Evaluation of a rapid coliform detection kit from clinical mastitis milk using colloidal gold nanoparticle-based immunochromatographic strips.

The accurate identification of mastitis-causing bacteria assists in effective management by both dairy farmers and veterinarians and can be used to implement the selective use of antimicrobials for treatment. The purpose of this study was to evaluate the ability of our developed anti-ribosomal protein-L7/L12 antibody-coated immunochromatographic strip (ICS) test to detect coliforms in milk by comparing the results with the bacteriological culture method. We investigated the performance of the ICS test as compared with the bacteriological culture method using 308 milk samples from clinical bovine mastitis. First, to determine the optimal ICS test cutoff point for detecting coliform mastitis, we developed a receiver-operating characteristic curve. The result showed that the cutoff point was at 0.5 of our index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the ICS test were 81.3%, 84.8%, 69.2%, and 91.54%, respectively. As the clinical signs increased in severity, the F-measure, a weighted harmonic mean of the sensitivity and overall PPV performance, increased. Because it is especially important to treat clinical mastitis appropriately in the early stages of detection, the ICS test, which can be used by both dairy farmers and veterinarians on dairy farms, is considered to be a useful tool for detecting coliform mastitis, which often presents with severe signs.

Intramammary infection caused by Staphylococcus aureus increases IgA antibodies to iron-regulated surface determinant-A, -B, and -H in bovine milk.

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BACKGROUND: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder. RESULTS: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01). CONCLUSION: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.

Rapid Staphylococcus aureus Detection From Clinical Mastitis Milk by Colloidal Gold Nanoparticle-Based Immunochromatographic Strips.

Rapid diagnostic technologies for bovine mastitis caused by Staphylococcus aureus (S. aureus) are urgently needed. In the current study, we generated an anti-ribosomal protein-L7/L12 antibody to detect S. aureus and an anti-ribosomal protein-L7/L12 antibody-coated immune-chromatographic strip (ICS) test. Moreover, we determined the ability of the ICS test to detect S. aureus from milk samples collected from cows with clinical mastitis. The developed ICS reacted to S. aureus in a bacteria load-dependent manner with a detection limit of ~104 CFU/mL. In the evaluation of possible cross-reactivity of the ICS test, six strains of coagulase-negative Staphylococci showed slightly positive reactions, although at a lower level; however, other bacteria were completely negative. Next, we investigated the sensitivity and specificity of the ICS test compared with the bacteriological culture method using milk samples from clinical bovine mastitis. The results of the experiments demonstrated that the ICS test had high sensitivity [100%, 95% confidence interval (CI): 91.3-100%] and specificity (91.9%, CI: 90.5-91.9%) compared with culture tests. In addition, the kappa statistic demonstrated that ICS tests showed substantial agreement (k = 0.77, CI: 0.66-0.87) with culture tests. Positive correlations were observed for the statistical analysis between S. aureus (nuc gene) copy numbers and ICS test scores in mastitic milk infected by S. aureus. Therefore, we assume that this new detection method using ICS may be useful as a highly sensitive S. aureus-screening method for the diagnosis of bovine mastitis. Our findings support the ongoing effort to develop an ICS method for bovine S. aureus-induced mastitis, which can contribute to the rapid diagnosis of this disease.

The bacterial load in milk is associated with clinical severity in cases of bovine coliform mastitis.

We evaluated the relationship between the severity of coliform mastitis and bacterial load in 106 quarter milk samples. We found no significant relationship between somatic cell count and coliform bacterial load in milk in bovine clinical coliform mastitis. Results of the Cochran-Armitage test for trend in milk bacterial load proportions indicated a significant decreasing low group (P<0.001), increasing medium group (P<0.002) and increasing high group (P<0.02) with increasing clinical grade. The present study indicates that the coliform bacterial load in milk is significantly associated with clinical severity states in cases of bovine coliform mastitis, and can be a useful indicator for optimal management of this disease.

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load.

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary-infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3-21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin-positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

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.