Development and analytical validation of a novel quantitative PCR assay for the detection of Trachemys herpesvirus 1.

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 107 to 1.0 × 101 copies per reaction with an R2 of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 101 copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.

Evaluation of the sedative effects and pharmacokinetics of detomidine gel administered intravaginally to horses.

OBJECTIVES: To determine the sedative effects and pharmacokinetic profile of detomidine when administered intravaginally as a gel formulation to horses. STUDY DESIGN: Randomized, crossover, masked experimental design. ANIMALS: A group of six healthy adult mares (494 ± 56 kg). METHODS: Mares were studied on two occasions and were administered either detomidine hydrochloride (10 µg kg-1) intravenously (treatment IV) or detomidine gel (40 µg kg-1) intravaginally (treatment IVG), separated by 1 week. Sedation, ataxia, muzzle-floor distance and heart rate (HR) were evaluated every 15 minutes for 240 minutes. Venous blood samples were collected at 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 and 360 minutes postadministration and were analyzed for detomidine and metabolites using liquid chromatography-tandem mass spectrometry. Measured variables were compared over time and between treatments using mixed model analysis. Correlation between drug plasma concentrations and muzzle-floor distance, and sedation and ataxia scores was determined using the Spearman correlation coefficient. Data are presented as mean ± standard error of the mean and p value was set at <0.05. RESULTS: Sedation was shorter with IV (119 ± 16 minutes) than with IVG (188 ± 22 minutes). Ataxia scores remained greater than baseline for 90 and 135 minutes for treatments IV and IVG, respectively. HR was lower than baseline for 45 and 30 minutes for IV and IVG, respectively, but did not differ between treatments. The mean maximum plasma concentration of detomidine, time to maximum concentration and bioavailability for treatment IVG was 8.57 ng mL-1, 0.37 hour and 25%, respectively. There was a significant correlation (r = 0.68) between plasma detomidine concentrations and sedation score. CONCLUSIONS AND CLINICAL RELEVANCE: Detomidine gel administered intravaginally resulted in clinically important sedation and is a viable method for detomidine gel delivery in mares.

Effect of fentanyl on the induction dose and minimum infusion rate of alfaxalone preventing movement in dogs.

OBJECTIVE: To determine the effect of fentanyl on the induction dose and minimum infusion rate of alfaxalone required to prevent movement in response to a noxious stimulus (MIRNM) in dogs. STUDY DESIGN: Experimental crossover design. ANIMALS: A group of six healthy, adult, intact female mixed-breed dogs, weighing 19.7 ± 1.3 kg. METHODS: Dogs were randomly administered one of three treatments at weekly intervals: premedication with 0.9% saline (treatment A), fentanyl 5 µg kg-1 (treatment ALF) or fentanyl 10 µg kg-1 (treatment AHF), administered intravenously over 5 minutes. Anesthesia was induced 5 minutes later with incremental doses of alfaxalone to achieve intubation and was maintained for 90 minutes in A with alfaxalone (0.12 mg kg-1 minute-1), in ALF with alfaxalone (0.09 mg kg-1 minute-1) and fentanyl (0.1 µg kg-1 minute-1) and in AHF with alfaxalone (0.06 mg kg-1 minute-1) and fentanyl (0.2 µg kg-1 minute-1). The alfaxalone infusion was increased or decreased by 0.006 mg kg-1 minute-1 based on positive or negative response to antebrachium stimulation (50 V, 50 Hz, 10 ms). Data were analyzed using a mixed-model anova and presented as least squares means ± standard error. RESULTS: Alfaxalone induction doses were 3.50 ± 0.13 (A), 2.17 ± 0.10 (ALF) and 1.67 ± 0.10 mg kg-1 (AHF) and differed among treatments (p < 0.05). Alfaxalone MIRNM was 0.17 ± 0.01 (A), 0.10 ± 0.01 (ALF) and 0.07 ± 0.01 mg kg-1 minute-1 (AHF) and differed among treatments. ALF and AHF decreased the MIRNM by 44 ± 8% and 62 ± 5%, respectively (p < 0.05). Plasma alfaxalone concentrations at MIRNM were 5.82 ± 0.48 (A), 4.40 ± 0.34 (ALF) and 2.28 ± 0.09 µg mL-1 (AHF). CONCLUSIONS AND CLINICAL RELEVANCE: Fentanyl, at the doses studied, significantly decreased the alfaxalone induction dose and MIRNM.

Bias due to Preanalytical Dilution of Rodent Serum for Biochemical Analysis on the Siemens Dimension Xpand Plus.

Clinical pathology testing of rodents is often challenging due to insufficient sample volume. One solution in clinical veterinary and exploratory research environments is dilution of samples prior to analysis. However, published information on the impact of preanalytical sample dilution on rodent biochemical data is incomplete. The objective of this study was to evaluate the effects of preanalytical sample dilution on biochemical analysis of mouse and rat serum samples utilizing the Siemens Dimension Xpand Plus. Rats were obtained from end of study research projects. Mice were obtained from sentinel testing programs. For both, whole blood was collected via terminal cardiocentesis into empty tubes and serum was harvested. Biochemical parameters were measured on fresh and thawed frozen samples run straight and at dilution factors 2-10. Dilutions were performed manually, utilizing either ultrapure water or enzyme diluent per manufacturer recommendations. All diluted samples were generated directly from the undiluted sample. Preanalytical dilution caused clinically unacceptable bias in most analytes at dilution factors four and above. Dilution-induced bias in total calcium, creatinine, total bilirubin, and uric acid was considered unacceptable with any degree of dilution, based on the more conservative of two definitions of acceptability. Dilution often caused electrolyte values to fall below assay range precluding evaluation of bias. Dilution-induced bias occurred in most biochemical parameters to varying degrees and may render dilution unacceptable in the exploratory research and clinical veterinary environments. Additionally, differences between results obtained at different dilution factors may confound statistical comparisons in research settings. Comparison of data obtained at a single dilution factor is highly recommended.

Influence of Genetic Background on Hematologic and Histopathologic Alterations during Acute Granulocytic Anaplasmosis in 129/SvEv and C57BL/6J Mice Lacking Type I and Type II Interferon Signaling.

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

Alterations due to dilution and anticoagulant effects in hematologic analysis of rodent blood samples on the Sysmex XT-2000iV.

BACKGROUND: Clinical pathology of rodents is hindered by sample volume limitations. A single whole heparinized blood sample is often submitted for hematologic and clinical chemistry analysis in exploratory research settings, and sample dilution may be required. Published information on the potential impact of sample dilution and heparin use on hematology variables in rodents is sparse. OBJECTIVES: The purpose of the study was to evaluate the effects of sample dilution and of anticoagulant on hematologic analysis of mouse and rat blood samples on the Sysmex XT-2000iV. METHODS: Mice and rats were obtained from various ends of study research projects, and whole blood was collected via terminal cardiocentesis in lithium heparin, and additionally in EDTA when paired samples were obtained from rats. Hematology analytes were measured on the Sysmex XT-2000iV straight and diluted from ×2 to ×5. RESULTS: Significant differences between heparinized samples analyzed straight vs diluted were found for MCV and MCHC, with a bias for several additional variables. Significant differences between paired heparinized and EDTA-anticoagulated samples at each dilution point were found for most variables, with the largest differences found in platelet count. Evidence of platelet clumping presumably due to heparin exposure was noted in numerous samples. CONCLUSIONS: Dilution-induced changes occur in some hematologic variables and may render dilution unacceptable in the exploratory research environment. Many variables, most notably platelet count, differ based on the anticoagulant used, and values from heparinized vs EDTA-anticoagulated samples should not be directly compared.