Molecular Characterization of Bacterial Pathogens Associated with Bovine Mastitis
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Date
2015
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MAFSU, Nagpur
Abstract
Mastitis is multi-etiological disease of dairy animals characterized by
physical, chemical and usually bacteriological changes in milk and pathological
changes in glandular tissues of udder. Mastitis has evolved as a disease of
highest economic significance particularly to the dairy industry worldwide. Financial losses arise from the costs incurred on treatment, culling, decreased
milk production, sometimes permanent loss of udder, decreased milk value and
even death of animal. For monitoring udder health, it is necessary to implicate the
reliable and affordable diagnostics method and constant need to improvise
them. In the present study easy, cheap and rapid MALDI-TOF MS technique was
applied for detection of pathogens associated with mastitis keeping PCR results as
reference particularly for major pathogens.
During present investigation a total of 235 dairy animals from unorganized
and organized farms located in Pune and Satara region were examined using
CMT to study prevalence of mastitis. All 136 CMT positive samples were further
processed for bacterial culture in enrichment medium which generated 127
culturally positive samples from which 203 pure bacterial colonies were isolated
culturally as single or as mixed infections. The major pathogens were identified
upon growing them on selective media like EMB and MacConkey’s agar for E. coli
and on MSA agar for S. aureus, respectively. They were confirmed by biochemical
catalase, oxidase and IMViC tests. Accordingly, total 80 (62.9 %) isolates were
detected as S. aureus and 53 ( 41.7 %) as E. coli.
All the isolates were processed for antibiotic sensitivity testing and overall
rate of sensitivity indicated, highest rate of sensitivity in 159 isolates (78.3 %) for
gentamicin followed by enrofloxacin 155 (76.3 %), ceftriaxone 122 (60 %),
ciprofloxacin 97 (47.7 %), chloramphenicol 73 (35.9 %), streptomycin 44 (21.6 %),
oxytetracycline 25 (12.3 %) and penicillin 9 (4.4 %) and overall rate of resistance
pattern of all isolates indicated highest rate of isolates i.e. 192 (94.5 %) being
resistant to Penicillin followed by oxytetracycline 149 (73.4 %), streptomycin 132
(65 %), chloramphenicol 88 (43.4 %), ciprofloxacin 78 (38.4 %), ceftriaxone 52
(25.6 %), gentamycin 36(17.7 %) and enrofloxacin 34(16.7 %).
Culturally detected E. coli and S. aureus were subjected to molecular
detection with PCR technique so as to use the results as reference for evaluating
MALDI TOF MS. Using femA gene PCR, 80 isolates identified culturally were
specifically (100 %) confirmed as S. aureus which generated 132bp size product
upon electrophoresis. Total 53 E. coli isolates when treated with 16SrRNA PCR
were also confirmed specifically (100 %) which yielded 232 bp size bands on electrophoresis of their PCR end product.
Randomly selected isolates of E. coli (n = 25) and S. aureus (n = 25)
were further processed with MALDI-TOF MS technique; keeping PCR result as
reference. Along with these the other gram positive (n = 32) and gram negative
(n=38) bacterial isolates were also processed which were generated during bacterial
culture of milk sample for their identification. MALDI-TOF MS technique was found
highly accurate and rapid in detection of major bacterial pathogens like E. coli and
S. aureus with highest specificity (100 %) other unidentified bacterial isolates
associated with mastitis from freshly cultured media were identified confirmed on
genus and probable species level with MALDI TOF MS as Salmonella anatum 4
(5.7%), Streptococcus dysgalactiae, Enterobacter cloacae, S. xylosus, Proteus
mirabilis, Clostridium difficile, Enterococcus faecium, all were 3 in no. ( 4.2 %), S.
hominis, Klebsiella oxytoca, Stenotrophomonas spp., S. schleiferi, Klebsiella
oxytoca, Pseudomonas flurescens, S. hominis, S. epidermidis, S. cohnii,
Proteus vulgaris, Streptococcus faecalis, Clostridium beijerinckii all were found 2.85
% ( 2 in no.) Lactobacillus agilis, Aeromonas encheleia, Lactobacillus aviarius,
Camphylobacter jejuni (all were 1.42 %). Some of the different species were also
detected during present studies which were Cuprividus hector, Azoarcus communis,
Arthrobacter polychromogen. While some organisms remained as unidentified as
peak score observed was not optimum.
From the present findings it may be concluded that, MALDI-TOF MS is a
robust assay in case of detection of disorders presenting polymicrobial etiologies
like in case of mastitis with high confidence and shortest possible time. MALDITOF
MS is a fast and truthful technique which has capability to replace
conventional identification of multiple bacterial strains usually associated with
udder environment and milk of bovine mastitis.
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