A screening test for subclinical liver disease in horses affected by pyrrolizidine alkaloid toxicosis

Objective: To evaluate various biochemical tests as indicators of subclinical liver disease in horses exposed to pyrrolizidine alkaloid toxicosis.
Design: A clinical pathology field study.
Animals: Twenty-two clinically normal horses from four properties in the Kimberley region of Western Australia.
Procedure: Serum samples from each horse were assayed for gamma glutamyltransferase, alkaline phosphatase and aspartate aminotransferase activities, and for serum bile acid concentration, albumin and total protein. Serum protein electrophoresis was performed and their amino acid profiles determined. Bromosulphophihalein halfclearance times were measured. Horses were then subjected to a single liver biopsy. Results were analysed by, variance of group means, the Fisher-Irwin exact test, and by sensitivity and specificity calculation.
Results: Horses were classified into 2 groups, of 10 unaffected and 12 subclinically affected, on the basis of liver histology. Significant differences between the unaffected and subclinical groups were observed for gamma glutamyltransferase and alkaline phosphatase activities (P < 0.01). Gamma glutamyltransferase had sufficient sensitivity (75%) and specificity (90%) to function as a primary screening test for subclinical liver disease in horses exposed to pyrrolizidine alkaloids. Alkaline phosphatase was useful, but with lower sensitivity (58%).
Conclusion: Serum gamma glutamyltransferase activity is a useful screening test for detecting subclinical liver disease in horses exposed to pyrrolizidine alkaloids under field conditions in northern Australia.     

Application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens

Objective A real-time polymerase chain reaction (PCR)/high-resolution melt (HRM) curve analysis protocol was developed in our laboratory to differentiate infectious bronchitis (IB) virus reference strains. In the current study, this method was used to detect and classify IB viruses in field submissions. Procedure Over an 11-month period samples from 40 cases of suspected IB virus were received and 17 submissions were positive for IB virus by polymerase chain reaction. HRM curve analysis classified each strain as subgroup 1, 2 or 3 strain (12 submissions) or a strain that was unable to be classified (5 submissions). The 3′ untranslated region (UTR) and partial S1 gene nucleotide sequences for the 17 IB virus strains were determined and their identity with those of the relative reference strains compared to confirm the classifications generated using the HRM curve analysis. Results Of the 12 IB field viruses classified as subgroup 1, 2, or 3 using HRM curve analysis, the 3′UTR and S1 gene nucleotide sequences had identities ≥99% with the respective subgroup reference strain. Analysis of the 3′ UTR and S1 gene nucleotide sequences for the five IB virus strains that could not be classified indicated that four belonged to one of the subgroups, and one was a potential recombinant strain (between strains from subgroups 2 and 3). A novel recombinant strain was also detected. Conclusion HRM curve analysis can rapidly assign the majority of IB viruses present in field submissions to known subgroups. Importantly, HRM curve analysis also identified variant genotypes that require further investigation.     

A non-invasive technique for standing surgical repair of urinary bladder rupture in a post-partum mare: a case report

An 11-year-old mare presented 36 hours after foaling with a ruptured bladder. Uroperitoneum was diagnosed on ultrasound and from the creatinine concentration of the peritoneal fluid. Bladder endoscopy demonstrated tissue necrosis and a rent in the dorsocranial aspect of the bladder. Following stabilisation, including abdominal drainage and lavage, the mare was taken to standing surgery. Under continuous sedation and epidural anaesthesia, and after surgical preparation, a Balfour retractor was placed in the vagina. Using sterile lubricant and moderate force, it was possible to insert a hand into the bladder. The tear was easily palpable on the dorsal portion of the bladder. Two fingers were inserted through the tear and used to provide traction to evert the bladder completely into the vagina where it could grasped with the surgeons other hand to prevent further trauma. A second surgeon could then visualise the entire tear and repaired this using a single layer of size zero PDS suture in a single continuous pattern. As soon as the bladder was repaired, it was replaced via the urethra. The mare did well after surgery and was discharged after 48 hours, apparently normal.This report is the first describing repair of the bladder without an abdominal incision or incision into the urethral sphincter. This greatly reduces the chance of possible complications such as urine pooling after surgery with the previously described standing technique or bladder trauma due to traction with abdominal surgery.     

In vitro Fertilizing Capacity of Frozen-thawed Bull Spermatozoa Selected by Single-layer (Glycidoxypropyltrimethoxysilane) Silane-coated Silica Colloidal Centrifugation

Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll™ (C2). Sperm recovery surpassed 50% for both C1–C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p < 0.05). In T, the mean (±SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 ± 23.3%, 24.5 ± 14.3% and 94.6 ± 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.