Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

Impact of 24-h Cooling Prior to Freezing on the Survival of Domestic Cat (Felis catus) Epididymal Sperm

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris–glucose–20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5°C at a rate of 0.5°C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5°C for 24 h (cooled group) and after freezing–thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5°C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze–thaw process.     

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 ×  g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 μm in diameter, with a dark ooplasm surrounded by three- or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70°C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO₂ at 38°C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.     

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

Amplification of Bovine Viral Diarrhoea Virus Introduced into an In Vitro Embryo Production System Via Oocytes from Persistently Infected Cattle

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus. Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2) oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus. Virus was not isolated from any samples in treatment group 1. As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.     

Biomechanical Comparison of Two Alternative Tibial Plateau Leveling Osteotomy Plates with the Original Standard in an Axially Loaded Gap Model: An In Vitro Study

Objective— To compare the axial compression stiffness of osteotomized canine tibiae stabilized with Slocum, Securos, or Synthes plates after a tibial plateau leveling osteotomy (TPLO) procedure. Study Design— In vitro, paired comparison of cadaveric tibial constructs subjected to mechanical testing under an axial load. Sample Population— Canine tibiae (n=16 pairs) from skeletally mature male and female dogs of various breeds (18–55 kg). Methods— Tibial pairs (n=16) were randomly assigned to 1 of 2 study cohorts (n=8 pairs/cohort): cohort 1, tibial osteotomy stabilization with a Slocum or a Securos plate, or cohort 2, tibial osteotomy stabilization with a Slocum or a Synthes plate. One tibia from each pair was stabilized with 1 of each plate design assigned to the cohort after TPLO. A 3.2 mm osteotomy gap was maintained during plate application in all constructs. Load and axial displacement were recorded while constructs were loaded to 2000 N in axial compression. Failure loads were not reported because no distinct yield point or failure point was evident within the load range for many specimens. Failure modes were recorded for each construct, and photographs of typical failures were obtained. Stiffness (N/mm) was calculated from load–displacement curves. Paired comparisons of mean stiffness were performed within study groups using a paired t‐test. Significance was set at P<.05. Results— The mean construct stiffnesses for the Slocum (383±183 N/mm) and Securos (258±64.1 N/mm) constructs were not significantly different (P=.164; power=0.566). The mean construct stiffness for the Synthes constructs (486±91.0 N/mm) was significantly greater than that of the Slocum constructs (400±117 N/mm); P=.0468. Modes of failure for the Slocum (16/16) and Securos (8/8) constructs included plastic deformation of the implant with valgus deformity combined with fibular luxation (2/16 Slocum; 1/8 Securos) or fibular fracture (2/16 Slocum; 4/8 Securos). Most Synthes constructs underwent elastic deformation (7/8). One Synthes construct fractured in the saggital plane through the tibial plateau depression at the point of load application. Conclusions— The Slocum and Securos plate/tibia construct have similar stiffness, whereas the Synthes/tibia constructs are significantly stiffer than the Slocum/tibia constructs. Modes of fixation failure observed in this model were consistent with TPLO fixation failures observed clinically. Clinical relevance— Construct stiffness in axial load varies with implant type. Implants that confer higher stiffness to the construct may result in greater fixation stability in tibial metaphyseal osteotomies.     

Effects of high and low inspired fractions of oxygen on horse erythrocyte membrane properties, blood viscosity and muscle oxygenation during anaesthesia

ObjectivesTo evaluate whether a period of hyperoxia or after a period of hypoxia produced changes attributable to reactive oxygen species in anaesthetized horses.Study designProspective randomized experimental study.AnimalsSix healthy (ASA I) geldings, aged 4.5–9.5 years and weighing 510–640 kg^?1.MethodsAfter 30 minutes breathing air as carrier gas for isoflurane, horses were assigned randomly to breathe air as carrier gas (CG0.21) or oxygen as carrier gas (CG1.00) for a further 90 minutes. After an interval of 1 month each horse was re-anaesthetized with the other carrier gas for the 90 minute test period. Ventilation was controlled throughout anaesthesia. Arterial blood was sampled to measure gas tensions, lactate, cholesterol, vitamin E, 4-hydroxy-alkenals, 8-epi-PGF_2α, half haemolysis time, half erythrolysis time, and erythrocyte membrane fluidity. Muscle blood flow and oxygenation were evaluated by near infrared spectroscopy and coloured Doppler.ResultsAfter the first 30 minutes horses were hypoxemic. Subsequently the CG1.00 group became hyperoxaemic (PaO₂～240 mmHg) whereas the CG0.21 group remained hypoxaemic (PaO₂～60 mmHg) and had increased lactate concentration. No significant changes in vitamin E, 4-hydroxy-alkenals, or 8-epi-PGF_2α concentrations were detected. During the 90 minute test period the CG0.21 group had increased resistance to free-radical-mediated lysis in erythrocytes, whereas the CG1.00 group had slightly decreased resistance of whole blood to haemolysis. CG0.21 induced a progressive muscle deoxygenation whereas CG1.00 induced an increase in muscle oxygen saturation followed by progressive deoxygenation towards baseline.Conclusions and clinical relevanceDuring isoflurane anaesthesia in horses, the hyperoxia induced by changing from air to oxygen induced minimal damage from reactive oxygen species. Using air as the carrier gas decreased skeletal muscle oxygenation compared with using oxygen.     

Outflow tract segmental arrhythmogenic right ventricular cardiomyopathy in an English Bulldog

A 5-year-old English Bulldog was presented for acute onset of syncope and fatigue caused by sustained ventricular tachycardia with left bundle block morphology and inferior axis. This arrhythmia had the electrocardiographic features of a ventricular tachycardia arising from the right ventricular outflow tract (RVOT), as described in an experimental canine model and in people. Since a RVOT aneurysm was identified by echocardiography, a segmental form of arrhythmogenic right ventricular cardiomyopathy (ARVC) was suspected. Gross examination of the heart confirmed the bulging of the RVOT and histological examination of the ventricular myocardium revealed segmental involvement of the RVOT with transmural fibro-fatty degeneration. To the authors' knowledge, this is the first reported case of AVRC in an English Bulldog and the first example of segmental AVRC described in the dog.