Cohort profile: The Golden Retriever Lifetime Study (GRLS).

The aim of this article is to provide a detailed description of the Golden Retriever Lifetime Study (GRLS), a prospective cohort study investigating nutritional, environmental, lifestyle, and genetic risk factors for cancer and other common diseases in dogs. Primary outcomes of interest include hemangiosarcoma, lymphoma, osteosarcoma, and high-grade mast cell tumors. Secondary outcomes of interest include other cancers, hypothyroidism, epilepsy, atopy, otitis externa, hip dysplasia, heart failure, and renal failure. A total of 3,044 United States Golden Retrievers aged 6 months to 2 years completed baseline enrollment from June 2012 to April 2015. As of May 31, 2021, 2,251 dogs remain engaged in the study, 352 have died, and 441 are lost to follow-up. Extensive annual questionnaires completed by owners and veterinarians gather information about lifestyle, environmental exposures, physical activity, reproductive history, behavior, diet, medications, and diagnoses. Dogs also have annual veterinary examinations and biospecimen collection (blood, serum, hair, nails, feces, urine) for biobanking. Additional reporting, including histology and tumor biobanking, is conducted for any malignancies or deaths. When an animal dies, full medical records are obtained, and necropsies are requested at owner discretion. Full or partial necropsies have been performed on 218 dogs. Questionnaire data are freely available to researchers with approved credentials who agree to a data use agreement. In addition, researchers can submit proposals to utilize biospecimens or obtain additional data.

Novel protective role for MAP kinase phosphatase 2 in inflammatory arthritis.

Objectives: We have previously shown mitogen-activated protein kinase phosphatase 2 (MKP-2) to be a key regulator of proinflammatory cytokines in macrophages. In the study presented here, we investigated the role of MKP-2 in inflammatory arthritis with a particular focus on neutrophils. Methods: To achieve this, we subjected MKP-2 deficient and wild type mice to collagen antibody induced arthritis, an innate model of arthritis, and determined disease pathology. To further our investigation, we depleted neutrophils in a prophylactic and therapeutic fashion. Last, we used chemotaxis assays to analyse the impact of MKP-2 deletion on neutrophil migration. Results: MKP-2-/- mice showed a significant increase in disease pathology linked to elevated levels of proarthritic cytokines and chemokines TNF-α, IL-6 and MCP-1 in comparison to wild type controls. This phenotype is prevented or abolished after administration of neutrophil depleting antibody prior or after onset of disease, respectively. While MCP-1 levels were not affected, neutrophil depletion diminished TNF-α and reduced IL-6, thus linking these cytokines to neutrophils. In vivo imaging showed that MKP-2-/- mice had an increased influx of neutrophils into affected joints, which was higher and potentially prolonged than in wild type animals. Furthermore, using chemotaxis assays we revealed that MKP-2 deficient neutrophils migrate faster towards a Leukotriene B4 gradient. This process correlated with a reduced phosphorylation of ERK in MKP-2-/- neutrophils. Conclusions: This is the first study to show a protective role for MKP-2 in inflammatory arthritis.

ABIN2 Function Is Required To Suppress DSS-Induced Colitis by a Tpl2-Independent Mechanism.

The A20-binding inhibitor of NF-κB 2 (ABIN2) interacts with Met1-linked ubiquitin chains and is an integral component of the tumor progression locus 2 (Tpl2) kinase complex. We generated a knock-in mouse expressing the ubiquitin-binding-defective mutant ABIN2[D310N]. The expression of Tpl2 and its activation by TLR agonists in macrophages or by IL-1ß in fibroblasts from these mice was unimpaired, indicating that the interaction of ABIN2 with ubiquitin oligomers is not required for the stability or activation of Tpl2. The ABIN2[D310N] mice displayed intestinal inflammation and hypersensitivity to dextran sodium sulfate-induced colitis, an effect that was mediated by radiation-resistant cells rather than by hematopioetic cells. The IL-1ß-dependent induction of cyclooxygenase 2 (COX2) and the secretion of PGE2 was reduced in mouse embryonic fibroblasts and intestinal myofibroblasts (IMFs) from ABIN2[D310N] mice. These observations are similar to those reported for the Tpl2 knockout (KO) mice (Roulis et al. 2014. Proc. Natl. Acad. Sci. USA 111: E4658-E4667), but the IL-1ß-dependent production of COX2 and PGE2 in mouse embryonic fibroblasts or IMFs was unaffected by pharmacological inhibition of Tpl2 in wild-type mice. The expression of ABIN2 is decreased drastically in Tpl2 KO mice. These and other lines of evidence suggest that the hypersensitivity of Tpl2 KO mice to dextran sodium sulfate-induced colitis is not caused by the loss of Tpl2 catalytic activity but by the loss of ABIN2, which impairs COX2 and PGE2 production in IMFs by a Tpl2 kinase-independent pathway.

PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters.

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death globally, and new immunotherapies developed and under development targeting PD-1/PD-L1 checkpoint inhibition require accurate patient selection to assure good clinical outcome. PD-L1 immunohistochemistry is the current biomarker assay used for patient selection, but still imprecise in predicting therapy response. Exploring this issue, we performed computational tissue analysis of PD-L1 immunostaining in procured NSCLC tissues (n = 50) using the Merck KGaA anti-PD-L1 clone MKP1A07310. Staining patterns and PD-L1 cut-off points were interrogated using relevant cancer immune-surveillance biomarkers. Groups with high PD-L1 expression levels (above 25/50% staining cut-off points) were enriched for a biomarker profile in the tumor-nest and microenvironment indicating escape from host-immunity, as represented by increased numbers of cells positive for CD8 and Granzyme B (immune-effectors), FOXP3 (immune-suppressive), and CD68 (P < 0.05). Manual analysis of PD-L1 staining patterns identified tumors with an immune-induced reactive pattern relevant for immunotherapy that would ordinarily be excluded by the arbitrary 25% staining threshold (P < 0.05). Conversely, some cases with completely or predominantly immune-independent constitutive PD-L1 staining patterns that indicate insensitivity to immunotherapy may have been incorrectly selected using this staining cut-off point criterion. Therefore, we propose differentiation of reactive vs constitutive PD-L1 staining patterns to improve the accuracy of this biomarker assay in selecting NSCLC patients for PD-1/PD-L1 immunotherapy.

Duchenne and Becker Muscular Dystrophies: A Review of Animal Models, Clinical End Points, and Biomarker Quantification.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular disorders that primarily affect boys due to an X-linked mutation in the DMD gene, resulting in reduced to near absence of dystrophin or expression of truncated forms of dystrophin. Some newer therapeutic interventions aim to increase sarcolemmal dystrophin expression, and accurate dystrophin quantification is critical for demonstrating pharmacodynamic relationships in preclinical studies and clinical trials. Current challenges with measuring dystrophin include the variation in protein expression within individual muscle fibers and across whole muscle samples, the presence of preexisting dystrophin-positive revertant fibers, and trace amounts of residual dystrophin. Immunofluorescence quantification of dystrophin can overcome many of these challenges, but manual quantification of protein expression may be complicated by variations in the collection of images, reproducible scoring of fluorescent intensity, and bias introduced by manual scoring of typically only a few high-power fields. This review highlights the pathology of DMD and BMD, discusses animal models of DMD and BMD, and describes dystrophin biomarker quantitation in DMD and BMD, with several image analysis approaches, including a new automated method that evaluates protein expression of individual muscle fibers.

Assessment of murine collagen-induced arthritis by longitudinal non-invasive duplexed molecular optical imaging.

OBJECTIVE: In the present study we evaluated the use of four commercially available fluorescent probes to monitor disease activity in murine CIA and its suppression during glucocorticoid therapy. METHODS: Arthritis was induced in male DBA/1 mice by immunization with type II collagen in Complete Freund's Adjuvant, followed by a boost of collagen in PBS. Four fluorescent probes from PerkinElmer in combination [ProSense 750 fluorescent activatable sensor technology (FAST) with Neutrophil Elastase 680 FAST and MMPSense 750 FAST with CatK 680 FAST] were used to monitor disease development from day 5 through to day 40 post-immunization. Fluorescence generated in vivo by the probes was correlated with clinical and histological score and paw measurements. RESULTS: The fluorescence intensity emitted by each probe was shown to correlate with the conventional measurements of disease. The highest degree of correlation was observed with ProSense 750 FAST in combination with Neutrophil Elastase 680 FAST; these probes were then used to successfully assess CIA suppression during dexamethasone treatment. CONCLUSION: We have demonstrated that longitudinal non-invasive duplexed optical fluorescence imaging provides a simple assessment of arthritic disease activity within the joints of mice following the induction of CIA and may represent a powerful tool to monitor the efficacy of drug treatments in preclinical studies.

Achilles tendon injuries in elite athletes: lessons in pathophysiology from their equine counterparts.

Superficial digital flexor tendon (SDFT) injury in equine athletes is one of the most well-accepted, scientifically supported companion animal models of human disease (i.e., exercise-induced Achilles tendon [AT] injury). The SDFT and AT are functionally and clinically equivalent (and important) energy-storing structures for which no equally appropriate rodent, rabbit, or other analogues exist. Access to equine tissues has facilitated significant advances in knowledge of tendon maturation and aging, determination of specific exercise effects (including early life), and definition of some of the earliest stages of subclinical pathology. Access to human surgical biopsies has provided complementary information on more advanced phases of disease. Importantly, equine SDFT injuries are only a model for acute ruptures in athletes, not the entire spectrum of human tendonopathy (including chronic tendon pain). In both, pathology begins with a potentially prolonged phase of accumulation of (subclinical) microdamage. Recent work has revealed remarkably similar genetic risk factors, including further evidence that tenocyte dysfunction plays an active role. Mice are convenient but not necessarily accurate models for multiple diseases, particularly at the cellular level. Mechanistic studies, including tendon cell responses to combinations of exercise-associated stresses, require a more thorough investigation of cross-species conservation of key stress pathway auditors. Molecular evidence has provided some context for the poor performance of mouse models; equines may provide better systems at this level. The use of horses may be additionally justifiable based on comparable species longevity, lifestyle factors, and selection pressure by similar infectious agents (e.g., herpesviruses) on general cell stress pathway evolution.

Morphological and cellular changes in secondary epidermal laminae of horses with insulin-induced laminitis.

OBJECTIVE: To determine cellular changes associated with secondary epidermal laminae (SEL) in forefeet and hind feet of ponies with insulin-induced laminitis. ANIMALS: 8 ponies. PROCEDURES: Laminitis was induced in 4 ponies by IV administration of insulin and glucose; 4 control ponies received saline (0.9% NaCl) solution IV. Laminar tissue samples obtained from the dorsal aspects of the hooves were histologically evaluated. Primary epidermal lamina (PEL) length and width and SEL length, width, and angle were determined. Numbers of epidermal cell nuclei per micrometer and per total length of SEL and numbers of apoptotic and proliferative cells in axial, middle, and abaxial laminar regions were determined. RESULTS: SEL in treatment group ponies were significantly longer, were significantly narrower, and had a smaller angle relative to PEL in all laminar regions versus control ponies. In treatment group ponies, the number of epidermal cell nuclei per SEL was typically higher and the number of cells per micrometer of SEL was lower in laminar regions, apoptotic cell numbers were higher in abaxial and middle regions in forefeet and hind feet, and proliferating cell numbers were higher in axial laminar regions in forefeet and all laminar regions in hind feet, versus control ponies. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated SEL elongation, narrowing, and alteration in orientation developed in all feet of ponies with insulin-induced laminitis. This was primarily attributable to cell stretching that developed at the same time as an accelerated cell death-proliferation cycle; differences in cell cycle responses among laminar regions between forefeet and hind feet may have been attributable to differences in load bearing.

Pituitary metastasis of pancreatic origin in a dog presenting with acute-onset blindness.

Pituitary metastases have rarely been recorded in dogs, and to date, none of those reported have been of pancreatic origin. MRI findings are available for only one of those cases. Herein the authors present an 11 yr old English springer spaniel diagnosed with pituitary metastasis of pancreatic origin with a 24 hr history of blindness and only a single lesion on MRI. Neurologic and ophthalmologic examinations localized the lesion to the optic nerves, optic tracts, or optic chiasm. MRI showed a single lesion characterized by a well-circumscribed pituitary mass with extrasellar extension, causing compression of the optic chiasm. Signal intensity was unusual as enhancement could not be appreciated after contrast administration. The dog was euthanized without further diagnostic tests. Histopathologic examination revealed a poorly differentiated exocrine pancreatic carcinoma with widespread metastasis involving the pituitary gland. To the authors' knowledge, this is the first such case reported in a dog. Pituitary metastases should be included as a differential diagnosis for dogs presenting with acute-onset blindness and for single brain masses affecting the pituitary gland.

Indicators of replicative damage in equine tendon fibroblast monolayers.

BACKGROUND: Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. RESULTS: We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. CONCLUSIONS: Researchers using tendon fibroblast monolayers should assess their "health" using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.

PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development.

Phosphoinositide-dependent kinase-1 (PDK1) controls the activation of a subset of AGC kinases. Using a conditional knockout of PDK1 in haematopoietic cells, we demonstrate that PDK1 is essential for B cell development. B-cell progenitors lacking PDK1 arrested at the transition of pro-B to pre-B cells, due to a cell autonomous defect. Loss of PDK1 decreased the expression of the IgH chain in pro-B cells due to impaired recombination of the IgH distal variable segments, a process coordinated by the transcription factor Pax5. The expression of Pax5 in pre-B cells was decreased in PDK1 knockouts, which correlated with reduced expression of the Pax5 target genes IRF4, IRF8 and Aiolos. As a result, Ccnd3 is upregulated in PDK1 knockout pre-B cells and they have an impaired ability to undergo cell-cycle arrest, a necessary event for Ig light chain rearrangement. Instead, these cells underwent apoptosis that correlated with diminished expression of the pro-survival gene Bcl2A1. Reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pro-B cells restored their ability to differentiate in vitro into mature B cells.

Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses.

Lamellar pathology in experimentally-induced equine laminitis associated with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (∼48h post-induction). However, lamellar pathology of the developmental, pre-clinical phase requires evaluation. The aim of this study was to analyse lamellar lesions both qualitatively and quantitatively, 6, 12 and 24h after the commencement of hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar pathology at each time-point included assessment of lamellar length and width, epidermal cell proliferation and death, basement membrane (BM) pathology and leucocyte infiltration. Archived lamellar tissue from control horses and those with acute, insulin-induced laminitis (48h) was also assessed for cellular proliferative activity by counting the number of cells showing positive nuclear immuno labelling for TPX2. Decreased secondary epidermal lamellar (SEL) width and increased histomorphological evidence of SEL epidermal basal (and supra-basal) cell death occurred early in disease progression (6h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6-48h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

Pellino1 is required for interferon production by viral double-stranded RNA.

Viral double-stranded RNA, a ligand for Toll-like Receptor 3 (TLR3) and the cytoplasmic RNA receptors RIG1 and MDA5, activate a signaling network in which the IKK-related protein kinase TBK1 phosphorylates the transcription factor Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1. IRF3 then translocates to the nucleus where it stimulates transcription of the interferonß (IFNß) gene, but the function of Pellino1 in this pathway is unknown. Here, we report that myeloid cells and embryonic fibroblasts from knock-in mice expressing an E3 ligase-deficient mutant of Pellino1 produce reduced levels of IFNß mRNA and secrete much less IFNß in response to viral double-stranded RNA because the interaction of IRF3 with the IFNß promoter is impaired. These results identify Pellino1 as a novel component of the signal transduction network by which viral double-stranded RNA stimulates IFNß gene transcription.

Disseminated lipid-rich peritoneal mesothelioma in a horse.

A 9-year-old Haflinger mare presented to the Liphook Equine Hospital with a history of weight loss, azotemia, and repeated episodes of ascites over a period of 10 days. The horse was euthanized after exploratory laparotomy revealed large numbers of variably sized masses distributed throughout the peritoneal cavity. Macroscopically, some masses were papillary, while others were nodular. Histologically, the masses were comprised of large to giant, variably shaped, and occasionally multinucleated neoplastic cells with marked anisokaryosis and anisocytosis and a high mitotic rate. Small to moderate numbers of neoplastic cells were swollen by 1 to several, moderately sized to large, clear, circular or ovoid vacuoles, which stained positive with oil red O. Immunohistochemically, the neoplastic cells co-expressed vimentin and cytokeratin. Electron microscopy demonstrated tumor cells with tight junctions, microvilli, and numerous intracytoplasmic lipid droplets. These findings are consistent with a lipid-rich form of mesothelioma, which should be considered as a differential diagnosis if lipid vacuoles are present in potentially neoplastic cells in equine abdominocentesis samples.

Determinants of bluetongue virus virulence in murine models of disease.

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.

Biochemical and hematological reference intervals for Krefft's turtles Emydura macquarii krefftii from the Burnett River Catchment, Australia.

Biochemical and hematological reference intervals have not previously been reported for Emydura macquarii krefftii. In 2009, 56 E. m. krefftii were captured by hand from the Burnett Catchment, clinically assessed to determine health status and blood sampled. Reference intervals were calculated from the 35 clinically healthy turtles using techniques established in other chelonid species. Aberrant blood results were identified from the 21 clinically unhealthy turtles. Low numbers of observed cases of creatine kinase, glucose, magnesium, phosphorus and uric acid outside of the blood biochemistry reference interval were recorded, as were high numbers of observed cases of estimated eosinophils, thrombocytes and total leukocyte counts outside of the hematological reference interval. Lesions of the shell and plastron (shell rot) were observed in 38% (21/56) of the examined healthy and unhealthy turtles. Microbiological assessment of a subsample (n=7) of these lesions grew Aeromonas veronii 100% (7/7), Aeromonas hydrophila 29% (2/7) and Acinetobacter baumannii 14% (1/7). Of the examined turtles, 13% (7/56) had evidence of opacity of the lens or anterior chamber of the eye and 70% (39/56) had erythema of the neck, axillary and inguinal soft tissues. Not all observed cases of erythema were associated with clinical ill-health. The anomalous blood results and clinical findings identified in this study suggest disease processes which may have resulted from causative agents in the surrounding environment.

Polyubiquitin binding to ABIN1 is required to prevent autoimmunity.

The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88(-/-) mice, demonstrating that toll-like receptor (TLR)-MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-α/ß, c-Jun N-terminal kinases, and p38α mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR-MyD88 pathways and prevent autoimmunity.

Reference intervals for plasma biochemical and hematologic measures in loggerhead sea turtles (Caretta caretta) from Moreton Bay, Australia.

Biochemical and hematologic reference intervals have been reported for loggerhead sea turtles (Caretta caretta, Linnaeus 1758), but low sample numbers and simple statistical analyses have constrained their diagnostic usefulness. During June 2007-May 2008, 101 loggerhead sea turtles in Moreton Bay, Queensland, Australia, were captured by hand from boats; clinically assessed to determine health status; blood was sampled; and biochemical and hematologic variables were measured. Of these turtles, 66 were classified as clinically healthy and 23 as unhealthy. Reference intervals were calculated using data from clinically healthy turtles. Of the clinically unhealthy turtles, 82 and 45% had at least one biochemical and hematologic result, respectively, outside of at least one of the calculated intervals. However, only low proportions of unhealthy loggerhead sea turtles had abnormal results for each variable. The highest percentage of unhealthy turtles that were outside at least one estimated reference interval was 35%, for thrombocyte counts. Neither sex nor maturity category (mature versus large immature) influenced the risk of being clinically unhealthy. These are the first plasma biochemical and hematologic reference intervals reported for loggerhead sea turtles from the southwestern Pacific Ocean. We conclude that, for loggerhead sea turtles in Moreton Bay, separate reference intervals are required for mature and immature turtles for thrombocyte counts and for male and female turtles for lymphocyte, heterophil, and total white cell counts; otherwise, a single reference interval can be used regardless of age or sex. When estimating reference intervals in loggerhead sea turtles, it is desirable to use both methods for calculating reference intervals used in this study because intervals can differ substantially between methods for some variables. Joint interpretation using reference intervals from both methods allows the categorization of results as "normal," "suspect," or "abnormal."

Health surveillance of stranded green turtles in southern Queensland, Australia (2006-2009): an epidemiological analysis of causes of disease and mortality.

Causes of disease and mortality in marine turtles are frequently based on opportunistic investigations producing results that may not contribute to knowledge on how to protect their survival rate. Over a 4-year period (2006-2009), the major causes of stranding and morbidity in 100 green turtles (Chelonia mydas) from southern Queensland on the east coast of Australia were determined by comprehensive postmortem examination. Lesions were characterized for analysis using descriptive and probability statistics. Spirorchiid parasitism was found to be the most frequently occurring cause of mortality (41.8%), followed by gastrointestinal impaction (11.8%), microbiological infectious diseases (5.2%), and trauma (5.2%). Spirorchiid parasitism with associated inflammation (75%) was the most frequently occurring disease, followed by gastrointestinal impaction (5.1%). All other diseases were observed at a low prevalence. Assessment of the likelihood of disease being influenced by risk factors (season, maturity, and gender) showed that: (i) there were more observed cases of spirorchiid infection in summer when compared with the other seasons (P = 0.029); (ii) immature turtles had more severe spirorchiid parasite infections than mature turtles (P = 0.032); and (iii) respiratory disorders were more likely (P = 0.01) in summer and autumn than winter or spring. Number of observed cases and severity of spirorchiid lesions were highest in the brain compared with other histologically examined organ systems (all P > 0.1). Further investigation is required to build on these findings, aid management decisions, and determine the significance of these diseases for green turtle survivorship in Queensland.

Development and application of biochemical and haematological reference intervals to identify unhealthy green sea turtles (Chelonia mydas).

Biochemical and haematological reference intervals (RIs) have been reported for sea turtles, but their value for ante-mortem disease diagnosis may be limited due to small sample sizes and outdated statistical analyses. In the present study, 290 green sea turtles (Chelonia mydas) were captured, clinically assessed and blood sampled. Of these, 211 were classified as 'clinically healthy' and 25 as 'clinically unhealthy'. RIs were estimated using data from the healthy turtles and compared with blood values from the unhealthy animals. All of the unhealthy animals had plasma biochemical and haematological values outside one or more RIs (albumin, 48% of unhealthy animals; alkaline phosphatase, 35%; aspartate transaminase, 13%; creatinine, 30%; globulin, 3%; glucose, 34%; lactic dehydrogenase, 26%; phosphorus, 22%; sodium, 13%; thrombocytes, 57%; and monocytes, 5%). Among small immature turtles, those with Chelonibia testudinaria plastron barnacle counts 20 were three times more likely to be unhealthy than those with no barnacles. In addition, small immature and mature turtles were more likely to be unhealthy than large immature turtles.