| A method for easy samples collection and disease detection is not only essential in monitoring dairy cow’s postpartum health but also in protecting the farm’s economic interests. Hepatic enzyme detection can offer direct evidence to postpartum cow’s liver function and is also related to some important metabolic diseases e.g. ketosis, which is due to excessive body fat mobilization. It is hard to detect and cure mastitis which caused by fungus factor in Gruangxi Autonomous Region. This research work includes two parts. rst, the relationship of some liver function enzymes between blood plasma and milk serum were analyzed during dairy cows lactation period so as to check whether the enzyme in milk whey can take the place of enzyme in plasma for the monitoring liver function. second, the development of a rapid diagnosis teststrip of cows mastitis caused by Candida albicans pathogen of was developed in dairy cows. All the study was done in order to make timely diagnosis and control the cow’s diseases. Part IALT, AST, GGT and ALP are four metabolic liver enzymes that play a vital role in animal liver diseases detection. In this assay we have measured the activity of ALT, AST, GGT and ALP in blood plasma and whey of healthy cows and analyzed their correlation. Method:35 Holstein cows were selected from a large-scale dairy farm and blood and milk samples were collected in 3～9 weeks after calving. The activity of ALT, AST, GGT and ALP were determined, respectively. Statistical correlation and regression analysis of the results were made.In the results:the activity of four enzymes in blood plasma and whey were extremely significant positively correlated. (ALT, r=0.852, P<0.001; AST, r=0.341, P<0.001, GGT, r=0.628, P<0.001; ALP, r=0.707, P<0.001). In blood plasma and whey, the ALT (P<0.05) activity was nearly same. The AST activities (P<0.001) in blood plasma were significantly higher than that in whey. However the activities of GGT and ALP (P<0.001) in blood plasma were significantly lower than those in whey. Further regression analysis was done for the activities of four enzymes in blood plasma and whey, (ALT, R2=0.72, P<0.001; AST, P<0.001; R2=0.11; GGT, R2= 0.43, P<0.001; ALP, R2=0.52, P<0.001), (with blood plasma as the dependent variable and whey as independent variable, described a unary function equation).The regression models are:ALT:y=1.78(x0.79), R2=0.72,P<0.001.AST:y=57.48e0.009x, R2=0.11, P<0.001.GGT:y=2.22(x0.34), R2=0.43,P<0.001.ALP:y=20.74 (x0.26), R2=0.52, P<0.001.Conclusion:In this study, both GGT and ALP activity in whey were higher than that in blood plasma. The activity of ALT in blood plasma was nearly same as that in whey. There were higher positive correlation between blood plasma and whey in 4 enzymes. In general, the detection of these enzymes except AST from whey could be a new approach with whey ALT activity analysis a possible alternative to blood plasma ALT activity analysis in monitoring liver function of dairy cows during early lactation. Part IIMastitis in dairy cow is one of the most serious diseases in the dairy industry and can lead to huge economic loss. In this paper, the infection rate and virulence of Candida albicans in cows within the Guangxi Autonomous Region China were investigated. The sensitivity of different drugs against Candida albicans was evaluated. At the same time, we conducted a study on the method of rapid diagnostic teststrip for detection of Candida albicans in milk for use in clinical practise and to take promptly measures.In this research, milk samples were collected from suspected Candida albicans infected cases in 6 farms of Guangxi Autonomous Region. Based on the characters of Candida albicans, identification of it were made though traditional pathogenic microbiology Gram staining, abacterial pellicle growth identification, pseudohypha identification, ink staining identification, TCTA color identification, PCR and biochemical tube identification. The incidence of Candida albicans mastitis were analyzed. Purified strains of Candida albicans were randomly selected to conduct poison test on mice. Six mice in the control group were injected physiological saline, 20 mice were underwent poison testing. The possible pathogenic capability of Candida albicans was examined by 48 hours.A variety of drug were tested:Western medicine (lincomycin, ciprofloxacin, kanamycin sulfate, gentamincin sulphate, micronomicin sulfate, ketoconazole, fluconzole), Chinese medicine (gallnut, cortex pseudolaricis, coptis chinensis, golden cypress, rhizoma anemarrhenae, syzygium aromaticum, cinnamon, garlic) and 16 kinds of drugs frequently used by cow farm (Lineo, kanamycin sulfate, Lincomycin Hydrochloride, etc). In the study about rapid diagnostic teststrip of Candida albicans: (1) Candida ablicans polyclonal antibody was made by immune New Zealand white rabbit with Candida ablicans as particulate antigen; immune Balb/c mice at the same time, positive hybridoma clones screened though cell fusion. The selected hybridoma conducted in vitro and vivo cultivation to get Candida ablicans monoclonal antibodies. (2) The preparation of colloidal gold particles and gold-labelled antibody, gold-labelled antibody with pad and the composition of the teststrip. (3) Testtrip in clinical application:detection of sensitivity and specificity of Candida ablicans and other bacteria. The results of this study:Identification of pathogenic microorganisms from the milk of cows who suffered from clinical intractable mastitis was used by microscopy, biochemical tube and other 5 kinds of methods. In addition 116 milk samples from 6 farms were checked,509 strains were obtained, among them 153 strains were fungus. The result of positive Candida ablicans from fungus:(1) Gram staining microscopy, comprising 20% of the total strain, and 67% from fungus. (2) Abacterial pellicle growth identification, comprising 20.08% from the total strain, and 96.73% from fungus. (3) Detection rate of pseudohyphae identification was 100%. (4) Detection rate of TCTA, comprising 20.43% from the total strain, and 67.97% from fungus. (5) From ink staining identification, comprising 23.97% of the total of strains and 79.74% from fungus. (6) By microbial PCR identification, comprising 23.18% from the total strain, and 77.12% from fungus. (7)Biochemical identification, from glucose identification, Candida ablicans produce acid and gas occupied 17.29% from the total strain, 57.51%, and Candida ablicans only produce acid occupied 5.11%, it occupied 16.99% from fungus; From mannose identification, Candida ablicans produce acid and gas occupied 18.27% from the total strain,60.78% from fungus and Candida ablicans only produce acid occupied 4.72%, comprising 15.69% from fungus; from galactose, maltose, fructose identification, it occupied 23.18%,22.00% and 22.The results of Candida albicans poison attack test and drug susceptibility test: in this study, a poison attack test of Candida ablicans were done on 20 mice with a fatality rate was 55% and pathogenicity rate was reached 100% (LD50 reached 1.97×105 cfu/g). Candida albicans were separated from all liver, ascites, lung and blood of mice, after autopsy, mice’s ascites increased and some organs were hyperemia and swelling. Different sizes of abscess nodules were showed in the surface of organs. By microscopic examination of the biopsy, inflammation and cell lesions were found on most organs. The results of the drug sensitive experiment showed:when the concentration of all kinds of traditional Chinese medicine (TCM) were at 10 mg/mL, the effect of garlic is the best. when the concentration of all kinds of traditional Chinese medicine (TCM) were at 20mg/mL, the effect of garlic and cortex phellodendri are the best, the extremely sensitive rate of them were 100% and 87.5%. When the concentration of all kinds of western medicine were at 10mg/mL, from the 7 kinds of western medicine in this study, ketoconazole and fluconazole are sensitive, the high sensitive rate of them were 37.5%,25%; From the 16 kinds of commonly used medicine in the farm, the moderate sensitive rate of Lineo, Gentamicin sulphate, Tong-Ru Xiao, Li-Tong Ru, Jun-DuKuang RuGong, Sheng-Zheng RuYan and Ru-YanKuang Ao Long were 43.75%,40.625%,15.625%, 9.375%,6.25%,6.25% and 3.125%. When the concentration of all kinds of western medicine were at 20mg/mL, from the 7 kinds of western medicine in this study, high sensitive rate of ketoconazole and fluconazole were 100% was and 96.88%, respectively; however the effect of 16 types medicine used in the farm were similar when the concentration at 10mg/mL. From the result of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of garlic and coptis chinensis against Candida albicans, it showed garlic has better effect than coptis chinensis.From polyclonal antibody by ELISA titer determination, rabbit serum still remained positive when diluted to 1:32000,1:1400000; After serum antibody purification, the results though nucleic acid spectrometer were 10.3mg/mL and 22.28mg/mL, respectively. The examination of rabbit serum via vertical SDS-PAGE electrophoresis showed specificity and highly purified antibody strip. Using Staphylococcus, Streptococcus and Escherichia coli as antigen, no cross reaction was fund by indirect ELISA method.(1) Hyperimmune serum can be obtain from Candida ablicans-immune Balb/c mice after 60 days. (2) 4 strains of stable hybridoma were obtained by cell fusion and screening; Ascites can be obtained after 7～8 days, its titer can reach more than 1:102400. Cross-reaction experiment results showed:no cross reaction was determined by using Staphylococcus, Streptococcus, Escherichia coli and other bacteria as antigen.2 times purified monoclonal antibody protein, its concentration reached 31.32mg/mL and 27.65mg/mL, respectively. Samples after purification were displayed by SDS-PAGE electrophoretic, the purification degree of samples were high.The results of teststrip experiment:Colloidal gold particles at 20nm are relatively stable. The optimum pH for protein antibodies combined with colloidal gold should be 8～9, while the optimal amount of combination antibody protein is 20～40μg/mL by Mey method. This experiment selected the best stabilizer 10% PEG20000. The optimal conditions are (Tris-HCl buffer,1% BSA,0.5% PEG20000,10% cane sugar and 2% Tween-20). This was used to dot immunogold filtration assay that determine the most suitable combination of NC membrane of quantity of package monoclonal antibody and sheep anti-rabbit IgG was 1:2 and 1:16, respectively. Analysis membrane of closed fluid was better effect by 3-5% skimmed milk powder and BSA, respectively. The sensitivity rate of the teststrip was 2×102cfu·mL-1. After storage for 10 days at 37℃, T line and C line of the test strip’s color will become weak, after storage for 30 days at room temperature, the coloration effect start decrease, but the stability stays good when storage at 4℃. The positive coincidence rate to clinical Candida ablicans of the test strip was 86.67%(13/15), Negative coincidence rate was 90%(9/10).Conclusion:A rapid diagnostic teststrip of candida ablicans was produced successfully in this study, this offers Candida ablicans mastitis of cows a rapid diagnostic methods, but it still remains to be test due to small samples of clinical experiment. |
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