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1. Using molecular typing to characterize bacterial pathogens associated with the dairy farm environment James A Higgins1, Christina Hohn2, and William R Hare1 USDA-ARS, Beltsville, MD1; NCSU Coll. Vet. Med., Raleigh, NC2 #197 Acknowledgements: Dr G. Sample, Dr. R. Joerger, J. Abell, L. Smith, Dr. J. Karns, Dr. J. Van Kessell Escherichia coli Staphylococcus aureus We are genotyping E. coli isolates from on-farm sources using a triplex PCR (Clermont et al. 2000). Based on the presence or absence of any of up to three amplicons, an isolate can be assigned to one of four genotypes: A, B1, B2, and D (gel image at left). When animals in the Beltsville Agricultural Research Center (BARC) herd contract mastitis, is the S. aureus responsible from outside the farm environment, or is the infection derived from strains already circulating in the herd as part of vaccine / therapeutics trials ? For molecular epidemiology analyses of S. aureus mastitis, we use spa (protein A) gene typing, common in clinical microbiology laboratories. This approach exploits nucleotide polymorphisms in the “X” region of the protein A gene (diagram above) and can resolve differences in S. aureus lineages with a fidelity comparable to PFGE. Key milk, manure, intestines n=34 ceca n= 51 trachea n= 16 6% 14% genotype A genotype B2 genotype B1 genotype D 37% 12% 29% 25% 44% 43% 37% 22% 31% Dairy Cow When E. coli isolated from different organs of chickens are genotyped, the data reveals that certain genotypes are predisposed to particular organs; for example, genotype B1 does not occur in the trachea, and B2, while present in the ingesta, does not occur in the mucosal lining of the cecum (pie charts at left). Genotype screeningof E. coli isolates from dairy cattle manure, internal organs, and milk indicates that genotype D (which includes E. coli O157:H7) is rare compared to other genotypes (pie chart above right). feces n = 40 mucosa n= 26 39% 25% 50% 46% 15% 15% 10% Chicken Bacillus spp. Results of spa typing PCR on S. aureus isolates from milk from two animals, and an isolate used in vaccine and transgenic animal trials (‘A’), are shown in the gel image above. When sequenced, the amplicons from these isolates segregate into two spa types, t267 (cow 178 and strain ‘A’) and t224 (cow 204 and other challenge strain ‘B’). BURP cluster analysis using Ridom StaphType™ software shows that these spa types cluster with clinical strains from Europe (above right). We also used PCR to screen our isolates for methicillin resistance (MRSA). As of Fall 2006, 11 of 12 suspected S. aureus isolates from 8 different animals have been typed as either t224 or t267 (table at right). This indicates that S. aureus associated with ongoing experimental trials is responsible for mastitis in our herd, rather than the introduction into the herd of strains from other sources. We use RAPD-PCR assays to genotype bacterial isolates and determine if distinct lineages are circulating in our animals. For example, four isolates of B. pumilus were recovered from the organs of a cow participating in a mastitis vaccine study that died due to infection with a contaminated inoculation of S. aureus. RAPD genotyping of the four isolates (A, B, C, D) revealed that they segregated into two clades (gel image left); thus, there are two sources or lineages of B. pumilus growing in the internal organs of the infected animal. These B. pumilus lineages are genetically distinct from those of B. licheniformis, also present in the same animal, and distinct from reference strain B. anthracis (gel at left). MRSA = methicillin resistant S. aureus; ND = not done