Detection of staphylococcus aureus in bovine sub clinical mastitis using polymerase chain reaction (PCR)
Abstract
The present investigation was undertaken to
standardize polymerase chain reaction (PCR) assay using
ten different sets of primers for detection of sub clinical
mastitis (SCM) directly from milk samples of apparently
healthy animals. A total of 1230 milk samples including
367 samples from cow farm, 228 samples from buffalo farm
and 635 samples brought in Veterinary College Central
Laboratory, CCS Haryana Agricultural University, Hisar,
were screened by PCR assay and other conventional tests
(cultural examination, California mastitis test, Electrical
conductivity test and Somatic cell count). Staphylococci
isolates were characterized phenotypically and
genotypically. Screening of cow farm using universal
primer yielded 56.52 per cent animals and 33.24 per cent quarters to be positive for SCM whereas18.85 per cent
quarters were found positive for Staphylococcus aureus by
16S-23S rRNA interspacer region primers. As many as
15.78 per cent animals and 5.70 per cent quarters were
found positive by using universal primer while 15.38 per
cent quarters were positive for Staphylococcus aureus by
16S-23S rRNA interspacer region primers from buffalo
farm. A total of 15.90 per cent samples of College Central
Laboratory were found positive for Staphylococcus aureus
by 16S-23S rRNA interspacer region primers and majority
of samples found negative by cultural examination and
positive by PCR were from animals who received prior
treatment with antibiotics. The Genus specific primers
encoding gap gene and 16S-23S rRNA region could detect
all Staphylococcus spp. Among species specific primers the
primers encoding 16S-23S rRNA region were found to be
100 per cent sensitive followed by aroA gene (98.41 per
cent), nuc gene (97.62 per cent) and coa gene (93.65 per
cent). A total of 99.20 per cent of isolates were found
positive by ubiquitous PCR assay. On the basis of results
of different conventional diagnostic tests, CMT was found
to be good animal side test for primary screening of large
herd to identify subclinically infected animals within a very
short period economically. Among phenotypic tests,
thermostable nuclease test was most sensitive in
comparison to latex and coagulase test. No false-positive
reactions were detected by any of the tests applied on
staphylococci other than Staphylococcus aureus. Genotypic
characterization of staphylococcal isolates yielded similar
results as were obtained by PCR of milk samples directly
indicating PCR assay as an important tool and an
alternative to phenotypic characterization. PCR was found
to be most rapid, sensitive and specific assay when
compared to bacteriological examination and other
conventional tests and can be adapted for screening of
large herd for detection of Staphylococcus aureus directly
from milk.