Describing the within laboratory and between laboratory agreement of a serum ELISA in a national laboratory network

Results from laboratory assays for detection of animal disease are often assessed for repeatability (agreement within laboratory) and reproducibility (agreement between laboratories). This work aimed to understand the strengths and limitations of available methods for describing these quantities. Five major veterinary laboratories in Australia volunteered to participate in a designed evaluation based on repeat testing of twenty bovine sera. Sampling was stratified so that ten of the sera were negative to the virus neutralisation test (VNT) for antibody to bovine herpes virus 1 (BHV-1) and the remaining ten sera were VNT positive. Each serum was divided into 50 replicates and each laboratory assayed one replicate of each serum on a weekly basis using a commercial ELISA for BHV-1. Laboratories were blinded to the identity of sera. The data on sample to positive control ratio (S/P) for these 1000 individual assays were collated, sources of variance analysed using a random effects model, and reliability coefficients (ρ) obtained from the variance estimates as quantitative measures of within and between laboratory agreement. Coefficient of variation (CV) was calculated for combinations of sera and laboratory. CV was found to be higher for sera with the lowest mean S/P values (VNT -ve sera). For VNT -ve sera, agreement of S/P within laboratory was low to moderate (ρ: 0.01-0.27) and the agreement between all labs was low (ρ=0.02). Reliability coefficients for VNT +ve sera were very high for agreement within laboratories (ρ: 0.63-0.92) and moderate for agreement between laboratories (ρ=0.52). As well, simulation demonstrated that sero-prevalence has a dramatic affect on the reliability coefficient if sampling were to be irrespective of VNT status. We conclude that there are some limitations with the available approaches for assessing agreement within and between laboratories. Although reliability coefficients have some drawbacks they are an attractive way of reducing reliance on subjective assessment of agreement.     

Applying molecular genetic tools to tiger conservation

The utility of molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Over the past decade, conservation genetic analyses based on mitochondrial DNA sequencing and microsatellite genotyping have provided powerful tools to resolve taxonomy uncertainty of tiger subspecies, to define conservation units, to reconstruct phylogeography and demographic history, to examine the genetic ancestry of extinct subspecies, to assess population genetic status non-invasively, and to verify genetic background of captive tigers worldwide. The genetic status of tiger subspecies and populations and implications for developing strategies for the survival of this charismatic species both in situ and ex situ are discussed.     

Carbon Dioxide Laser Keratectomy as a Treatment Option for Equine Corneal Stromal Abscessation: A Comparison to Lamellar Keratectomy and Penetrating Keratoplasty

Carbon dioxide (CO₂) laser keratectomy was compared to lamellar keratectomy (LK) and penetrating keratoplasty (PK) as a potential treatment option for equine corneal stromal abscessation. The medical records of 35 client owned horses undergoing surgical management of stromal abscessation were reviewed. Among those 17 underwent LK, 3 PK and 15 CO₂ laser keratectomy. Healing of the stromal abscess was achieved in all treated eyes. Those horses undergoing CO₂ laser keratectomy demonstrated few complications, and the time to healing and cosmetic outcome was comparable to those horses undergoing LK and PK.     

Differential expression of TAR DNA-binding protein (TDP-43) in the central nervous system of horses afflicted with equine motor neuron disease (EMND): a preliminary study of a potential pathologic marker

Equine motor neuron disease (EMND) is a neurodegenerative disorder of unknown etiology affecting horses worldwide. Trans-Active Response DNA Binding Protein of 43?kDa (TDP-43) has been reported in the central nervous system (CNS) of several neurodegenerative conditions in humans including Amyotrophic Lateral Sclerosis (ALS) and assumed to play role in the disease. We examined whether horses afflicted with EMND express the TDP-43 in CNS. Ten horses with EMND and 6 controls of different ages and breed we enrolled. Detection of presence of TDP-43 protein in the CNS was analyzed by immunohistochemical staining using rabbit anti-human TARDBP (TDP-43) polyclonal antibody. Formalin fixed neuronal tissues from medulla, cervical, and lumbar spinal cord were harvested from EMND and from control horses. Sections were assigned randomly to TDP-43 treated or rabbit anti-IgG as control. Nuclear staining of TDP-43 was detected in one of the neural tissues of 75?% of EMND-positive and 0 of 0?% of control horses in the central nervous system (medulla, and/or cervical spinal cord and/or lumbar spinal cord). TDP-43 antibody was detected in the nucleus of EMND horses and no cytoplasmic staining was noted. As in ALS, there was no pattern of age clustering associated with the detection of TDP-43. This is the first report on the staining of TDP-43 in neuronal tissues of horses and suggests that TDP-43 may play a role in the pathogenesis of EMND. Further studies are needed to elucidate the etiologic role of this protein in the diseases.     

Clinical,pathologic, and toxicologic characterization of Salvia reflexa (lance-leaf sage) poisoning in cattle fed contaminated hay

Salvia reflexa (lance-leaf sage)-contaminated alfalfa hay was fed to ~500 mixed-breed beef cattle. Within hours of exposure, nearly half of the cattle developed lethargy, anorexia, depression, and recumbency, followed by bellowing, colic, and death. Even though the uneaten contaminated hay was removed the first day, nearly 100 animals died within the first 48 h. Three of these cattle were examined postmortem, and tissues and hay samples were collected for microscopic and chemical analysis. Several days later, a smaller number of the clinically poisoned cattle developed neurologic disease with aberrant behavior, aggression, icterus, blindness, exhaustion, and death. A total of 165 cattle were fatally poisoned. Poisoned cattle had swollen, dark, mottled livers that had a prominent nutmeg-like lobular pattern on cut section. Histologically, there was severe centrilobular-to-panlobular hepatic necrosis with marked hepatocellular swelling, degeneration, and necrosis. The surviving cattle developed liver disease characterized by altered serum biochemical analyses and microscopic hepatocellular degeneration and necrosis. In subsequent biopsies and analysis, these lesions resolved within 6–7 mo. After confirming toxicity of the hay in cattle, goats, and mice, followed by a mouse bioassay–guided chemical fractionation process, Salvia reflexa was identified as the contaminant in the hay responsible for the hepatotoxicity. S. reflexa has not been reported previously to cause fatal hepatotoxicity in livestock in North America, to our knowledge.     

Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.     

Evaluation of the effects of ischemic injury and ureteral obstruction on delayed graft function in cats after renal autotransplantation

OBJECTIVE: To determine the relative importance of ischemic injury to delayed graft function (DGF) in cats. STUDY DESIGN: Experimental study. ANIMALS: Six intact female cats. METHODS: Cats had renal autograft transplantation without ureteral transection and reimplantation and a contralateral nephrectomy. Serum creatinine and blood urea nitrogen (BUN) concentrations were measured regularly and abdominal ultrasound was performed before surgery, the day after surgery and twice weekly thereafter. Ultrasound-guided renal biopsy was performed on day 7. Cats were euthanatized on day 21. Histology of the autograft, ureter, bladder, vascular anastomoses sites, and contralateral kidney were performed. Observations were compared with those from an historic group of research cats that had extravesicular ureteroneocystostomy and contralateral nephrectomy. RESULTS: Five cats completed the study. Serum creatinine and BUN concentrations increased after surgery, peaking at 3.2+/-0.8 and 77.6+/-15.9 mg/dL, respectively, 1-2 days after surgery. Serum creatinine concentration returned to the reference interval by 6 days after surgery. BUN gradually decreased in all cats but did not return to the reference interval by study end. Serum creatinine and BUN concentrations were consistently lower but not significantly so (P=.29 and .56, respectively) compared with the historic ureteroneocystostomy group. No ultrasonographic abnormalities or renal biopsy histologic abnormalities were observed. At necropsy, 1 autograft had generalized interstitial fibrosis. CONCLUSION: Harvesting the renal graft and the ischemia before revascularization causes impaired renal function after engraftment. CLINICAL RELEVANCE: The process of harvesting and reimplanting the renal graft can contribute to DGF in cats, independent of ureteral obstruction.     

Epidemiological features and association with crib‐biting in horses with neurological disease associated with temporohyoid osteoarthropathy (1991–2008)

A retrospective study of 43 cases of temporohyoid osteoarthropathy was performed to evaluate the epidemiological features and a possible association with crib‐biting. Data collected from records included case details, what diagnostics were utilised, whether medical or surgical treatment was administered, and outcome. Owners were contacted via telephone and asked whether the horse had displayed crib‐biting behaviour. Forty‐three horses were diagnosed with neurological disease associated with temporohyoid osteoarthropathy, 62.8% of which were Quarter Horse‐types. Median age at presentation was 10 years and median duration of neurological signs prior to presentation was 3 days. Skull radiographs and guttural pouch endoscopy were used to definitively diagnose temporohyoid osteoarthropathy in 72% of the cases. Of 43 horses, 21 received medical treatment and 15 surgical treatment, with an overall survival rate of 55.8%. Crib‐biting was observed in 31.3% of cases and there was a significant association between being afflicted with THO and likelihood of possessing the behaviour. Horses with neurological disease associated with THO were 8 times more likely to be crib‐biters compared to the general population.