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Date
2023-01-23
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Abstract
Subclinical mastitis (SCM) is a major concern of the present day as its prevalence has increased, compared to clinical mastitis. Among the causative organisms, coagulase negative staphylococci (CNS) are of great interest because of their ability to cause persistent infection. Moreover, they tend to be more resistant to antimicrobials than Staphylococcus aureus (S. aureus) and are associated with biofilm formation. Hence detection of SCM and a better understanding on CNS with respect to their antimicrobial resistance (AMR) and biofilm formation are essential for development of livestock sector.
Hundred apparently healthy milch animals from university farm and a private farm near Thrissur district were selected for the study. Milk from all the four hundred quarters excluding seven blind quarters were separately screened using California mastitis test (CMT), electrical conductivity (EC) and somatic cell count (SCC). A high occurrence of SCM with 48.34 per cent on quarter basis and 91 per cent on animal basis was noted in the study. The SCC estimation by direct microscopic count (DMC) and DeLavel cell count (DCC) methods were statistically significant. No association was found between the occurrence of SCM and risk factors viz., age, parity, stage of lactation and quarter affected.
All the milk samples from 393 quarter samples were subjected to bacterial isolation and identification. Among which 109 samples yielded pure bacterial growth with 86 Gram positive cocci and 23 Gram negative bacilli. Based on the cultural and biochemical characterisation, the organisms isolated were CNS (44.95 per cent), S. aureus (9.17 per cent), Micrococci spp. (22 per cent), Streptococci spp. (2.8 per cent), Klebsiella spp. (12.8 per cent) and Escherichia coli (E. coli) (8.3 per cent). Various CNS organisms isolated included Staphylococcus epidermidis (S, epidermidis), Staphylococcus scuiri (S. scuiri), Staphylococcus haemolyticus (S. haemolyticus), Staphylococcus chromogenes (S. chromogenes), Staphylococcus schleiferi (S. schleiferi) and Staphylococcus xylosus (S. xylosus).
On antimicrobial susceptibility testing, 42.85 per cent of the CNS isolates were found to be resistant to gentamicin followed by methicillin (32.6 per cent), ceftriaxone tazobactam (24.48 per cent), enrofloxacin (20.4 per cent), tetracycline (16.32 per cent) and cotrimoxazole (4 per cent).
Biofilm formation of CNS isolates were phenotypically identified using Congo red agar method (CRA), tube adherence method (TA) and tissue culture plate method (TCP). Significant difference was noted between CRA and TCP and also between TA and TCP. No significant difference was noted between CRA and TA.
Genotypic confirmation of the CNS was done by polymerase chain reaction (PCR) using 16SrRNA and cns gene. All the 49 identified CNS isolates were subjected to PCR targeting genes for AMR (mecA, aacA-aphD and norA) and biofilm formation (bap, icaA and icaD). The percentage of CNS isolates with mecA, aacA-aphD and norA were 14.28, 44.89 and 32.65 respectively. The biofilm associated protein (bap) and intracellular adhesion genes (icaA and icaD) were found in 26.53 per cent, 91.83 per cent and 55.1 per cent of the CNS isolates.
There was significant difference between the phenotypic and genotypic antimicrobial resistance of the CNS isolates against methicillin. Significant difference was also noted for both CRA and TA with the presence of biofilm formation genes.
From the study it was clear that CNS was the major pathogen causing subclinical mastitis and its occurrence had increased at an alarming rate. Majority of the CNS were resistant to commonly used antibiotics with a higher proportion being resistant towards gentamicin. All the isolates were capable of biofilm formation.