| Bovine mastitis is the most costly disease in the dairy industry and can be caused by at least 135 different agents, mostly bacteria. Antibiotic treatment has been widely used for mastitis treatment and control, but it has been a hotly debating issue all over the world concerning drug-resistant bacteria and the safety of residue antibiotics to human health.To optimize the genetic drug for treating bovine mastitis, the cDNA for human lysozyme (hLYZ) was subcloned into a mammary gland-specific expression vector p215C3 and a eukaryotic expression vector pcDNAK, respectively. The three recombinant expression vectors p205C3LYZ, p215C3LYZ and pcDNAKLYZ were injected into lactating mice via vein route. Micrococcal lysis assay and Remazol Brilliant Blue R (RBB-R)-labeled colorimetric assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands.To test whether the recombinant vectors can express active lysozyme in bovine mammary glands, eukaryotic expression vector pcDNAKLYZ or p215C3LYZ was injected into milk cisterna of the normal mammary gland by different means and expression of gene of interest was demonstrated by using RBB-R-labeled colorimetric assay. The results indicate that both the two recombinant vectors can express active enzyme in milk, which lasted for at least 20 days. The treatments with different routes, doses and injection times showed that effectiveness of quarter base injection was better than that of intracisternal injection, which was dose- and injection time-dependent. Fermentation experiments showed that the low level of recombinant lysozyme expressed in milk had no significant influence on milk fermentation and thus yogurt production.To evaluate the feasibility of recombinant vecotrs for treating dairy cow mastitisresistant to antibiotic treatment, the mammary gland-specific expression vector p215C3LYZ was injected into the milk cisterna or base of each quarter of the dairy cows with clinical or subclinical disease and the effectiveness of the treatment was evaluated on the basis of clinical changes and milk somatic cell counting using the standard C.M.T assay. After daily injection of 400(μg of the recombinant vector for successive 2 days, >90% of the treated cows become clinically normal with about 30% cure rate at 10 day post-treatment.To explore the feasibility of preventing cow mastitis during drying off, another eukaryotic expression vector pcDNAKLYZ was injected into the base of each quarter of the dairy cows. The result showed that the prevention effectiveness of the genetic drug was better than that of antibiotic treatment.To evaluate the biosafety of recombinant vectors for clinical use, different doses of the recombinant vector were injected into mice, calves and dairy cows with mastitis, respectively, and its dynamics of degradation in mouse stomach was investigated by agarose gel electrophoresis, polymerase chain reaction and transformant isolation from feces samples. The results showed that injection of the vector had no obvious side effect on the animals tested. The vector was not detectable in mouse stomach one minute after oral administration even by sensitive PCR and no vector-transformant was isolated from the feces samples. These data indicate that the recombmant vector is safe for dairy cow use without evidence of horizontal transmission and thus can be further developed as a new drug for dairy cow mastitis.These results indicate that injection of the recombinant plasmid containing hLYZ cDNA can be used as an alternative strategy for treating and preventing dairy cow mastitis.
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