Creatine kinase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines.

BACKGROUND: Creatine kinase (CK) exists as three isoenzymes (CK-MM, CK-MB, and CK-BB) that are predominantly expressed in specific tissues and can be detected in both the serum and cerebrospinal fluid (CSF). CSF CK has been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total CSF CK activity can indicate neurologic abnormalities. OBJECTIVES: The purpose of this study was to establish reference intervals for CK and its three major isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive reference interval for this enzyme in healthy canines, the diagnostic use and possible significance of CK in clinical disease can be studied. METHODS: Serum and/or CSF were collected from healthy dogs. Total CK activity was measured spectrophotometrically, and isoenzyme distributions were determined using the QuickGel CK Vis Isoenzyme Kit and a densitometric scanner. Total CK and CK isoenzyme activities were determined within 8 h of collection. RESULTS: The median serum total CK in healthy canines was 159.0 U/L (range: 53.0-539.0 U/L), while the median CSF total CK was 3.7 U/L (range: 2.0-84.0 U/L). CK-BB and CK-MM were approximately equal in the serum, while CK-MM was the predominant isoenzyme in the CSF. CONCLUSIONS: Knowledge of the normal distribution and concentration of CK in canine serum and CSF will set the foundation for future studies of canine CK as a potentially clinically useful biomarker.

The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission.

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV's alternating-host transmission cycle on the virus's genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV's genome. To understand the role of alternating hosts in BTV's genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

Bovine viral diarrhea in captive Rocky Mountain bighorn sheep associated with administration of a contaminated modified-live bluetongue virus vaccine.

In late summer 2017, we observed acute, fatal cases of bovine viral diarrhea in captive Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) in Colorado following use of a contaminated modified-live bluetongue virus vaccine. Following vaccination, at least 14 of 28 (50%) vaccinated bighorn sheep developed hemorrhagic diarrhea, and 6 of 28 (21%) vaccinated bighorn sheep died. Autopsy findings were predominantly necroulcerative-to-necrohemorrhagic gastrointestinal lesions. Less frequent lesions included suffusive hemorrhages of serosal surfaces of abdominal viscera, and lymphoid necrosis in gut-associated lymphoid tissues. All of the 6 bighorn sheep that died were positive on real-time PCR (rtPCR) for bovine viral diarrhea virus (BVDV) in multiple tissues. Seroconversion to BVDV-1 and immunohistochemistry for BVDV in affected tissues confirmed rtPCR results. Next-generation sequencing confirmed a match between the infecting strain of BVDV-1b and the contaminated vaccine.

Effects of Low-level Brodifacoum Exposure on the Feline Immune Response.

Anticoagulant rodenticides have been implicated as a potential inciting factor in the development of mange in wild felids, but a causative association between anticoagulant rodenticide exposure and immune suppression has not been established. Specific-pathogen-free domestic cats were exposed to brodifacoum over a 6-week period to determine whether chronic, low-level exposure altered the feline immune response. Cats were vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses. Measures of immune response included delayed-type hypersensitivity tests and cell proliferation assays. IgE and antigen-specific antibodies were quantified via ELISA assays, and cytokine induction following exposure to vaccine antigens was also analyzed. While cats had marked levels of brodifacoum present in blood during the study, no cats developed coagulopathies or hematologic abnormalities. Brodifacoum-exposed cats had transient, statistically significant decreases in the production of certain cytokines, but all other measures of immune function remained unaffected throughout the study period. This study indicates that cats may be more resistant to clinical effects of brodifacoum exposure than other species and suggests that the gross impacts of environmentally realistic brodifacoum exposure on humoral and cell-mediated immunity against foreign antigen exposures in domestic cats are minimal.

Reference intervals for the activity of lactate dehydrogenase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines.

BACKGROUND: Lactate dehydrogenase (LD) exists as 5 isoenzymes (LD-1 through LD-5) that are expressed throughout the body and can be detected in both serum and cerebrospinal fluid (CSF). LD and its isoenzymes have been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total LD activity and atypical isoenzyme patterns can indicate disease processes, including neurologic abnormalities. OBJECTIVES: The purpose of this study was to establish RIs for LD and its isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive RI for this enzyme in healthy canines, further study of the clinical and diagnostic usefulness of LD can be undertaken. METHODS: Serum and atlantoaxial CSF were collected from clinically healthy dogs. Total LD activity was measured spectrophotometrically immediately after collection. Isoenzyme distributions were also determined within 8 hours of collection using the QuickGel LD Isoenzyme technique and a densitometric scanner. RESULTS: The median serum total LD in healthy canines was 69.0 U/L (n = 41; range: 21.0-217.0 U/L), while the median CSF total LD was 10.0 U/L (n = 40; range: 6.0-19.3 U/L). LD-5 is the predominant isoenzyme in canine serum (n = 40), contributing over half of the total enzyme activity. Conversely, in canine CSF (n = 42), LD-1 is the predominant isoenzyme, followed by LD-2 and LD-3. CONCLUSIONS: Knowledge of the distribution and concentration of LD in the serum and CSF of healthy dogs will set the foundation for future studies of canine LD as a potentially clinically useful biomarker.

Replacement, Refinement, and Reduction in Animal Studies With Biohazardous Agents.

Animal models are critical to the advancement of our knowledge of infectious disease pathogenesis, diagnostics, therapeutics, and prevention strategies. The use of animal models requires thoughtful consideration for their well-being, as infections can significantly impact the general health of an animal and impair their welfare. Application of the 3Rs-replacement, refinement, and reduction-to animal models using biohazardous agents can improve the scientific merit and animal welfare. Replacement of animal models can use in vitro techniques such as cell culture systems, mathematical models, and engineered tissues or invertebrate animal hosts such as amoeba, worms, fruit flies, and cockroaches. Refinements can use a variety of techniques to more closely monitor the course of disease. These include the use of biomarkers, body temperature, behavioral observations, and clinical scoring systems. Reduction is possible using advanced technologies such as in vivo telemetry and imaging, allowing longitudinal assessment of animals during the course of disease. While there is no single method to universally replace, refine, or reduce animal models, the alternatives and techniques discussed are broadly applicable and they should be considered when infectious disease animal models are developed.