Thesis (Ph.D.)--Chulalongkorn University, 2007

The uses of natural non-toxic antibacterial agents from plant source are expected to replace the limited uses and contaminated residues of excessive antibiotics uses in dairy farm. This research aimed to find a novel antibacterial agent against bacterial isolates from mastitic cows. Bactericidal activity against mastitis-causing bacteria and mastitis preventing efficacy of teat antiseptic of polysaccharide gel (PG) from fruit-rinds of durian (Durio zibethinus Murr.) were investigated. PG teat dip was formulated as a teat sanitizer by using 2.5% PG (w/v) in Ringer’s solution together with 10% glycerin and 10% propylene glycol. The PG teat dip was tested for bactericidal activity against nine field bacterial isolates causing mastitis by in vitro time-kill analysis. The PG teat dip illustrated good killing effect to all tested isolates including Staphylococcus aureus, S. chromogenes, S. simulans, Streptococcus agalactiae, S. dysagalactiae, S. acidominimus, S. uberis, S. bovis, S. porcinus, Escherichia coli, Klebsiella sp. and Pseudomonas sp. The reduction in cfu/ml values of tested Streptococci more than 90% was observed within 1 minute. The efficacy of PG teat dip was evaluated for mastitis prevention in vivo by experimental challenge and natural exposure field trials. The PG teat dip reduced percentage incidence density for new intramammary infection (IMI) by 100% (p < 0.05) with both S. aureus and S. agalactiae as demonstrated by using the experimental challenge protocol. The results of natural exposure study showed that the incidence rates between PG teat dip and iodophor (positive control) group was not different (p > 0.05). Soft teat skin and no irritation of teat end were also observed during the study period. PG teat dip effectively prevented mastitis infection against contagious bacteria was observed by natural exposure study in vivo. The results indicated that the antiseptic teat dip of PG from durian rinds has potential to prevent bovine IMI in lactating cows. Immunomodulatory activity of PG was also evaluated. Cytotoxic effect of PG was tested by XTT reduction method together with flow cytometric procedure with propidium iodide on bovine mammary leukocytes, no cytotoxic effect was observed with 1% w/v PG, however, at 2.5% w/v PG showed moderate cytotoxic effect. The immunostimulating potential of PG on bovine mammary gland was assessed by in vitro phagocytosis assay using nitroblue tetrazolium (NBT) reduction. The result indicated that at 1% w/v PG caused the highest phagocytic activation of bovine mammary leukocytes compared with 0.5, 2.5% w/v PG treated groups and its control. The scanning electron micrograph of macrophages after exposured with PG also showed a similar result, pseudopodal formation of macrophage was high with 1% w/v PG. Therefore 1% PG was used for in vivo study, intramammary infusion of 1% PG in non-lactating cows was performed to evaluated the immune system stimulation. The results showed that a single intramammary infusion of three different doses at 12.5, 25 and 62.5 ml of 1% PG caused a transient elevation of the somatic cell count by increasing number of CD14 and MHC class II-positive leucocytes in udder secretions. The total white blood cells count was significantly increased at day 8 post-infusion; however, differential counts of peripheral blood samples were not significantly different throughout the study in comparison with normal control cows. Intramammary infusion of PG into the bovine udder potentially enhanced some local immune responses in non-lactating cows. Bactericidal and immunostimulating activity of PG may benefit in reduction the risk of udder infections during the dry period. The results suggested that PG may be use as an alternative attractive teat antiseptic for routine use in dairy farm, since using of antibiotic and chemical agents in livestock will be restricted in near future.