Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.     

RADIOGRAPHIC FEATURES OF PRIMARY AND CONCOMITANT FLEXOR ENTHESOPATHY IN THE CANINE ELBOW

Primary flexor enthesopathy is a recently recognized elbow disorder and should be considered in the differential diagnosis of elbow lameness. For treatment planning purposes, it is important to make a distinction between primary and concomitant forms of the disease. The purpose of this prospective study was to compare radiographic findings for dogs with primary flexor enthesopathy (n = 17), concomitant flexor enthesopathy (n = 24), elbow dysplasia (n = 13), and normal dogs (n = 7). All dogs underwent a complete radiographic examination and each radiographic image was evaluated for the presence or absence of following characteristics: irregular medial humeral epicondyle, spur and calcified body. Additionally, the presence or absence of other elbow disorders (medial coronoid process disease, osteochondritis dissecans, ununited anconeal process, incongruity, subtrochlear sclerosis, and osteoarthritis) was recorded. Radiographic characteristics of flexor enthesopathy were found in 86% of painful joints in the primary flexor enthesopathy group and in 100% of painful joints in the concomitant flexor enthesopathy group. Radiographic characteristics of flexor enthesopathy were not found in sound elbow and elbow dysplasia groups. Frequencies and details of individual radiographic characteristics did not differ between primary and concomitant flexor enthesopathy groups. Findings support the use of radiography as a first screening method for detection of flexor enthesopathy, but not as a technique for distinguishing primary vs. concomitant forms.     

Congenital cataracts in an Ayrshire herd: a herd case report

Background

Microbiological standards within pork slaughter processing plants in the European Union are currently governed by Commission Regulation (EC) 2073/2005, which describes detailed performance criteria at specific stages of the procedure (following carcass dressing and before chilling) for total viable counts (TVC), Enterobacteriaceae (EB) and Salmonella spp. In this study, 95 carcasses from an Irish pork slaughter plant were sampled by swabbing 100 cm² of surface at three sites (belly, ham, jowl) to examine the effects of eight processing stages (stunning, bleeding, scalding, singeing, polishing, evisceration, final inspection and chilling) on contamination levels.

Results

TVC ranged from approximately 1.7–6.3 log cfu cm² during sampling. There were significant reductions in TVC for all sites after scalding and singeing (p < 0.05), whilst there was a significant increase in counts after polishing and evisceration (p < 0.05) compared with preceding stages. EB counts indicated hygienic weak points in the examined slaughter plant leading to faecal (cross)-contamination, with elevated counts after stunning, bleeding and evisceration (p < 0.05), compared with final counts after chilling.

Conclusions

Although the bacterial numbers reported in this study may reflect specific plant practices and temporal influences, results show that contamination can be introduced at various steps in the process and highlight the importance of monitoring locations other than those required by legislation within the process. Monitoring can be used to establish baseline levels for high-risk stages specific to each plant and to assess the effectiveness of additional interventions.     

A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis

A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.     

Reclassification of small intestinal and cecal smooth muscle tumors in 72 dogs: clinical, histologic, and immunohistochemical evaluation

Objectives— To reclassify canine small intestinal and cecal leiomyoma (LM) and leiomyosarcoma (LMS) into smooth muscle and gastrointestinal stromal tumors (GIST) using histologic and immunohistochemical (IH) analysis and to report clinical findings and survival data.
Study Design— Retrospective review of cases.
Animals— Dogs (n=47) with small intestinal (40 LMS; 7 LM) and 25 dogs with cecal tumors (23 LMS; 2 LM).
Methods— Clinical and survival data were reviewed. Tissue sections were reevaluated for light-microscopic malignancy criteria and examined for expression of SMA, desmin, vimentin, S-100, and CD117 (KIT) by immunohistochemistry.
Results— Reclassification resulted in 2 LM, 9 LMS, 19 GIST, and 17 GIST-like tumors in the small intestine and 23 GIST and 2 GIST-like tumors in the cecum. GIST-like tumors were morphologic and IH identical to GIST but lacked KIT expression. No significant difference in survival was observed for tumor type, location, histologic, or IH characteristics; however, dogs with cecal tumors were significantly older in age, presented more commonly with intestinal perforation and peritonitis, and less commonly with weight loss. Cecal tumors had more histologic malignancy criteria than small intestinal tumors. After excision, 1 and 2 year recurrence-free periods were 80.1% and 67.2% for small intestinal and 83.3% and 61.9% for cecal tumors.
Conclusion— Prognosis for intestinal tumors with histologic smooth muscle appearance is good after excision and not related to tumor type, location, histologic, or IH characteristics.
Clinical Relevance— Clinical importance could not be demonstrated for reclassification, but may be for future treatment, of intestinal smooth muscle or stromal tumors.     

A keratoprosthesis prototype for the dog

Objective  To describe the technique for implantation of a novel keratoprosthesis (KP) prototype and evaluate its application for the treatment of corneal blindness in dogs.
Animals studied  Seven dogs, all of them being clinically blind before surgery as a result of severe corneal endothelial disease (5/7) or chronic superficial keratitis (2/7) that were unresponsive to prior therapy.
Procedures  A silicone KP was implanted unilaterally, just anterior to Descemet's membrane, after creating a stromal pocket by deep stromal lamellar dissection.
Results  Implantation of the KP was accomplished without complication in six of seven operated dogs. In the remaining case, an intra-operative complication (perforation of Descemet's membrane) was associated with extrusion of the KP 8 weeks postoperatively. All operated eyes regained limited vision after surgery. Three to six months after implantation purulent keratitis occurred in all five eyes with endothelial disease, necessitating surgical removal of the KP 6 months postoperatively in 5/7 eyes.
Conclusions  This KP prototype shows promise as a treatment for certain blinding corneal diseases. However, changes in the design of this KP, allowing improved stromal integration, will be necessary before its clinical application can be approved.     

Estimating direct genetic and maternal effects affecting rabbit growth and feed efficiency with a factorial design

The aim of this experiment was to evaluate the significance of neonatal environment on feed efficiency. For that purpose, rabbits from a line selected for residual feed intake (RFI) during 10 generations (G10 kits) were cross‐fostered with non‐selected control does (i.e., G0 line), and reciprocally. In parallel, sibs were fostered by mothers from their original line. Nine hundred animals were raised in individual (N = 456) or collective (N = 320) cages. Traits analysed in this study were body weight at 32 days and at 63 days, average daily gain (ADG), feed intake between weaning and 63 days (FI), feed conversion ratio (FCR) and RFI. The maternal environment offered by does from the line selected for RFI deteriorated the FCR of the kits, independently of their line of origin, during fattening (+0.08 ± 0.02) compared to FCR of kits nursed by G0 does. The line, the type of housing and the batch were significant effects for all the measured traits: G10 kits were lighter than their G0 counterparts at 32 days (?82.9 ± 9 g, p < 0.0001) and at 63 days (?161 ± 16 g, p < 0.0001). They also had a lower ADG (?2.36 ± 0.36 g/day, p < 0.0001), RFI (?521 ± 24 g/day, p < 0.0001) and a lower FI (?855 ± 31 g, p < 0.0001), resulting in a more desirable feed efficiency (FCR: ?0.35 ± 0.02). There was no significant difference in the contrast of G10 and G0 performances between collective and individual/digestive cages (p > 0.22): ?2.35 g/day versus 2.94 g/day for ADG, ?0.39 versus ?0.40 for FCR, ?577 g versus ?565 g for RFI and ?879 g versus ?859 g for FI, respectively). Thus, no genotype‐by‐environment (housing) interaction is expected at the commercial level, that is, no re‐ranking of the animals due to collective housing.     

Antimicrobial susceptibility testing of German Campylobacter fetus subsp. venerealis isolates by agar disk diffusion method

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis and is transmitted by asymptomatic carrier bulls via contaminated semen during artificial insemination. The aim of the present study was to determine the in vitro susceptibility of Campylobacter fetus subsp. venerealis isolated from bovine specimens in the years from 2000 to 2009 in Germany to antibiotics generally used in semen treatment. The susceptibilities of 50 strains to spectinomycin (10 microg), gentamicin (10 microg), streptomycin (25 microg), penicillin (10 microg), lincomycin (10 microg), ciprofloxacin (5 microg), erythromycin (30 microg) and tetracycline (30 microg) were determined using a disk diffusion susceptibility test. All strains were susceptible to gentamicin. A considerably reduced susceptibility to one or more antimicrobial agents was detected in seven out of 50 isolates (14%) with the most frequent reduction in susceptibility to lincomycin and spectinomycin. Furthermore, strains with reduced susceptibility to more than one antimicrobial agent were always associated with reduced susceptibility to lincomycin. It is recommended to determine the antimicrobial susceptibility of Campylobacter fetus subsp. venerealis isolates in order to evaluate the efficacy of the generally used antibiotic treatment of bull semen and to detect possible resistances.