Identification, Virulent Gene Detection And Gene Therapy Of Mastitis-causing Bacteria In Dairy Cows

Bovine mastitis remains to be one of the most important diseases of dairy industry. To investigate the prevalence of bovine mastitis pathogens,100 milk samples were collected from cows with clinical or sub-clinical mastitis in 5 farms in the Jiangsu Province. All the samples were submitted to bacterial isolation and biotyping. The experiments showed that Escherichia coli had a highest isolation rate (82%) in mastitis milk samples, followed by Streptococcus uberis (53%), Staphylococcus aureus (41%), Streptococcus dysgalactiae (29%) and Streptococcus agalactiae (27%). In addition, Str. uberis and S. aureus were more frequently associated with clinical mastitis than sub-clinical case, while the infection rates of other bacteria were similar. Interestingly, Staphylococcus epidermids and Staphylococcus saprophyticus, which were considered as environmental bacteria, had relatively high (15% and 10%, respectively) in the milk samples tested.To investigate the prevalence of antibiotics-resistant mastitis-causing bacteria in Jiangsu Province,30 Streptococcus uberis isolates,20 Streptococcus agalactiae isolates,10 Streptococcus dysgalactiae isolates and 40 Staphylococcus aureus isolates were submitted to antibiotics sensitivity test. Among 16 different antibiotics tested, most of the Streptococcus were highly sensitive to minocycline and ciprofloxacin, but resistant to chemitrim and oxacillin. While most of Staphylococcus aureus were highly sensitive to vancomycin and spectinomycin, but resistant to furantoin, amphemycin and minocycline.To investigate the prevalence of the virulent genes of mastitis-causing Escherichia coli, 123 islates from different dairy farms were submitted to PCR detection for genes coding for toxins, pathogenicity islands, adhesin, hemolysin and outer membrane protein. The results showed that 76 (61.8%) isolates were positive for toxins gene, among them ST2 was the most common gene (24.4%), followed by Cnf2 (20.3%) and LT1 (17.1%). Among 123 isolates tested,68 isolates (55.3%) were positive for PAI-associated genes, including 36.6% for ETT2,14.6% for HPI and 4.1% for aer, respectively. Adhesin-associated gene detection showed that only 8 isolates were positive, aomong which 3 isolates carried P fimbriae,3 contained S fimbriae and 2 had F17a fimbriae. None of the isolates were PCR-positive for ST1, Cnf1, afaD8, afaE8, CS31A and F17b genes. In addition, aomong 123 isolates tested, 8 isolates produced hemolysin and 21 isolates produced serum resistance factor. To facilitate clinical application of the recombinant vector for treating bovine mastitis, 500μg of the recombinant plasmid was injected into each quarter of milk-dryng or diseased cows using a needle-free injector. After repeat injection on the following day, their milk samples were collected and somatic cells numbers were counted by CMT assay. The results showed>90% prophylactic efficacy for healthy cows and 81.8% cure rate for the diseased animals. These data suggest that the needle-free injector can replace conventional injection method for treating bovine mastitis without animal restraint and advantage of quick and massive injection.To further evaluate the biosafety of gene therapy for treating bovine mastitis, the recombinant plasmid harboring human lysozyme cDNA was injected into 18 lactating cows with subclinical mastitis with an injection dose of 400μg,600μg or 800μg/quarter or into 6 healthy cows with an injection dose of 400μg/quarter. After repeat injection with the same daose, the experimental animals were observed for 28 days for changes in psychosis, appetite, heart rate, respiratory rate and anus temperature and therapeutic efficacy was determined based on somatic cell numbers of the milk samples. The data showed that all animals were normal in terms psychosis, appetite, heart rate, respiratory rate and anus temperature. Three additional lactating cows with subclinical mastitis, as well as two healthy cows, were injected twice with 1600μg (400μg/quarter) of the recombinant vector and their milk, feces, urine, litte and water samples were collected. On day 20 after the secondary injection, the experimental animals were sacrified and their heart, liver, spleen, lung, kidney, muscle and mammary gland samples were collected. PCR detection showed that, except for the milk samples to be positive 24 h after secondary injection, all other samples were negative. These data suggest that intramammary injection of the recombinant vector is safe for both healthy and diseased cows without in vivo transmission and enviorment contamination of the plasmid.