# Presentation: 317887

## 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**

- 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.

- 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 StaphTypeTM 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.

- 	**genotype A****	**		**genotype B2**
- 	**genotype B1****	**	**genotype D**

- 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 screening* *of *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).

- 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).

- Acknowledgements: Dr G. Sample, Dr. R. Joerger, J. Abell, L. Smith, Dr. J. Karns, Dr. J. Van Kessell

- MRSA = methicillin resistant S. aureus; ND = not done

- Chicken

- ceca n= 51

- trachea n= 16

- mucosa n= 26

**37%**

**12%**

**29%**

**22%**

**31%**

**44%**

**25%**

**39%**

**46%**

**15%**

- 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).

- Dairy Cow

- milk, manure, intestines n=34

**43%**

**37%**

**14%**

**6%**

**Key**

***Staphylococcus aureus***

***Escherichia coli***

***Bacillus***** spp.**

**50%**

**25%**

**15%**

**10%**

- feces n = 40