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Campylobacteriosis is the most frequent food-borne human enteritis. The major source for infection with Campylobacter spp. is broiler meat. Risk assessments consider the reduction of Campylobacter in primary production to be most beneficial for human health. The aim of this study was to test the efficacy of a bacteriophage application under commercial conditions which had proved to be effective in previous noncommercial studies under controlled experimental conditions. A phage cocktail for Campylobacter reduction was tested on three commercial broiler farms each with a control and an experimental group. Colonization of Campylobacter was confirmed prior to phage application in fecal samples. Subsequently, a phage cocktail was applied via drinking water in the experimental group (log 10 5.8 to 7.5 PFU/bird). One day after phage application, Campylobacter counts of one experimental group were reduced under the detection limit (<50 CFU/g, P ‫؍‬ 0.0140) in fecal samples. At slaughter, a significant reduction of >log 10 3.2 CFU/g cecal content compared to the control was still detected (P ‫؍‬ 0.0011). No significant reduction was observed in the experimental groups of the other trials. However, a significant drop in cecal Campylobacter counts occurred in a phage-contaminated control. These results suggest that maximum reduction of Campylobacter at the slaughterhouse might be achieved by phage application 1 to 4 days prior to slaughter.

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The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

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In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan mgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10 2 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, costeffective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culturebased detection of M. avium subsp. paratuberculosis is timeconsuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12,43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7,8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a onestep and closed-tube reaction (13).As a molecular reference marker for the confirm...

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.The use of PCR as a diagnostic tool for the detection of bacterial pathogens has become more frequent during the past few years. The PCR technique allows the amplification of preselected, species-specific DNA regions which can be used for genotypic identification of bacteria. A molecule most suited for these purposes appears to be the gene encoding the 16S rRNA. According to Bentley et al. (4) and Bentley and Leigh (6), the sequence variability of the V2 region of the 16S rRNA allows a differentiation of 31 species of the genus Streptococcus, including the species S. uberis and S. parauberis. Both species, formerly classified as S. uberis types I and II (19), are well known as causative agents of bovine mastitis. According to biochemical and serological characteristics, the two species are almost indistinguishable (19). A molecular identification of both species can be performed by analysis of restriction fragment length polymorphisms (RFLPs) of the 16S rRNA gene (13,14,18) or by the use of species-specific oligonucleotide probes (5, 6). A major disadvantage of these procedures is the additional manipulation of the samples subsequent to the PCR and the time-consuming preparation of the gene probes. In the present study, species-specific regions of the 16S and 23S rRNA genes and the 16S-23S rRNA intergenic spacer region of S. uberis and S. parauberis were used to construct speciesspecific oligonucleotide primers. These oligonucleotide primers were used to identify and differentiate both species.A total of 17 S. uberis and 2 S. parauberis strains were used in this study, all of which were obtained from the Institute's strain collection. The cultures were identified and differentiated into both species as described previously (15, 18). In addition, 51 streptococci obtained from routine mastitis diagnostics and also S. parauberis strain 94/16 were included. The latter, kindly obtained from J. F. Fernández-Garayzábal (Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain), was originally isolated from a diseased turbot (9). The cultures isolated from routine diagnostics were preliminarily identified as S. uberis by using conventional methods (15, 16). These methods included carbohydrate fermentation tests for arabinose, fructose, inulin, lactose maltose, mannitol, raffinose, ribose, saccharose, salicin, sorbitol, and trehalose; esculin and sodium hippurate hydrolysis; determination of arginine hydrolysis; and analysis of the enzymes ␤-D-glucuronidase, pyrrolydonylaminopeptidase, and hyaluronidase. The ␤-D-glucuronidase and pyrrolydonylaminopeptidase enzyme activities were inves...