterize serum concentrations of haptoglobin (Hp) and serum amyloid A (SAA), as well as electrophoretic profiles of common bottlenose dolphins (Tursiops truncatus) in free-ranging (n = 33) and professional care (n = 27) settings. Results were correlated to commonly utilized inflammatory indices including erythrocyte sedimentation rate (ESR), fibrinogen, total white blood cell count (WBC), and absolute neutrophil count. SAA levels, measured with a dolphin-specific enzyme linked immunosorbent assay (ELISA), were significantly higher (P = 0.05) in free-ranging dolphins (mean = 4.26; SE = 1.12) when compared with those under professional care (mean = 1.82; SE = 0.45). For dolphins under professional care, a statistically significant correlation was identified between ESR and Hp (P < 0.001; r = 0.69), ESR and SAA (P < 0.001; r = 0.67), fibrinogen and Hp (P = 0.001; r = 0.58), and fibrinogen and SAA (P = 0.002; r = 0.56). In addition, there was a significant correlation between WBC and SAA (P = 0.01; r = 0.38) and absolute neutrophil count and SAA (P = 0.04; r = 0.32). There were no significant correlations between study variables observed in free-ranging dolphins. The variable correlation of APPs with commonly utilized inflammatory indices demonstrates that these proteins are independent measures of inflammation with unique sensitivity, specificity, and timeline of expression. The results of this study contribute to improved health monitoring of dolphins and have the potential to assist in identification of compromised health.

REFERENCE INTERVALS FOR ACUTE PHASE PROTEINS FOR KOALAS (PHASCOLARCTOS CINEREUS) AT THE SAN DIEGO ZOO.

The synthesis and circulating concentrations of acute phase proteins (APPs) are regulated in response to inflammation, infection, trauma, and neoplasia in many domestic and nondomestic species. The APP response is species specific; thus, assays must be validated, and reference intervals must be determined for each species. Koalas (Phascolarctos cinereus) are a vulnerable species, threatened by infectious and inflammatory diseases both under human care and in the wild. The ability to diagnose, treat, and provide prognosis for common koala health problems is challenged by the paucity of sensitive diagnostic tests. Assays for C-reactive protein, serum amyloid A, and haptoglobin were validated for use in koalas. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology based on serum samples from 26 healthy koalas at the San Diego Zoo. The reference intervals are as follows: C-reactive protein, 3.2-24.1 mg/L; serum amyloid A, 0.10-0.45 mg/L; haptoglobin, 0.10-0.64 mg/ml.

REFERENCE INTERVALS FOR ERYTHROCYTE SEDIMENTATION RATE, LACTATE, FIBRINOGEN, HEMATOLOGY, AND PLASMA PROTEIN ELECTROPHORESIS IN CLINICALLY HEALTHY CAPTIVE GOPHER TORTOISES ( GOPHERUS POLYPHEMUS).

Currently available tests for the diagnosis of inflammatory disease in reptiles are limited and poorly sensitive. However, a number of hematological and plasma biochemical analytes are validated in the diagnosis of inflammation in mammals. The objective of this study was to establish reference intervals for erythrocyte sedimentation rate, lactate, heat-precipitated fibrinogen, hematology, and plasma protein electrophoresis based on total protein by biuret method in 23 clinically healthy, captive gopher tortoises ( Gopherus polyphemus) after successful rehabilitation and to determine differences by age, sex, and season. In order to investigate biological differences, samples were collected in April, July, and November. There were no sex differences in any measured analyte; however, there were significant differences by age and season. Immature animals (<2 kg) had significantly higher total protein, albumin : globulin ratio, pre-albumin, albumin, and α-1 globulin than adults. Tortoises sampled in the spring season had significantly higher total solids (refractometer) and lower eosinophils compared with animals sampled in the summer. Further investigation is required to determine the clinical utility of these analytes in the diagnosis of inflammation in this species.

DIAGNOSTIC PERFORMANCE OF INFLAMMATORY MARKERS IN GOPHER TORTOISES ( GOPHERUS POLYPHEMUS).

The lack of sensitive and specific markers of inflammation poses a diagnostic challenge in sick or injured reptile patients. The objective of this study was to investigate the diagnostic performance of blood analytes associated with inflammation in healthy ( n = 24) and sick ( n = 38) gopher tortoises ( Gopherus polyphemus). Receiver operating characteristic analysis identified the following as the best-performing diagnostic tests: erythrocyte sedimentation rate (area under the curve [AUC] = 0.812; 95% confidence interval [CI] = 0.693, 0.900), absolute mature heterophils (AUC = 0.771; 95% CI = 0.646, 0.869), total leukocytes (AUC = 0.767; 95% CI = 0.642, 0.866), lactate (AUC = 0.766; 95% CI = 0.641, 0.864), and absolute immature heterophils (AUC = 0.755; 95% CI = 0.628, 0.856). These results support the clinical application of additional tools for the diagnosis and monitoring of inflammatory disease in gopher tortoises. Clinicians may consider adding erythrocyte sedimentation rate and lactate to the minimum database for this species.

Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.

Comparison of Serum Protein Electrophoresis Values in Wild and Captive Whooping Cranes ( Grus americana ).

Protein electrophoresis of serum samples from endangered, wild whooping cranes ( Grus americana ) was performed to help assess the health of the only self-sustaining, migratory population in North America. Serum samples from wild adult cranes (n = 22) were taken at Aransas National Wildlife Refuge, Texas, USA during winter. Wild juvenile cranes (n = 26) were sampled at Wood Buffalo National Park, Northwest Territories, Canada, in midsummer. All captive crane samples were acquired from the International Crane Foundation, Baraboo, WI, USA. Captive adult cranes (n = 30) were sampled during annual examinations, and archived serum samples from captive juvenile cranes (n = 19) were selected to match the estimated age of wild juveniles. Wild juveniles had significantly lower concentrations of all protein fractions than wild adults, except for prealbumin and γ globulins. All protein fraction concentrations for wild juveniles were significantly lower compared with captive juveniles, except for prealbumin and γ globulins, which were higher. Wild adults had significantly greater γ globulin concentrations than captive adults. Captive juveniles had significantly lower prealbumin and albumin concentrations and albumin : globulin ratios than captive adults. The higher γ globulin concentrations in wild versus captive cranes are likely because of increased antigenic exposure and immune stimulation. Protein fraction concentrations vary significantly with age and natural history in this species. Reference intervals for serum protein electrophoresis results from captive adult whooping cranes are provided in this study.

Health and Reproductive Assessment of Selected Puerto Rican Parrots ( Amazona vittata ) in Captivity.

The Puerto Rican parrot ( Amazona vittata ) has become an iconic and high-profile conservation species. The cornerstone of the recovery plan for this critically endangered species is an active captive breeding program, management of the wild population, and a long-term reintroduction program. In 2002, 40 adult Puerto Rican parrots that had not produced viable offspring were selected for reproductive assessment at 2 aviary populations in Puerto Rico (Iguaca and Río Abajo), which are the only sources of parrots for release. The goal was to enhance reproductive potential and produce productive pairings in an attempt to augment the population growth and provide ample individuals for reintroduction. Seven Hispanolian Amazon parrots ( Amazona ventralis ) that were used as surrogate parents for the Puerto Rican parrots were also included in the study. This assessment included physical examination, endoscopic evaluation, hematologic and plasma biochemical profiles, viral screening, and hormonal assays. Results of general physical examination and hematologic and plasma biochemical testing revealed overall good health and condition of this subset of the population of Puerto Rican parrots; no major infectious diseases were found. Endoscopic examination also revealed overall good health and condition, especially of females. The apparent low fertility of male birds warrants further investigation. The findings helped to define causes of reproductive failure in the selected pairs and individual birds. New pairings resulting from the assessment helped to augment reproduction of this critically endangered species.

Assessment of hemoglobin binding protein in loggerhead sea turtles (Caretta caretta) undergoing rehabilitation.

The acute phase response is an important component of the early reaction of the immune system to insults including infection, inflammation, trauma, neoplasia, and stress. Acute phase proteins are valuable prognostic indicators in many mammalian species but have been poorly studied in reptiles thus far. This study examined 18 paired samples from loggerhead sea turtles (Caretta caretta) for changes observed during the rehabilitation period. Analyses performed included packed cell volume (PCV), hemoglobin binding protein, and plasma protein electrophoresis. Significant differences were observed in all of the protein electrophoresis values. Notably, the concentration of hemoglobin binding protein (as determined by a haptoglobin assay) increased in conjunction with rising total protein (by refractometry) and PCV. The results indicate that this assay may have the potential to be a useful tool in assessing the health of sea turtles.

Reference intervals, longitudinal analyses, and index of individuality of commonly measured laboratory variables in captive bald eagles (Haliaeetus leucocephalus).

The objectives of this study were to determine reference intervals, perform longitudinal analyses, and determine the index of individuality (IoI) of 8 hematologic, and 13 biochemical and electrophoretic variables for a group of captive bald eagles (Haliaeetus leucocephalus). Reference intervals were determined from blood samples collected during annual wellness examinations for 41 eagles (23 male and 18 female) with ages ranging between 6 and 43 years (18.7 +/- 7.4, mean +/- SD) at the time of sample collection. Longitudinal analyses and IoI were determined for measured hematologic, biochemical, and protein electrophoretic variables, both individually and as a group, for a subset of 16 eagles (10 male and 6 female) during a 12-year period. This smaller group of eagles ranged in age between 2 and 20 years at the start of the study period, and between 14 and 32 years (21.9 +/- 5.0, mean +/- SD) at the end of the study period. Significant increases with age within the group of 16 eagles were observed only for red blood cells, percent heterophils, total protein, and beta-globulin protein fraction, while albumin:globulin decreased significantly with age. A low IoI (> or = 1.4) was determined for all hematologic and biochemical variables except gamma globulins, which had high IoI (< or = 0.6) for 3 individuals within the subset of 16.

Monoclonal hyperglobulinemia in lymphosarcoma in a cockatiel (Nymphicus hollandicus) and a blue and gold macaw (Ara ararauna).

A 15-yr-old cockatiel (Nymphicus hollandicus) presented with decreased activity and appetite. A presumptive diagnosis of lymphoproliferative disease was made because of a monoclonal band observed on plasma protein electrophoresis. The owner declined further diagnostics or treatment, and the bird died 2 yr later. In a second case, a 4-yr-old blue and gold macaw (Ara ararauna) presented with lethargy and anorexia. Despite supportive treatment, the bird died 7 days later. Blood studies also demonstrated a monoclonal band on plasma protein electrophoresis. The histologic review of tissues from both birds found extensive neoplastic lymphoplasmocytic cells in the liver and supported a diagnosis of lymphosarcoma.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive Chlamydiaceae antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and coastal waters of Charleston (CHS), South Carolina, USA, were tested for antibodies to Chlamydiaceae as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables was evaluated in Chlamydiaceae-seropositive dolphins (n = 43) and seronegative healthy dolphins (n = 83). Fibrinogen, lactate dehydrogenase, amylase, and absolute numbers of neutrophils, lymphocytes, and basophils were significantly higher, and serum bicarbonate, total alpha globulin, and alpha-2 globulin were significantly lower in dolphins with positive Chlamydiaceae titers compared with seronegative healthy dolphins. Several differences in markers of innate and adaptive immunity were also found. Concanavalin A-induced T lymphocyte proliferation, lipopolysaccharide-induced B lymphocyte proliferation, and granulocytic phagocytosis were significantly lower, and absolute numbers of mature CD 21 B lymphocytes, natural killer cell activity and lysozyme concentration were significantly higher in dolphins with positive Chlamydiaceae antibody titers compared to seronegative healthy dolphins. Additionally, dolphins with positive Chlamydiaceae antibody titers had significant increases in ELISA antibody titers to Erysipelothrix rhusiopathiae. These data suggest that Chlamydiaceae infection may produce subclinical clinicoimmunopathologic perturbations that impact health. Any potential subclinical health impacts are important for the IRL and CHS dolphin populations, as past studies have indicated that both dolphin populations are affected by other complex infectious and neoplastic diseases, often associated with immunologic perturbations and anthropogenic contaminants.

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.

Lipid Panel Reference Intervals for Amazon Parrots (Amazona species).

The lipoprotein panel is a useful diagnostic tool that allows clinicians to evaluate blood lipoprotein fractions. It is a standard diagnostic test in human medicine but is poorly understood in avian medicine. Amazon parrots (Amazona species) are popular pets that frequently lead a sedentary lifestyle and are customarily fed high-fat diets. Similar to people with comparable diets and lifestyles, Amazon parrots are prone to obesity and atherosclerosis. In human medicine, these conditions are typically correlated with abnormalities in the lipoprotein panel. To establish reference intervals for the lipoprotein panel in Amazon parrots, plasma samples from 31 captive Amazon parrots were analyzed for concentrations of cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The data were also grouped according to sex, diet, body condition score, and age. Aside from HDL levels, which were significantly different between male and female parrots, no intergroup differences were found for any of the lipoprotein fractions.

Diagnosis and treatment of diffuse large B-cell lymphoma in an orangutan (Pongo pygmaeus).

Lymphoma is a common malignancy observed in companion animals. This type of naturally occurring neoplasia has been uncommonly reported in great apes. Diffuse large B-cell lymphoma was diagnosed in an 8-yr-old captive orangutan (Pongo pygmaeus) with gastrointestinal disease by histologic and immunohistochemical methodologies. The orangutan was treated with three cycles of combination chemotherapy (intravenous Rituxan, cyclophosphamide, doxorubicin, and vincristine). The primate has been in good health and exhibiting normal behaviors for more than 15 mo following treatment.

Use of an indirect sampling method to produce reference intervals for hematologic and biochemical analyses in psittaciform species.

As with other animal species, comprehensive reference intervals (RI) for psittaciform species are rare and plagued by common issues, including sparse information regarding methods used to analyze specimens, low sample sizes, and improper statistical analyses. The purpose of this study was to examine the use of an indirect sampling method of RI generation from several years of data collected from specimens of multiple psittaciform species submitted to a veterinary diagnostic laboratory. These data were unselected for health status. A previously published method for indirect RI generation was applied to data collected for routine hematologic and biochemical analyses. Seven species groups were examined, and sample size ranged from 346 to 2358. Results showed that RI varied by species and appeared to represent a broader range than expected compared with other RI and traditional clinical expectations for core health assessments, such as total white blood cell count and white blood cell differential results. Some biochemical results reflected more narrow ranges, and a few were consistent with other published ranges. The intervals were likely influenced by changes related to stress and underlying disease. The results of the current study reflect the imprecision of this method related to data obtained from the population served by this laboratory. Overall, this method is not suitable for the production of comprehensive RI, although it may provide rough estimates for some limited analyses until traditional RI can be generated.

Acute phase proteins in animals.

Acute phase proteins (APP) were first identified in the early 1900s as early reactants to infectious disease. They are now understood to be an integral part of the acute phase response (APR) which is the cornerstone of innate immunity. APP have been shown to be valuable biomarkers as increases can occur with inflammation, infection, neoplasia, stress, and trauma. All animals--from fish to mammals--have demonstrable APP, but the type of major APP differs by species. While the primary application of these proteins in a clinical setting is prognostication, studies in animals have demonstrated relevance to diagnosis and detection and monitoring for subclinical disease. APP have been well documented in laboratory, companion, and large animals. With the advent of standardized and automated assays, these biomarkers are available for use in all fields of veterinary medicine as well as basic and clinical research.

Differences in protein fractions of avian plasma among three commercial electrophoresis systems.

Previous studies have defined the presence of 6 protein fractions in plasma from many psittaciform species. Additionally, extensive reference intervals have been published for many of these species with the Beckman Paragon electrophoresis system, which had been commonly used in clinical laboratories to analyze the protein fractions of avian plasma. In mid-2009, Beckman discontinued the Paragon product line, leaving 2 primary alternative systems: Helena and Sebia. To compare electrophoresis results from the 3 commercial protein electrophoresis systems, specimens from 40 African grey parrots (Psittacus erithacus) were analyzed with the electrophoresis systems from Beckman, Helena, and Sebia. Marked differences in fraction migration were found between the Beckman/Helena and Sebia systems, which manifested as a large decrease in prealbumin and an increase in alpha1 globulins in the latter system. Both proportional and constant errors were observed among the fraction quantitation data of both the Helena and Sebia systems compared with the Beckman system. Based on Bland-Altman plot data and imprecision studies, the Helena system appears more similar with the Beckman system, although neither the Helena nor the Sebia systems are identical to the Beckman system. Because of the differences in electrophoresis methods, clinicians should be careful to consistently use particular clinical laboratories. For best application, reference intervals should be established based on both species and electrophoresis system.