Preliminary Studies On Gene Therapy For Dairy Cow Mastitis Using Human Lysozyme Gene

Dairy cow mastitis is one of the most harmful and common cow diseases. It causes great loss every year in the world. Because of the complexity of the causative bacteria, dairy cow mastitis was very difficult to treat.To obtain a functional cDNA for human lysozyme (hLYZ), PCR was carried out using the pooled first-stranded cDNA from human mammary gland as the template and 1.5 kb dscDNA was amplified by PCR. The cDNA was subcloned into pGEM-T vector and submitted to sequence analysis. The deduced amino acid sequence alignment showed that the cDNA was 100% identical to that cloned from human placenta, macrophages and colon, and differed by 1 and 6 amino acids from that cloned from histiocytes and Chinese placenta, respectively. The cDNA was subcloned into one eukaryotic expression vector and one mammary gland-specific expression vector. The recombinant vector, pcDNASLYZ, was transfected into COS-1 cells following mixing with branched 25 kDa polyethylenimine. Expression of hLYZ was revealed by indirect immunofluorecemce using specific antibody against hLYZ. Another recombinant vector, pCXLYZ, was injected into lactational mice via tail vein route. Expression of hLYZ at level of 69.3μg/ml was detected in the milk samples from the gene-injected mouse using micrococcal lysis assay.To treat cow mastitis by gene therapy, the hLYZ cDNA was subcloned into self-constructed mammary gland-specific expression vector p205C3 and the recombinant vector p205C3LYZ was injected into milk pools of the mammary gland. The hLYZ activity at level of >1.18μg/ml was detected in the milk samples from the plasmid-injected mammary gland, which maintained for at least 8 days. In treatment 1, 3x200μg of p205C3LYZ was injected into the milk pools of cows with mastitis and the effectiveness of the treatment was evaluated by C.M.T. of the milk samples, with the efficiency of 4 714, 14 714, 12/14 at the 2nd, 6th and 10th day post injection, respectively. In treatment 2, the effectiveness of gene therapy was compared with that of antibiotics treatment and Chinese herbs treatment, which showed that gene therapy and antibiotics treatment had an effectiveness of 84.6%(19/22) and 88.9%(8/9), respectively, both of which were significantly higher than that of ruyanxiao (69.2%,9/13), a preparation of Chinese herbs. After the treatments, total bacterial numbers, as well as representative causative bacterial numbers (such as staphylococcus, streptococcus and E.coli), of the samples of the three groups decreased significantly. The milk yields of the three treatment groups increased significantly during and post treatment compared to pretreatments. The clinical cow mastitis was treated by the same way with the effectiveness of 55.6%(10/18). After treatment, the milk recovered to normal color and the clinical syndrome disappeared. These dada demonstrate that the recombinant plasmid containing hLYZ cDNA can be used as a new drug for preventing and curing dairy cow mastitis.