Performance evaluation of a rapid immunochromatographic test kit in detecting bovine mastitis-causing streptococci.

Mastitis causes significant economic losses to the dairy industry due to decreased milk production in infected cows. Identification of mastitis-causing pathogens, such as streptococci, is necessary for selecting an effective antibiotic for treating mastitis. Although bacterial cultivation is widely used for pathogen identification, it requires more than 24 hr to complete. Contrarily, Lateral flow assays are simple, rapid, and inexpensive testing procedures. In this study, the effectiveness of an immunochromatographic test kit for detecting streptococci in milk samples from cows with clinical mastitis was evaluated as an alternative to bacterial cultivation. The performance of the immunochromatographic test kit for detecting mastitis-causing pathogens was compared with that of bacterial cultivation and real-time quantitative polymerase chain reaction (qPCR). The sensitivity and specificity of the immunochromatographic test kit were 0.800 and 0.875, respectively, compared with bacterial cultivation. Additionally, the κ statistic values of the immunochromatographic test kit was 0.667, indicating substantial agreement with the results of bacterial cultivation. Statistically, sensitivity and specificity of the immunochromatographic kit and real-time qPCR did not differ significantly; thus, the immunochromatographic test kit detected mastitis-causing streptococci as effectively as real-time qPCR. Therefore, the immunochromatographic kit is a rapid, inexpensive, and simple method for detecting streptococci and contributes to the timely selection of appropriate antibiotics for treatment and promotes early recovery from mastitis.

Differences in antimicrobial components between bacterial culture-positive and culture-negative bovine clinical mastitis milk.

A bacterial culture of milk is the most common test to determine the presence of mastitis-causing pathogens, which informs appropriate treatment. However, a certain proportion of clinical mastitis milk shows no growth of any mastitis-causing pathogens. We hypothesized that bacterial culture-negative clinical mastitis milk is associated with the activity of antimicrobial components contained in the milk. In this study, the differences in antimicrobial components (lactoferrin, transferrin, lysozyme, lactoperoxidase, and lingual antimicrobial peptide [LAP]) between bacterial culture-positive and culture-negative bovine clinical mastitis milk were investigated using Holstein cows. Our results showed that 37 out of 71 samples of clinical mastitis milk had negative bacterial cultures. The LAP concentration in bacterial culture-negative milk was lower than that in positive milk (31.95 ± 1.64 nM vs. 42.85 ± 4.01 nM). In contrast, the lysozyme concentration in bacterial culture-negative milk was higher than that in positive milk (0.76 ± 0.15 µg/ml vs. 0.42 ± 0.06 µg/ml). In conclusion, the concentration of antimicrobial components was different between bacterial culture-positive and culture-negative bovine clinical mastitis milk, which suggests that antimicrobial components are related to bacterial culture results.

Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter.

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10(6) cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10(6) cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10-fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.

Relationship between concentration of lingual antimicrobial peptide and somatic cell count in milk of dairy cows.

Lingual antimicrobial peptide (LAP) belongs to the ß-defensin family in cattle and is found in milk. LAP concentrations increase in milk from mastitic udders; however, the relationship between LAP concentrations and the somatic cell count (SCC) in milk remains to be elucidated in detail. Therefore, the present study was undertaken to investigate the relationship between LAP concentrations and the SCC in bovine milk to assess whether LAP may be used as an indicator of SCC. Milk was collected from 66 udders showing various SCCs. The SCC and LAP concentrations were measured in the milk. A significantly higher LAP concentration was observed in milk having 500-5000 × 10(3)cells/ml and >5000 × 10(3)cells/ml SCC groups than in lower SCC groups (<50 × 10(3)cells/ml and 50-500 × 10(3)cells/ml). A significantly positive correlation between LAP concentrations and SCCs in milk was observed (r=0.68). In milk samples with >26 nM of LAP, 92.0% of milk samples had high SCCs (>200 × 10(3)cells/ml). The concentration of LAP in milk infected with Staphylococcus aureus, Streptococcus bovis, Streptococcus dysgalactiae, and Escherichia coli was significantly higher than that in uninfected milk. These results suggest that the concentration of LAP can be a useful indicator of the SCC in dairy cows.

Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan.

This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species.

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle.

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.