| Filter paper was widely used to collect and store liquid biological samples for its portable, safety and economic properties. This study was conducted in order to systematically evaluate the suitability, sensitivity and specificity of milk samples collected on filter paper disks. The filter paper disks (5.0 mm in diameter) were made from Xinhua No.3 Chromatographic filter paper. Each filter paper disks can absorb 24.99±0.05μL milk samples. A series diagnosis protocols based on lactose, protein and bacteria information of milk samples collected on filter paper disks were proposed.A regional investigation was conducted in Hangzhou, China to estimate the prevalence of bovine mastitis (BM) and its association with the seasons, parity, period postpartum, milk fat, milk protein and milk production. A total of 4388 milk samples were analyzed for the SCC. A linear mixed model with restricted maximum likelihood (REML) was applied to analyse the association between somatic cell counts (SCC) with the factors mentioned above. The results presented that month (p < 0.01), parity (p < 0.01), milk production (p < 0.01), milk fat (p = 0.049), and milk protein (p < 0.01) show significantly associated with the SCC. A SCC response model was established. It is turned out an efficient model tested with a colineartic diagnostics. The predicted values by it were similar to the origin (p > 0.05). A logistic regression analysis demonstrated that the factors (month, parity, milk fat, milk protein, milk production) including in this survey contribute to the risk of mastitis in Holstein cows (p < 0.05). Based on these analysis, the related risk estimation models were worked out. The models revealed that in Aug and Sept the cows are prone to develop mastitis, while in the suitable milk production (in 20-30 kg/d), milk fat(in 3.5-5.0%) and milk protein(2.6-3.1%) patterns, the mastitic risk are very low.To evaluate the influence of time and temperature for storage on the lactose of milk samples on filter paper disks, a DNS assay was employed to analyze lactose concentration under different stored conditions. The results indicated that intra-sample reproducibility of lactose concentration in the milk samples collected on filter papers from the same individual was found to be 13.6 percent, while for the tube sampling milk was 4.7 percent. There was no significant difference between results obtained from tube sampling milk and filter paper disk collected milk (p > 0.05) . Storage at different temperatures (p > 0.05) and for various periods (p > 0.05) showed little change in lactose concentration of milk sample preserved in filter paper disks. There was a little decrease in concentrations of lactose in milk on the filter paper when compared to tubes collected milk and a high standard deviation probably because of loss in the extraction process. Milk collected on filter paper disks could be used to screen subclinical mastitis cases using milk lactose as an indicator.Bradford protein assay was employed to evaluate the accuracy, suitability and coefficient of variation of the milk protein collected on filter paper disks. The intra-sample coefficient of variation (CV) was 4.41%. The protein level significantly decreased in comparison with the results of raw milks and only 83.6% protein could be recovered. The results presented that the protein concentration was significantly changed by time for storage and pretreatment with media. The protein levels on filter paper disks gradually decreased with the time increasing. The results presented that the filter paper disks pretreated with different media give a great variation in the protein level of milk samples kept on it.To acquire the viable Strep. agalactiae and Staph. aureus and PCR to use artificially contaminated milk samples collected on filter paper disks, milk samples to analyze were dried and kept one to four weeks at room temperature. After that, they were cultured for PCR analysis. The results revealed that the recovery rate of Strep. agalactiae and Staph. aureus on dried filter paper disks was influenced by the pretreatment with media and storage period. The recovery test suggested that not all the bacteria artificially contaminated milk samples on the filter paper disks could be recovery. The recovery rate was 86.6% and 83.5%, respectively. After that, a PCR based on 16-23S intergenic spacer region of Strep agalactiae was performed. The methods can distinguish the Strep. agalactiae from major pathogens of bovine mastitis at a 2×10~3 colony forming units ( CFU)/mL level, which show similar sensitivity to the liquid milk samples. It also presented that the milk samples collected on filter paper disks could be kept at room temperature for one to four weeks and had little negative effect on its sensitivity and specificity. The field test showed that the sensitivity and specificity of diagnosis was 96.15% and 98.60% respectively. The primers SAISR1 and SAISR2 based on 16-23S intergenic region can distinguish the Staph. aureus from major pathogens of bovine mastitis at 2×10~4 and 2×10~3 CFU/mL level, which show similar sensitivity to the method by liquid milk samples, respectively. It also presented that the milk samples collected on filter paper disks can be kept at room temperature for one to four weeks and have little negative effect on its sensitivity and specificity. The field test based on 193 milk samples showed that the sensitivity and specificity of SAISR2 was 89.19% and 98.08%, respectively. A Kappa analysis presented that PCR method based on milk samples collected on filter paper disks was in good consistent with the bacterial culture in vitro based on correspondent liquid milk. Thus, there can be no doubt that the PCR protocol based on milk samples collected paper disks will be a rapid and economic procedure for the detection of bovine mastitis.SDS-PAGE based on milk samples collected on filter paper disks were conducted to evaluate the modification of milk protein components under subclinical mastitis. The results revealed that the protein with small MW can be good separated (R_f > 0.2), while the molecules with large MW presented low R_f and near the loading portion. The bands with a value of R_f>0.2 formed six main band groups in the PAGE patterns: 0.51(A), 0.62(B) , 0.72(C) , 0.78(D) , 0.84(E) and 0.98(F) . Band groups A, B and F presented no significant correlation with the SCC and major pathogens, while the band groups C, D and E show marked correlation with SCC(r = 0.795, p < 0.01), HMT(r = 0.471, p < 0.01) and the major pathogens(r = 0.389, p < 0.01). The sensitivity and specificity of PAGE was 100% and 55.66% respectively. The PAGE methods is in a moderate consistent with pathogens culture (kappa=0.515, p < 0.05). If the CNS was included in the bacterial culture results, the sensitivity and specificity was 90% and 70.59% respectively, with a Kappa value of 0.622 (p < 0.05). PAGE method showed similar sensitivity and specificity with SCC and more sensitive than HMT in the diagnoses of subclinical bovine mastitis.As described above, lactose level, PCR on major pathogens and PAGE of protein could be used to detect BM using milk samples collected on filter paper disks. In addition, the specificity of PCR and the sensitivity of PAGE were discussed. |
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