Isolation, molecular characterization and antimicrobial susceptibilities of isolates of Mycoplasma agalactiae from bulk tank milk in an endemic area of Spain.

AIM: To isolate and characterize strains of Mycoplasma agalactiae from bulk tank and silo ewes' milk. METHODS AND RESULTS: Thirteen mycoplasma isolates were obtained from samples of sheep milk taken from bulk tank and large silos and identified as Myc. agalactiae by PCR-DGGE. The isolates were typed by pulsed field gel electrophoresis (PFGE), SDS-PAGE and immunoblot. The in vitro activity of 13 antimicrobials of veterinary interest was tested against these isolates. Results showed that the most effective compounds against Myc. agalactiae in vitro were clindamycin, an antibiotic not previously described as a suitable contagious agalactia (CA) treatment, with Minimum Inhibitory Concentration (MIC) values of <0·12 µg ml(-1) , and quinolones, with MIC values <0·12-0·5 µg ml(-1) , which are used as standard treatments against CA. CONCLUSIONS: Based on the in vitro assay, clindamycin, quinolones, tylosin and tilmicosin would be appropriate antimicrobials for CA treatment. The isolates were mostly resistant to erythromycin, indicating that it would not be a suitable choice for therapy. The isolates showed common molecular and protein profiles by PFGE and SDS-PAGE, with minor differences observed by immunoblot analysis, suggesting a clonal relationship among them. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of the appropriate selection of antimicrobials for treatment of CA.

Relationship among specific bacterial counts and total bacterial and somatic cell counts and factors influencing their variation in ovine bulk tank milk.

To analyze the relationship among the counts of different organisms and total bacterial count (BTTBC) and somatic cell count (BTSCC) as determined in dairy laboratories in ovine bulk tank milk, 751 bulk tank milk samples from 205 dairy sheep flocks belonging to Consortium for Ovine Promotion (CPO) were collected between January and December 2011. Four samplings were carried out in each flock, once per season, throughout 1 yr. Variables analyzed were bulk tank counts of thermoduric, psychrotrophic, coliform, and gram-positive catalase-negative cocci (GPCNC) bacterial groups. Thermoduric, psychrotrophic, and coliform species were significantly related to BTTBC, whereas GPCNC were correlated with both BTTBC and BTSCC variables. Highest counts were for psychrotroph and coliform groups, and a moderate to high correlation (r=0.51) was found between both variables, indicating that poor cleaning practices in the flocks tend to select for less-resistant organisms, such as gram-negative rods. In addition, BTTBC correlated with BTSCC (r=0.42). Some variation factors for specific bacterial counts, such as breed, season, milking type, dry therapy, and milk yield, were also analyzed. Flock information was collected from flock books, annual audits, and the CPO traceability system. Psychrotrophs and coliforms had elevated counts in winter, whereas GPCNC were higher in summer and in hand-milked flocks. Dry therapy contributed to the reduction in psychrotrophic bacteria; therefore, some strains of mammary pathogens could also be psychrotrophic bacteria. Results of this study would be helpful for troubleshooting milk quality problems and developing premium payment systems in dairy sheep.

Multilocus sequence typing of Mycoplasma agalactiae.

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

Influence of storage and preservation on microbiological quality of silo ovine milk.

This study was designed to analyze the effects of the storage and preservation conditions on counts of mesophilic, thermoduric, psychotrophic, coliform, Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus organisms in silo ovine milk. A total of 910 analytical determinations were conducted from aliquots of 10 silo ovine milks. The conditions tested were unpreserved and azidiol-preserved milk stored at 4°C, and unpreserved milk stored at -20°C. Milk aged 2, 24, 48, 72, and 96 h post-collection for refrigerated aliquots, and 7, 15, and 30 d post-collection for frozen aliquots. The factors silo and storage conditions significantly contributed to variation of all microbiological variables, although milk age effect within storage was only significant for mesophilic, psychrotrophic, and coliform bacteria counts. In refrigerated raw milk, mesophile, psychrotroph, and coliform counts significantly increased over 96 h post-collection, whereas the other groups and bacteria species tested maintained their initial concentration. In all cases, azidiol preservation maintained the initial bacterial concentration in raw sheep milk under refrigeration throughout 96 h. Thus, azidiol was a suitable preservative for microbiological studies in sheep milk. Smallest counts were registered for frozen samples, particularly for coliforms, E. coli, Strep. agalactiae and Staph. aureus. Estimates of mesophilic, thermoduric and psychrotrophic organisms showed similar values on both azidiol-preserved and frozen milk samples. Coliforms and E. coli counts significantly decrease over time after freezing. Consequently, freezing at -20°C could also be appropriate for analysis of mesophilic, thermoduric, and psychrotrophic bacterial groups, but not for coliforms or mammary pathogens.