Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene

The aim of this study was to determine if fluorescent PCR could be used instead of nested PCR, for the detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in clinical specimens, to improve the sensitivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratuberculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagnostic work. The sensitivity of each PCR system was assessed by analysing a serial dilution of M. paratuberculosis DNA. To improve the reliability of the PCR and to facilitate the interpretation of the PCR results, a positive internal control molecule ("mimic") was developed and used for single and fluorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extraction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on seventeen ileum mucosa specimens, previously found positive for M. paratuberculosis by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis organisms, illustrating the effect of inhibitory substances in combination with small amounts of M. paratuberculosis DNA. We conclude that the extraction method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimens, but that the method is useful for identifying positive samples. Fluorescent PCR was a suitable alternative to both single PCR and nested PCR for the detection of M. paratuberculosis.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

Paratracheal air cyst in a foal

A 1-day-old American Paint Horse was presented for a large air-filled mass along the ventral aspect of the neck. Bronchoscopy and oesophagoscopy revealed no sign of communication with the trachea or oesophagus. Radiographs and a computed tomography scan of the neck identified a communicating tract between the lumen of the cystic mass and mid-trachea. The foal was systemically healthy at this initial presentation, and delayed removal of the cyst was recommended to allow further maturation of the foal prior to undergoing general anaesthesia. Upon discharge, the cyst continued to grow in size and became more fluid than air-filled requiring repeat centesis and draining. The foal was then re-presented at 3 weeks of age for surgical removal. In surgery, direct communication with the trachea was identified and ligated. Histopathology demonstrated that the cyst lining was composed of squamous epithelium with goblet cells and occasional ciliated cells. The location, morphological features and congenital presentation of the mass were consistent with a paratracheal air cyst (PAC). Surgical resection resulted in excellent functional and cosmetic outcome. Although not previously reported in horses, PAC should be included in the differential diagnosis of an air-filled ventral neck mass in equine neonates. Complete surgical excision may result in a successful outcome.     

Thin-slice three-dimensional gradient-echo magnetic resonance imaging of the pituitary gland in healthy dogs

OBJECTIVE: To evaluate thin-slice 3-dimensional gradient-echo (GE) magnetic resonance imaging (MRI) of the pituitary gland in healthy dogs. ANIMALS: 11 healthy dogs. PROCEDURES: By use of a 0.2-Tesla open magnet, MRI of the skull was performed with T1-weighted GE sequences and various protocols with variations in imaging plane, slice thickness, and flip angle before and after administration of contrast medium; multiplanar reconstructions were made. The pituitary region was subjectively assessed, and its dimensions were measured. Image quality was determined by calculation of contrast-to-noise and signal-to-noise ratios. RESULTS: Best-detailed images were obtained with a T1-weighted GE sequence with 1-mm slice thickness and 30 degrees flip angle before and after administration of contrast medium. Images with flip angles > 50 degrees were of poor quality. Quality of multiplanar reconstruction images with 1-mm slices was better than with 2-mm slices. The bright signal was best seen without contrast medium. With contrast medium, the dorsal border of the pituitary gland was clearly delineated, but lateral borders were more difficult to discern. CONCLUSIONS AND CLINICAL RELEVANCE: MRI of the canine pituitary gland with a 0.2-Tesla open magnet should include a T1-weighted GE sequence with 1-mm slice thickness and flip angle of 30 degrees before and after administration of contrast medium. The neurohypophysis was best visualized without contrast medium. The MRI examination permitted differentiation between the pituitary gland and surrounding structures.     

Effects of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected immunological and histological measurements in the spleen of gilts

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of immunological effects of Fusarium toxins to porcine spleen cells. Contaminated diets were fed to 36 Landrace prepubertal gilts for 35 d. Concentrations (as-fed basis) of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON), respectively, were 210 and 4 (control--group I), 3,070 and 88 (group II), 6,100 and 235 (group III), and 9,570 and 358 microg/kg (group IV). No signs of hyperestrogenism or uterotrophic effects were observed because of dietary treatments. The feeding of mycotoxin-contaminated diets did not cause gross pathological findings in the spleens of animals. In vivo, no inhibitory effects were detected on concanavalin A-stimulation of blood lymphocytes; however, the proliferation rate of splenocytes was inhibited (P < 0.05) in pigs fed the diet with the highest DON/ZON concentration. With in vitro studies, lower proliferation rates of blood lymphocytes and splenocytes preexposed to DON were detected. Serum IgA concentrations of pigs in group II were increased (P < 0.05) compared with the baseline value before feeding the DON/ZON diet. The histopathological data indicated elevated (P < 0.05) iron staining in the red pulp of spleens in gilts from groups I to IV after 35 d of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopic investigation. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in the absence of clinical signs, especially in pigs fed higher concentrations (groups III and IV) of Fusarium toxin-contaminated wheat.     

Digital cushions in horses comprise coarse connective tissue, myxoid tissue, and cartilage but only little unilocular fat tissue

Digital cushions were studied in horses with particular reference to vascularization, tissue constituents and matrix components. The cushions mainly resembled a network of coarse collagen bundles. The areas inbetween the bundles were replenished with loosely woven interstitial connective tissue, myxoid tissue, and fibrocartilage. Expected masses of fat lobules were missing: only solitary adipocytes or small groups of adipocytes were seen. Vascular supply to the cushions was remarkably poor. The mucinous myxoid matrix largely consisted of hyaluronan with little sulphated glycosaminoglycans. Myxoid cells were stellate or ramified in shape and showed a tendency to store glycogen and lipid droplets. Myxoid cells reacted for vimentin and stained for S-100 protein. Moreover, myxoid cells often reacted for neuron specific enolase and glial fibrillary acidic protein. Myxoid tissue continuously transformed into loosely organized interstitial connective tissue with fibroblasts, which remained unreactive when tested for neuroectodermal markers. Myxoid tissue also was not clearly demarcated against irregularly interspersed islets of fibrocartilage or hyaline cartilage. Chondrocytes did not stain for neuron specific enolase but reactivity for S-100 protein and glial fibrillary acidic protein was noted in peripheral regions of fibrocartilage. Single or grouped unilocular fat cells were rarely placed into myxoid areas. Unilocular fat cells stained for vimentin, S-100 protein, and occasionally for glial fibrillary acidic protein but not for neuron specific enolase. Continuous transformation of myxoid tissue into cartilage together with corresponding reactivity for neuroectodermal marker proteins of myxoid cells and peripherally located chondrocytes suggest close relationship between myxoid cells and chondrocytes. The same criteria indicate relationship between myxoid cells and adipocytes. Coarse connective tissue, myxoid tissue, fibrous cartilage, and fat cells are functionally combined to absorb mechanical shock in the horse digital cushions.     

Meat inspection education in Finnish veterinary curriculum

This article describes the Finnish meat-inspection curriculum and presents an expert-panel evaluation of meat-inspection education. The work tasks of the meat-inspection veterinarian are challenging and include classical meat inspection, meat hygiene, hygiene control, and animal disease and welfare. The meat-inspection veterinarian is not only an inspector, which by itself is very demanding, but also an expert or "consultant" on food safety. The significant role of the meat-inspection veterinarian in society puts high demands on meat-inspection education, which should provide veterinary students with sufficient tools to perform meat inspection and hygiene control in slaughterhouses, cutting premises, and further processing plants. To be of high quality, such education must be evaluated from time to time. An expert panel evaluated Finnish undergraduate meat-inspection education and found that it provides veterinary students with good knowledge of meat inspection. The structure of the curriculum, with theoretical studies followed by four weeks of practice in a slaughterhouse, was considered vital for learning and for creating interest in meat inspection. The evaluation also revealed that certain subjects should receive greater emphasis and some new subjects should be introduced. Hygiene-control tasks, in particular, have increased and should receive more emphasis in education. Personnel management and interaction skills should be introduced into the curriculum as these skills influence all the duties of the meat-inspection veterinarian. This article outlines the subjects to be included in the modern, high-quality meat-inspection curriculum recommended by the expert panel.