Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Streptococcus suis serotype 2 is the main cause of zoonotic S. suis infection despite the fact that other serotypes are frequently isolated from diseased pigs. Studies comparing concurrent invasive human and pig isolates from a single geographical location are lacking. We compared the population structures of invasive S. suis strains isolated between 1986 and 2008 from human patients (N = 24) and from pigs with invasive disease (N = 124) in the Netherlands by serotyping and multi locus sequence typing (MLST). Fifty-six percent of pig isolates were of serotype 9 belonging to 15 clonal complexes (CCs) or singleton sequence types (ST). In contrast, all human isolates were of serotype 2 and belonged to two non-overlapping clonal complexes CC1 (58%) and CC20 (42%). The proportion of serotype 2 isolates among S. suis strains isolated from humans was significantly higher than among strains isolated from pigs (24/24 vs. 29/124; P<0.0001). This difference remained significant when only strains within CC1 and CC20 were considered (24/24 vs. 27/37,P = 0.004). The Simpson diversity index of the S. suis population isolated from humans (0.598) was smaller than of the population isolated from pigs (0.765, P = 0.05) indicating that the S. suis population isolated from infected pigs was more diverse than the S. suis population isolated from human patients. S. suis serotype 2 strains of CC20 were all negative in a PCR for detection of genes encoding extracellular protein factor (EF) variants. These data indicate that the polysaccharide capsule is an important correlate of human S. suis infection, irrespective of the ST and EF encoding gene type of S. suis strains.

A novel PCR-based restriction fragment length polymorphism analysis of the Brachyspira nox gene was developed. The restriction patterns for Brachyspira hyodysenteriae, B. pilosicoli, B. intermedia, B. murdochii, and B. innocens were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust.The porcine intestinal tract is frequently colonized by different Brachyspira species. Brachyspira hyodysenteriae is the causative agent of swine dysentery, and B. pilosicoli causes intestinal spirochetosis (9). The other species, namely, B. intermedia, B. murdochii, and B. innocens, are considered nonpathogenic (9). Due to differences in pathogenicity (9) and the zoonotic potential of B. pilosicoli (15), rapid and user-friendly differentiation between species is important with respect to the economic use of medication, preharvest food safety, and animal trade.Currently, porcine Brachyspira isolates are differentiated based on phenotypic criteria, including intensity of hemolysis, indole production, hippurate hydrolysis, and activities of ␣-galactosidase, ␣-glucosidase, and ␤-glucosidase (6, 14). In addition, a positive ring phenomenon is indicative of B. hyodysenteriae (11). Due to variable results in these tests and difficulty in correctly assessing the intensity of hemolysis, it seems desirable to replace phenotypic identification schemes with genotypic methods. The currently used sequencing analyses of 16S or 23S ribosomal DNA (rDNA), the nox gene, or unidentified DNA segments or PCR methods based on these data (1,4,5,10,12,13) either are not practical for routine veterinary diagnostic purposes or are confined to the identification of selected species only. Therefore, the purpose of the present study was to develop a robust and user-friendly method for the identification of all porcine Brachyspira species and to evaluate its performance in routine diagnostic work.

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