Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.     

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.     

Interpretation of serum antibody response to Anoplocephala perfoliata in relation to parasite burden and faecal egg count

REASON FOR PERFORMING STUDY: Increased knowledge is needed to assist in the interpretation of presently available diagnostic techniques for infection by the tapeworm Anoplocephala perfoliata in horses. HYPOTHESIS: The suggested cut-off level of an A. perfoliata specific ELISA may not adequately reflect the actual infection level. Hence, faecal egg counts may be a more useful diagnostic test for individual horses than previously reported. METHODS: Eighty-four horses admitted for slaughter at a Danish abattoir were examined for the presence of A. perfoliata. The number of tapeworms, their stage of development and gross pathological mucosal lesions were recorded and compared with serum antibody responses and faecal egg counts. Faecal egg counts were determined in samples from A. perfoliata infected horses using a semi quantitative centrifugation/flotation technique. Blood samples collected at slaughter were analysed by ELISA to determine serum antibody levels against A. perfoliata 12/13 kDa excretory/secretory antigens. RESULTS: Macroscopically visible tapeworms were detected in 24 (29%) of the horses. The overall sensitivity of the faecal egg count was found to be 0.46; however, if the detection limit was increased to above 20 tapeworms, sensitivity increased to 0.89. There was a correlation of 0.71 between worm burden and egg count. The antibody levels correlated significantly with infection intensity despite a wide variation among horses with similar levels of infection. The optimal cut-off value was determined using receiver operating characteristic analysis. If cut-off was chosen at optical density (OD) = 0.7, sensitivity was 0.68 and specificity 0.71. CONCLUSIONS: Both diagnostic methods were capable of revealing potentially pathogenic infections, with the faecal egg count being more applicable on the individual horse level. POTENTIAL RELEVANCE: In the population of Danish horses investigated the serum ELISA test should be interpreted such that horses in need of anti-Anoplocephala treatment have an OD = 0.7 or above.     

The clinical utility of the right lateral intercostal ultrasound scan technique in dogs

When performing abdominal ultrasonography in dogs, the right aspect of the liver, porta hepatis, right kidney, right adrenal gland, pancreas, and duodenum are often not fully visible from a ventral, or subcostal, approach. The right lateral intercostal plane is an alternative approach that allows evaluation of these structures. This report provides multiple case examples that demonstrate the sonographic anatomy via the right intercostal approach. Other cases are included to demonstrate indications for this approach. Animals in which the right intercostal approach may prove most useful include large- and giant-breed dogs; deep-chested dogs; dogs with gas distention of the stomach, duodenum, and colon; dogs with microhepatia; and those with abdominal effusion and pain.     

Tibial plateau symmetry and the effect of osteophytosis on tibial plateau angle measurements

A novel technique was developed to estimate the caudal medial tibial plateau landmark in the face of osteophytosis to improve accuracy in tibial plateau angle measurements. Using this technique, tibial plateau angles were evaluated in 31 normal dogs before and 8 months after right cranial cruciate ligament transection. There was no significant difference in mean tibial plateau angle before or after induction of osteophytosis. Additionally, it was determined that 90% of dogs had a difference of =2 degrees between right and left tibial plateau angles, which was considered symmetrical.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Effect of prostaglandin F2alpha on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares

The effect of a single injection of prostaglandin F2alpha (PGF) during mid-diestrus on systemic concentrations of progesterone, LH, FSH, estradiol, and cortisol and on blood flow to the corpus luteum was studied in 10 controls and 10 PGF-treated mares. Blood flow was assessed by estimating the percentage of corpus luteum with color-Doppler signals of blood flow during real-time scanning of the entire structure and by the diameter of the vascular pedicle near its attachment to the ovary. Treatment was done 8 days after ovulation and 0 h was immediately before the treatment. Examinations and collection of blood samples were done at 0 h, every 5 min until 1h, and then at 1.5, 2, 4, 8, 12, 24, 48, and 72 h. The concentrations of estradiol did not change, but progesterone, LH, FSH, and cortisol increased significantly within 5 min. Concentrations of LH and FSH in the PGF group remained elevated until a temporarily lower concentration at 8 or 4h, respectively, rebounded to 12h, and then slowly decreased. Cortisol remained elevated, until a decrease between 1 and 4h. Progesterone in the PGF group increased significantly until 10 min after 0 h and then decreased by 40 min to below the concentrations in controls. Within the PGF group, progesterone decreased significantly by 45 min to below the concentrations at 0 h. The values for each of the two indicators of blood flow did not differ significantly between the PGF and control groups until a decrease at 24h in the PGF group. Results did not support the hypothesis that the immediate transient post-PGF increase in progesterone was associated with an increase in luteal blood flow. Luteolysis, as indicated by decreasing progesterone, began well before the beginning of a decrease in luteal blood flow.     

Effects of a veterinary student leadership program on measures of stress and academic performance

Assuming leadership roles in veterinary student governance or club activities could be considered an added stressor for students because of the impact on time available for personal and academic activities. The study reported here evaluated the effects of participation in a leadership program and leadership activity across two classes of veterinary students on measures of stress, using the Derogatis Stress Profile (DSP), and on veterinary school academic performance, measured as annual grade-point average (GPA) over a three-year period. Program participants and their classmates completed the DSP three times across the first three years of veterinary school. On average, participating students reported self-declared stress levels that were higher and measured DSP stress levels that were lower than those of the general population. Students were more likely to assume elected or appointed leadership roles while in their first three years of the veterinary degree program if they participated in the optional leadership program and demonstrated lower stress in several dimensions. Some increased stress, as measured in some of the DSP stress dimensions, had a small but statistically significant influence on professional school GPA. The study determined that the most important predictors of students' cumulative GPA across the three-year period were the GPA from the last 45 credits of pre-veterinary coursework and their quantitative GRE scores. The results of the study indicate that neither participation in the leadership program nor taking on leadership roles within veterinary school appeared to influence veterinary school academic performance or to increase stress.