Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk

Abstract

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.

Genetic diversity and virulence genes in Streptococcus uberis strains isolated from bovine mastitis

“…Likewise, similar results were obtained for S. uberis isolated from mastitis samples in other geographic regions (KHAN et al, 2003;ROSEY et al, 1999;SHOME et al, 2012;LEIGH, 2004). Perrig et al (2015) related the presence of the pauA gene in 94.9% of the strains studied.…”

“…Perrig et al (2015) related the presence of the pauA gene in 94.9% of the strains studied. Other studies conducted in India showed that the prevalence of the pauA gene was 100% (KHAN et al, 2003;SHOME et al, 2012). In contrast, Reinoso et al (2011) showed a slower frequency (61.5%) of this gene in strains isolated from bovine mastitis samples in Argentina.…”

“…Additionally, this illness causes economic losses for producers due to presence of antibiotic residues in the milk caused by it indiscriminate use, beyond the emergence Genetic diversity and virulence genes in Streptococcus uberis strains isolated from bovine mastitis mastitis. It has a significant impact on cow's udder health, milk quality, and herd productivity (SHOME et al, 2012). According Pullinger et al (2006), this bacterium is one of the main mastitis causative pathogens in many countries, and it has been associated with economic losses for dairy producers.…”

“…A successful vaccine against S. uberis should confer broad protection against the predominant strains. For this, additional epidemiological data and an understanding of virulence mechanisms in this bacterium are necessary (PERRIG et al, 2015;SHOME et al, 2012).…”

“…Among the different molecular typing methods, the Pulsed Field Gel Electrophoresis (PFGE) technique has been used to study pathogens involved in the etiology of bovine mastitis because it is highly discriminatory and highly reproducible (BASEGGIO et al, 1997;DOUGLAS et al, 2000;LASAGNO et al, 2011;PHUEKTES et al, 2001;RATO et al, 2008RATO et al, , 2013. According to Shome et al (2012), PFGE is the technique of choice for short-term or local epidemiological analyses. Aside from the advances in molecular tools, there are few reports about the population diversity and virulence profiles of the S. uberis strains that cause bovine mastitis in Brazilian dairy herds.…”