Development of multiplex PCR assays for the molecular characterization of Streptococcus uberis strains isolated from bovine mastitis

Streptococcus uberis is an important environmental udder pathogen in the modern dairy industry. It ranks among the main causes of mastitis in countries around the world, including Australia, Brazil, Canada, the Netherlands, New Zealand, the United Kingdom, and the United States (Zadoks et al., 2011). S. uberis has a great ability to survive in cow habitat (soil, water, hay, faeces) and cattle are constantly exposed to risk of infections (Zadoks et al., 2005). This spreading ability could be due to hasA (hyaluronate synthase A), hasB (UDP-glucose dehydrogenase), and hasC (UDP glucose pyrophosphorylase) genes, which are involved in capsulation (Reinoso et al., 2017). Indeed, hasA positive bacteria could be associated with clinical mastitis, while hasA negative strains were correlated with subclinical cases (Pullinger et al., 2006). Among virulence factors, S. uberis adhesion molecule (SUAM), encoded by sua gene, play a key role in the invasion of mammalian epithelial tissue, due to its high affinity for lactoferrin, a protein required for iron assimilation (Almeida et al., 2006). Further, the Plasminogen activator A (PauA), responsible for degradation of milk proteins into small peptides and free amino acids useful for the growth (Loures at al., 2017), was considered from several authors as potential target for the development of new vaccine therapy due to its high prevalence in S. uberis population (Perrig et al., 2015; Leigh et al., 1999). The knowledge of the distribution of virulence genes is required to understand the route of transmission and the pathogenesis of S. uberis. To date, molecular characterization has improved our knowledge of epidemiology of S. uberis identifying molecules that have roles in the establishment of the intramammary infection (Loures et al., 2017). Therefore, the aim of this study was to develop two multiplex PCR assays for the simultaneous detection of 10 virulence factors of S. uberis, useful both for a rapid characterization of S. uberis strains isolated from bovine milk and for epidemiological studies. S. uberis ATCC 9927 was used as reference strain for the development of mPCR assays. Genomic DNA was extracted from pure cultures as described by Cremonesi et al. (2006). We developed two different mPCR assays called reaction 1 and reaction 2. The first one included: tuf, cpn60 (Dmitriev et., al 2006), pauA, sodA, sua, oppF and gapC genes, while reaction 2 amplified the three genes responsible for the synthesis of capsule, i.e. hasA, hasB and hasC. All primer pairs, apart those for cpn60 gene, were designed using PRIMER3 (http://primer3.ut.ee/) and their specificity was in silico and in vivo evaluated. Once the specificity of the primer pairs has been determined, both the PCR conditions and the buffer and primer concentrations were optimised in order to combine the primers into the two multiplex PCR reactions, without affecting the ability of the primer pairs to generate gene specific amplicons. In this way the mPCR assays were successfully optimized and the desired amplicons were obtained in both reactions. In reaction 1, it is possible distinguished 7 bands with size of 143 bp, 205 bp, 280 bp, 350 bp, 369 bp, 400 bp and 505 bp, corresponding to tuf, pauA, soda, sua, cpn60, oppF and gapC gene, respectively. While in reaction 2, three bands size of 193, 400, 599 bp were obtained, corresponding to hasC, hasB and hasA, respectively. All of the bands were well differentiated on gel. Afterwards, the limit of detection (LoD) for each mPCR assay was determined using 2-fold serial dilution of the genomic DNA extracted from the reference strain, starting from 48 ng/?L of the DNA template. The sensitivity of the two mPCR reactions was comparable with other studies (Wang et al., 2015; Cremonesi et al., 2006): 12 pg and 6 pg of template DNA for reaction 1 and 2, corresponding approximately to 12 x 101 CFU/ml e 6 x101 CFU/ml, respectively. Phuektes and co-workers (2001) reported 50 pg of DNA as threshold of detection of the multiplex PCR assay set up for the identification of S. uberis, while Shome and co-workers (2011) developed a really sensitive mPCR reaching 10 fg as LoD. Finally, to check the reliability of the two mPCR assays, we screened 33 pure cultures of S. uberis strains isolated from bovine mastitis milk. All S. uberis strains have been previously identified by colony morphology, Gram stain, catalase reaction, esculin hydrolysis and growth in 6.5% sodium chloride. Further, all streptococcal isolates were identified to the species level by the API 20 Strep. We found that the most common pattern of virulence-associated genes was hasA+ hasB+ hasC+ tuf+ cpn60+ pauA+ sodA+ sua+ oppF+ gapC+ (82%, 27 out of 33). Moreover, our results showed the high presence of pauA (97%, 32 out of 33) and sua (91%, 30 out of 33) genes in the S. uberis strains analysed, confirming the data previously reported (Loures et al., 2017; Perrig et al., 2015;Yuan et al., 2014). To our knowledge only few studies described mPCR assays developed for the molecular characterization of the S. uberis strains. Therefore, owing to specificity, rapidity and ease of use, the mPCR methods described in this study could be a powerful tool for studying the virulence and pathogenicity of S. uberis isolates. In conclusion, our mPCR assays will be of value in the dairy sector for S. uberis mastitis diagnosis and in epidemiological investigations.