A novel phage-displayed MilA ELISA for detection of antibodies against Myc. bovis in bovine milk.

AIMS: The aim of this study was to assess a phage-displayed MilA protein of Myc. bovis in an indirect ELISA for the detection of Myc. bovis antibodies in milk samples. METHODS AND RESULTS: The desired sequence of milA gene was synthesized and cloned into pCANTAB-F12 phagemid vector. The expression of the MilA on the phage surface was confirmed by Western blotting. The recombinant phage was used in the development of an indirect ELISA to detect Myc. bovis antibodies in milk samples. There was a significant agreement between the results of phage-based ELISA and recombinant GST-MilA ELISA for the detection of Myc. bovis antibodies in milk samples. CONCLUSIONS: The inexpensive and convenient phage-based ELISA can be used instead of recombinant protein/peptide ELISA as an initial screening of Myc. bovis-associated mastitis. SIGNIFICANCE AND IMPACT OF STUDY: Mastitis associated with Myc. bovis is a continuous and serious problem in the dairy industry. Sero-monitoring of Myc. bovis infection cases are one of the key factors for surveillance of the infections in dairy farms. Despite the existence of some commercially serological assays for Myc. bovis antibodies, they have some limitations regarding their sensitivity and availability. The development of accurate diagnosis tools could contribute to control programmes of Myc. bovis-associated mastitis in the dairy herds.

Genomic characterization of coagulase-negative staphylococci including methicillin-resistant Staphylococcus sciuri causing bovine mastitis.

Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have recently emerged as a significant cause of bovine mastitis worldwide. Here we describe the isolation of MRCoNS from cases of bovine mastitis from a single dairy farm in Australia. Fourteen CoNS isolates were identified as MRCoNS on the basis of having an oxacillin MIC of ≥0.5 µg/mL. The isolates were speciated as S. chromogenes (n = 1) S. fleurettii (n = 1), S. haemolyticus (n = 2), S. sciuri (n = 5), S. simulans (n = 1) S. succinus (n = 2) and S. xylosus (n = 2). Five of the isolates (S. fleuretti, S. haemolyticus S. sciuri and two S. succinus) were mecA-positive. We also detected a previously described S. sciuri mecA homolog in four oxacillin-resistant S. sciuri isolates. The remainder of the putative MRCoNS did not contain any mecA-related resistance determinants in their genomes. Comparative genomic analysis of three previously published S. sciuri isolates, from humans, a squirrel and a cereal crop (rice), and a representative isolate from our study demonstrated clustering and a high degree of genetic homogeneity (>95%), suggesting S. sciuri has low host specificity. In conclusion, CoNS, in particular S. sciuri, may act as a reservoir for SCCmec elements that can easily be spread between different host species by direct cross-infection.

Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions.

OBJECTIVES: Currently, there is no consensus protocols regarding the combination of glycerol (GLY), gelatin or foetal bovine serum (FBS) with dimethyl sulphoxide (DMSO) as cryoprotectants for Mycoplasma bovis in bovine milk samples. This study aimed to compare different cryopreservation compounds and storage temperatures for M. bovis. RESULTS: There were significant differences in the survival of M. bovis on different media. Differences were also observed between different storage conditions. All additives improved the survival of M. bovis in comparison to control (CON). The combination of GLY and DMSO was shown to be significantly different to CON with 57.1% (95% CI = 21.43-133.34) and 19.1% (95% CI = 11.73-60.27), respectively at week 16, and its use should be encouraged as a cryoprotectant for M. bovis at - 20 and - 80 °C. GEL/DMSO showed the highest survival rate for M. bovis with 57.14% (95% CI = 21.43-133.34) at 4 °C in comparison with CON 14.29% (95% CI = 9.60-50.39). FBS/DMSO showed the highest survival rate for the short-term preservation similarly to other additives. The evaluated cryopreservative compounds would improve survivability of M. bovis in milk for both transport and long-term storage. Hence, it is recommended to use the mentioned methods for routine transportation or storage purposes for suspicious M. bovis milk samples.

Discrimination between some Mycoplasma spp. and Acholeplasma laidlawii in bovine milk using high resolution melting curve analysis.

OBJECTIVES: This study aimed to provide a rapid, accurate and cost-effective diagnostic real time polymerase chain reaction-high resolution melting curve assay (PCR-HRM) to identify and distinguish between four different mycoplasmas and Acholeplasma laidlawii isolated at cow-level from a single commercial dairy farm in South Australia. One set of genus-level universal primers was designed targeting the 16S ribosomal RNA gene. RESULTS: Real time PCR-HRM analysis was able to identify and distinguish between five different mollicutes, namely A. laidlawii, M. arginini, M. bovirhinis, M. bovis and uncultured Mycoplasma. Results were confirmed through sequencing. Our developed assay provides rapid and accurate screening for Mycoplasma mastitis detection.

Evaluation of effects of Mycoplasma mastitis on milk composition in dairy cattle from South Australia.

BACKGROUND: Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. RESULTS: Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. CONCLUSIONS: Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels.