Objectives: To identify the staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine milk, and examine the genetic relatedness between MRSA from bovine milk and MRSA from human isolates. Methods: Antimicrobial susceptibility tests were performed on MRSA isolated from bovine milk. PCR and sequencing analysis were performed to determine the SCCmec type of MRSA, and to confirm their toxin carriage. Genetic relatedness among the bovine isolates and between bovine and human isolates was detected with PFGE and multilocus sequence typing (MLST). Results: Fourteen MRSA and a silent mecA-carrying methicillin-susceptible S. aureus (smMSSA) were isolated from the milk of cows with an isolation ratio of 0.18%. SCCmec of 14 MRSA strains were designated as new subtype IVg, and one smMSSA strain was not classified. All 14 MRSA strains shared Panton-Valentine leucocidin (PVL) and staphylococcal enterotoxin D (SED), SEI and SEJ; the smMSSA strain had only PVL. All MRSA and smMSSA isolates showed no multidrug resistance and had community-acquired MRSA (CA-MRSA) characteristics. PFGE revealed that all isolates except the smMSSA belonged to the same genetic lineage, and MLST analysis showed that they had no genetic relatedness with CA-MRSA which had caused human infection in Korea. Conclusions: MRSA isolated from bovine milk harboured a unique SCCmec subtype, and they may not be correlated with the emergence of CA-MRSA in human infection in Korea.

An ileal cannulation model was developed in conjunction with a flow cytometric assay to gain a better understanding of the mechanisms of immunopathogenesis of Johne's disease caused byMycobacterium aviumsubsp.paratuberculosis. Initial studies with calves showed thatM. aviumsubsp.paratuberculosisDNA is detectable by PCR in ileal biopsies during the first months following experimental infection. Inflammatory lesions were not detected on endoscopic evaluation up to 8 months postexperimental infection.M. aviumsubsp.paratuberculosisDNA was detected in multiple tissues at necropsy 8 months postinfection. Examination of the activation status of epithelial lymphocytes from the jejunum and ileum from infected and control animals at necropsy revealed that none of the major subsets of lymphocytes (NK, CD2⁺, and CD2⁻γδ T lymphocytes, or CD4 and CD8 αβ T lymphocytes) expressed activation molecules CD25, CD26, CD71, ACT1, or ACT16. Subsets of CD4 and CD8 T lymphocytes from control and infected animals expressed CD26. The majority of CD4 and CD8 T lymphocytes expressed CD45R0, the memory T-lymphocyte marker. An immune response toM. aviumsubsp.paratuberculosiswas detected by 3 months postinfection, dominated by a strong proliferative response of CD4 memory T lymphocytes. The findings indicate an immune response develops following initial exposure toM. aviumsubsp.paratuberculosisthat controls but does not eliminate the pathogen. This persistence ofM. aviumsubsp.paratuberculosispossibly leads to erosion and dysregulation of protective immunity at later time points postinfection. Continuous access to the ileum offers an opportunity to elucidate the cellular and molecular events leading to immune dysregulation and development of chronic inflammatory ileitis.

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10 ⁻⁷ to 2.9 × 10 ⁻⁷ . Three genes were selected for mutagenesis: pknG and relA , which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis , and lsr2 , a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis ( M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp . The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.