Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

Evaluation of a novel quantitative canine species-specific point-of-care assay for C-reactive protein.

BACKGROUND: Species-specific point-of-care tests (POCT) permit a rapid analysis of canine C-reactive protein (CRP), enabling veterinarians to include CRP in clinical decisions. Aim of the study was to evaluate a novel POCT for canine CRP (Point Strip™ Canine CRP Assay) run on a small in-house-analyzer (Point Reader™ V) using lithium heparin plasma and to compare assay performance to an already established canine CRP assay (Gentian Canine CRP Immunoassay) run on two different bench top analyzers serving as reference methods (ABX Pentra 400, AU 5800). Linearity was assessed by stepwise dilution of plasma samples with high CRP concentrations. Limit of quantification (LoQ) was determined by repeated measurements of samples with low CRP concentrations. Coefficient of variation (CV) at low (10-50 mg/l), moderate (50-100 mg/l), and high (100-200 mg/l) CRP concentrations was investigated as well as possible interferences. Method comparison study was performed using 45 samples of healthy and diseased dogs. Quality criteria were fulfilled if the total observed error (TEobs = 2CV% + bias%) was below the minimal total allowable error of 44.4% (TE min). Additionally, a reference range (n = 60 healthy dogs) was established. RESULTS: Linearity was present at CRP concentrations of 10-132 mg/l (≙ 361 mg/l CRP with reference method) with a LoQ set at 10 mg/l. At moderate to high CRP concentrations, intra- and inter-assay CVs were ≤ 8% and ≤ 11% respectively, while CVs ≤ 22% and ≤ 28% were present at low concentrations. No interferences were observed at concentrations of 4 g/l hemoglobin, 800 mg/l bilirubin and 8 g/l triglycerides. Method comparison study demonstrated an excellent correlation with both reference methods (r = 0.98 for ABX Pentra 400; 0.99 for AU 5800), though revealing a proportional bias of 19.7% (ABX Pentra 400) and 10.7% (AU 5800) respectively. TEobs was 26.7-31.9% and 16.7-21.9% and thus < TEmin. Healthy dogs presented with CRP values ≤11.9 mg/l. CONCLUSIONS: The POCT precisely detects canine CRP at clinically relevant moderate and high CRP concentrations. The assay correlates well with both reference methods. Due to the bias, however, follow-up examinations should be performed with the same assay and analyzer.

Method Validation and Establishment of Reference Intervals for an Insulin-like Growth Factor-1 Chemiluminescent Immunoassay in Cats.

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats.

Oxidative damage of canine erythrocytes after treatment with non-steroidal anti-inflammatory drugs.

OBJECTIVE: To investigate oxidative erythrocyte damage in dogs treated with different non-steroidal anti-inflammatory drugs. MATERIAL AND METHODS: Case-controlled prospective observational study using blood obtained from dogs presenting for lameness examinations or standard surgical procedures to a private referral clinic. Sampling was performed from April 2018 to July 2019. Groups comprised dogs receiving either metamizole (dipyrone) (22 dogs), carprofen (20 dogs) or meloxicam (20 dogs) for a minimum of 10 days. Dogs with gastrointestinal hemorrhage were excluded from the study. A complete hematological, as well as a basic biochemical profile were performed in every dog. Pappenheim stained blood smears were evaluated for eccentrocytes and brilliant cresyl blue stained smears for Heinz bodies. EDTA blood was frozen at -80°C immediately after sampling for measurement of superoxide dismutase and gluthathione peroxidase activity at an external laboratory. Hemoglobin concentration, superoxide dismutase and gluthathione peroxidase activities, reticulocyte count, eccentrocyte and Heinz body numbers were determined prospectively as key parameters for further statistical assessment with Kruskal-Wallis test and Dunn's multiple comparisons test. RESULTS: Dogs receiving metamizole showed a significant increase in eccentrocyte (median 14.5/500 cells vs. 0/500 cells in the other groups, p < 0.0001) and reticulocyte number (median 191.4 × 109/l vs. 31.6-37.9 × 109/l, p < 0.0001) and a significant decrease in hemoglobin concentration (median 8.4 mmol/l vs. 10.1-10.5 mmol/l, p < 0.0003). No significant difference in superoxide dismutase and gluthathione peroxidase activities was observed between dogs receiving metamizole and the other groups. Heinz bodies were not found in any of the dogs. CONCLUSION: Treatment with metamizole for 10 or more days resulted in decreased hemoglobin concentration, eccentrocytosis and reticulocytosis in dogs in this study. This might be a sign of increased oxidative damage caused by this drug. CLINICAL SIGNIFICANCE: Prolonged metamizole therapy should be evaluated critically in patients already affected by severe illness or underlying anaemia.

Nonregenerative immune-mediated anemia associated with a diffuse large B-cell lymphoma in a captive jaguar (Panthera onca).

An 18-year-old male castrated jaguar (Panthera onca) was presented with anorexia and continuous bleeding from the oral cavity after a history of fighting with the partner animal. Clinical evaluation revealed ulcerating lesions on the gingiva and hard palate and a hematoma on the tongue. Computed tomography of the head and endoscopic examination of the esophagus and stomach were unremarkable. Hematology and clinical chemistry revealed severe nonregenerative anemia, mild thrombocytopenia, and moderate azotemia. Several PCRs for feline hemotropic mycoplasmas (Mycoplasma haemofelis, M heamominutium, M turicensis), Babesia felis, and Bartonella spp., as well as an FeLV antigen test were negative. The cytologic examination of a bone marrow aspirate was consistent with ineffective erythropoiesis, most likely due to immune-mediated destruction of the erythroid precursor cells. Prednisolone therapy was initiated (1.25 mg/kg/day), and the CBC returned to normal 16 days after the initiation of the therapy. Anemia relapsed after 4 months and severe splenomegaly was noted. A repeat bone marrow aspirate revealed active erythropoiesis in the presence of erythroid precursor phagocytosis suggesting an immune-mediated process. Splenic fine-needle aspiration and tissue biopsies were taken, and all findings including histology and immunohistochemistry were consistent with a diffuse large B-cell lymphoma (DLBCL). Five days later, the clinical condition deteriorated and the jaguar died. Histopathology following necropsy showed infiltration with neoplastic lymphoblasts in the spleen, liver, and abdominal lymph nodes. This case report describes a nonregenerative immune-mediated anemia associated with a DLBCL in a jaguar.

Feline paraneoplastic alopecia associated with metastasising intestinal carcinoma.

CASE SUMMARY: A 10-year-old male neutered British Shorthair cat was presented with a 6 month history of lethargy, weight loss and alopecia. Clinical examination revealed widespread alopecia of the ventral abdomen and hindlimbs. The skin in these areas was smooth and shiny and hairs could be easily epilated. Spontaneous pruritus was observed. Cytological examination of superficial impression smears showed a severe Malassezia species dermatitis and pyoderma. Ectoparasites could not be detected and no sign of dermatophytosis was visible in trichograms and Wood's lamp analysis. Abdominal ultrasound found a focally thickened wall of the large intestine and multiple nodules in the liver. Fine-needle aspirates from lymph nodes, liver and altered colonic wall were consistent with an undifferentiated malignant neoplasia. The cat was euthanased at the owners' request, owing to potential neoplasia with metastatic spread. At necropsy a metastasising carcinoma of the colonic wall was found, as well as a paraneoplastic alopecia. RELEVANCE AND NOVEL INFORMATION: Feline paraneoplastic alopecia has been reported in association with pancreatic carcinoma, bile duct carcinoma and hepatocellular carcinoma, as well as with neuroendocrine pancreatic carcinoma and hepatosplenic plasma cell tumour. This is the first reported case of feline paraneoplastic alopecia associated with a colon carcinoma.

The speed of kill of fluralaner (Bravecto™) against Ixodes ricinus ticks on dogs.

BACKGROUND: Pathogens that are transmitted by ticks to dogs, such as Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi sensu latu, and Ehrlichia canis, are an increasing problem in the world. One method to prevent pathogen transmission to dogs is to kill the ticks before transmission occurs. Fluralaner (Bravecto™) is a novel isoxazoline insecticide and acaricide that provides long persistent antiparasitic activity following systemic administration. This study investigated the speed of kill of fluralaner against Ixodes ricinus ticks on dogs. METHODS: A total of 48 dogs were randomized to 8 groups of 6 dogs and each dog was infested with 50 female and 10 male I. ricinus ticks. Two days later (day 0), 4 groups received a single treatment of 25 mg fluralaner/kg body weight as Bravecto™ chewable tablets; the dogs in the other 4 groups were left untreated. Separate control and treatment groups were paired at each time point (4, 8, 12, or 24 hours after treatment) for assessment of tick-killing efficacy. At 4, 8, and 12 weeks after treatment, all dogs were re-infested with 50 female I. ricinus ticks and subsequently assessed for live or dead ticks at either 4, 8, 12, or 24 hours after re-infestation. Efficacy was calculated for each assessment time point by comparison of the treatment group with the respective control group. RESULTS: Tick-killing efficacy was 89.6% at 4 hours, 97.9% at 8 hours, and 100% at 12 and 24 hours after treatment. Eight hours after re-infestation, efficacy was 96.8%, 83.5%, and 45.8% at 4, 8, and 12 weeks after treatment, respectively. At least 98.1% tick-killing efficacy was demonstrated 12 and 24 hours after re-infestation over the entire 12 week study period. CONCLUSIONS: Fluralaner kills ticks rapidly after treatment at 4 hours, and over its entire 12-week period of efficacy, it achieves an almost complete killing effect within 12 hours after tick infestation. The rapid tick-killing effect together with the long duration of efficacy enables fluralaner to aid in the prevention of tick borne diseases.