Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Staphylococcus aureus and Streptococcal Causes of Bovine Mastitis

Abstract

To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. The target sequence was the 16S to 23S rRNA spacer regions. The performance of the assay was examined with 117 milk samples collected from a subclinically infected herd, and the diagnostic specificities and sensitivities of the multiplex PCR were compared with conventional culture. PCR was significantly more sensitive than culture for detection of S. aureus and S. uberis, but there were no significant differences in sensitivities between PCR and culture for the detection of S. agalactiae and S. dysgalactiae. The results suggest that this multiplex PCR assay could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection of S. aureus, S. agalactiae, S. dysgalactiae, and S. uberis in milk samples.

Incidence of Staphylococci and Streptococci During Winter in Mastitic Milk of Sahiwal Cow and Murrah Buffaloes

“…and second multiplex PCR reaction for streptococci group (S. agalactiae, S. dysgalactiae and S. uberis). The oligonucleotide primers used in this study for staphylococcal group and streptococcal group were used as explained by Morot-Bizot et al [17] and Phuektes et al [15], respectively. The oligonucleotide sequences and amplification conditions are shown in Table 1.…”

“…Grampositive cocci and catalase negative test isolates cocci were further biochemically tested with Christie, Atkins and Munch-Petersen reaction (CAMP) and esculin hydrolysis [15] to distinguish the types of streptococci.…”

“…DNA was extracted as per procedure of Phuektes et al [15] with modification. In brief, milk samples were enriched in BHI broth by incubating at 37°C for 12 h. Bacterial cells were harvested by centrifugation at 5,000 rpm at 4°C for 15 min.…”

“…Among various PCR based assay, multiplex is quiet useful for in vitro detection of more than one pathogen within a single tube reaction. Multiplex PCR has been explored for detection of mastitis pathogens [14,15]. The information is scanty regarding the detection of occurrence of mastitis pathogens (especially staphylococci and streptococci) in the tropical region like India.…”

Correlation Between Bacteria Isolated From Bovine Milk Samples and Human Throat Swabs

“…S.aureus is one of the most prevalent bacteria in subclinical mastitis in dairy cows and in our investigation may be return to milker hands which consider the main tool in distribution of microorganisms from teat to teat and from cow to cow, in addition to lack of hygiene (El-Balkemy et al, 1997) Many of disease causing pathogens are founded in milk along with numerous foodborne outbreaks, clearly demonstrate the risk associated with drinking raw milk (Lin et al, 1991;Cohen et al, 2005;Bessen., 2009). The streptococcus is the main cause of sore throat in human especially in young ages and is the main bacteria transmitted from the milk to human and also can transmitted from the human to animals (Siddique et al, 1988;Phuektes, et al, 2001;Kalorey et al, 2007;Salasia et al, 2004). Pharyngitis caused by strept.pyogens is peaked in late winter and early spring (Vincent et al, 2004;Bessen, 2009).The cases of pharyngitis caused by strept.pyogens may reach to 616 million cases worldwide each year.…”