Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance

Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.</p> <p>Methods</p> <p>Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.</p> <p>Results</p> <p>MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.</p> <p>Conclusions</p> <p>The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC <it>in vivo</it>. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.</p

Evaluation of PCR Methods for Rapid Identification and Differentiation of Streptococcus uberis and Streptococcus parauberis

Streptococcus uberis and Streptococcus parauberis reference strains and isolates obtained from routine diagnostics were investigated by PCR with oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene, the 23S rRNA gene, and the 16S-23S rRNA intergenic spacer region of both species. All three primer pairs allowed an identification of 67 isolates as S. uberis and 4 isolates as S. parauberis

Diagnosis of paratuberculosis in cattle from a dairy region in Columbia

ABSTRACT In Colombia serum and fecal samples from 307 asymptomatic lactating Holstein cows over three years of age from 14 herds with no previous diagnosis of paratuberculosis were taken. All serum samples were analyzed with a lipoarabinomannan based-ELISA (ELISA A). Positive and doubtful samples in ELISA A were analyzed with a protoplasmic antigens based-ELISA (ELISA B), including pre-absorption with Mycobacterium phlei. Fecal samples from animals positive in ELISA A were analyzed using a nested IS900-PCR and a F57 / ISMav2-real-time PCR. Fecal samples of animals from ELISA A-seropositive herds were decontaminated with 0.75% Hexadecylpyridinium Chloride and cultured on Herrold´s Yolk Agar medium. The same samples were decontaminated later with 4% NaOH and 5% oxalic acid and cultured on Lowestein-Jensen medium. Ten percent (31/315) of the samples and 70% (10/14) of the herds were positive with ELISA A. Only two animals of two different herds were positive with ELISA B. Six fecal samples were positive with PCR and only one was simultaneously positive in the two PCR types. Serological and PCR results did not always coincide. Cultivation was negative for paratuberculosis in all samples inoculated. However Mycobacterium engbaekii was isolated from LJ medium. Results confirm the presence of paratuberculosis in dairy herds in Colombia and demonstrate the limitations of available diagnostic tests for detection of subclinical infections, the determinant influence of ELISA type used, the low bacterial shedding in the cattle feces examined, the possible roll of other mycobacteria and the effect of conservation on the diagnosis of paratuberculosis

Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia

The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme–linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis

Identification and Molecular Characterization of Beta-Hemolytic Streptococci Isolated from Harbor Seals (Phoca vitulina) and Grey Seals (Halichoerus grypus) of the German North and Baltic Seas

Bacteriological investigations of seals of the German North and Baltic seas resulted in the isolation of bacteria of the genus Streptococcus belonging to Lancefield's serological groups C, F, and L. According to biochemical, serological, and 16S ribosomal DNA analysis, the group C and group F streptococci were identified as Streptococcus phocae. The group L streptococci could be classified as Streptococcus dysgalactiae subsp. dysgalactiae