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20201
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Subject: Veterinary Gynaecology and Obstetrics × Author: LEEBA CHACKO × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    DETECTION OF BIOFILM FORMING BACTERIA AND THERAPEUTIC EFFICACY OF BIOFILM DISRUPTING AGENTS WITH ANTIBIOTICS IN ENDOMETRITIS OF CROSSBRED COWS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2020-07-24) LEEBA CHACKO; K. Promod
    The present study was carried out to investigate the presence of biofilm forming bacteria in uterine samples of postpartum cows with endometritis and evaluate the efficacy of different therapeutic protocols incorporating biofilm disrupting agents along with the investigation for the presence of VF genes, Agn43aCFT073 and Agn43bCFT073, in E. coli isolates. Crossbred dairy cows (n=137) at 28-35 days postpartum, aged between three to eight years, parity between two to six without any postpartum reproductive complications were selected for the study. Screening for endometritis was carried out by the evaluation of vaginal mucus discharge using Metricheck® device and endometrial cytology by cytobrush technique. Postpartum cows with VMS score ≥ 1 or more than five per cent PMN cells in the EC smears by cytobrush technique were considered as positive for endometritis. Isolation and identification of bacteria and in vitro antibiotic susceptibility tests were performed by disc diffusion method. The antibiotic to which most of the bacteria were highly sensitive was used for the in vivo treatment for endometritis. Biofilm disrupting agents (EDTA-Tris and NAC) were also used for treatment incorporated with antibiotic or alone. Biofilm forming ability of isolates were assessed using microtitre plate assay. The E. coli isolates were screened for biofilm VF genes viz., Agn43aCFT073 and Agn43bCFT073 by PCR employing suitable primers. Sixty postpartum cows with endometritis were randomly allotted into five groups (n=12). Group I were treated with intrauterine infusion of ciprofloxacin to which most bacteria were sensitive, Group II were treated with 30 mL of 3.5 mM EDTA, 50 mM Tris, (pH 8.0) and ciprofloxacin, Group III were treated with 30 mL of 3.5 mM EDTA, 50 mM Tris (pH 8.0), Group IV were treated with 30 mL of 3.3 per cent NAC and ciprofloxacin and Group V were treated with 30 mL of 3.3 per cent NAC as intra uterine infusions. In the present study, vaginal mucus discharge was collected from 137 dairy cows and among that 20.44, 45.26, 15.33, 10.95 and 8.03 per cent cows exhibited VMS no discharge, zero, one, two and three, respectively. Cows with purulent vaginal discharge (PVD) had lowest reproductive performance at 35 days postpartum was a better criterion for the diagnosis of endometritis. Out of 137 animals, 47 cows exhibited VMS ranging from one to three at 31.58 ± 0.13 dpp were diagnosed positive for CE and the prevalence recorded was 34.31 per cent. The prevalence of subclinical endometritis detected by cytobrush technique in the present study was 10.95 per cent. The overall prevalence of endometritis by VMS and EC at 31.58 ± 0.13 dpp was 34.31 and 43.79 per cent, respectively EC detected more number of cows with endometritis than VMS. The results of the present study indicate that by measuring the VMS and EC (five per cent cut off) at 31.58 ± 0.13 dpp predicted animals with endometritis. Hence, it could be concluded that the cytobrush samples were superior and this method of sample collection is more consistent and reliable in postpartum dairy cows. The most frequently isolated bacteria in the present study were E. coli, Staphylococcus spp., Bacillus spp., Streptococcus spp., Enterobacter spp., Enterococcus spp. and Pseudomonas spp. Heirarchichal cluster anlaysis of antibiotics under present study revealed highest sensitivity for ciprofloxacin followed by enrofloxacin. In the present study, biofilm forming ability was detected in 80.52 per cent of bacterial isolates on microtitre plate assay. Maximum number of isolates (77.42%) exhibited moderate biofilm forming ability while, 8.06 and 14.52 per cent exhibited strong and weak biofilm forming ability, respectively. No significant (p>0.05) was noted in the biofilm forming ability of different bacterial isolates. None of the E. coli isolates recorded the VF genes, Agn43aCFT073 and Agn43bCFT073. Highest recovery rate (100%) and conception rate (83.33%) was noticed after treatment with NAC and ciprofloxacin intrauterine. There was no significant difference (p>0.05) was noticed between the treatment groups in the conception rate. Recovery from endometritis was more with combination biofilm disrupting agents and antibiotics than using antibiotics or biofilm breaking agents alone. Disruption of biofilm resulted in better penetration of antibiotics which aid in recovery. Collection of vaginal mucus discharge using Metricheck® device and endometrial cytology samples by cytobrush technique help detection of endometritis during early postpartum period under field conditions. It was concluded that resistance to antibiotics could be attributed to the presence of biofilm forming bacteria which form a layer and resist the action of antibiotics. Biofilm disruption aid in better penetration of antibiotics and hence, recovery. Further studies in a wider population in different dosage regimens and combinations needed to be undertaken to elucidate the efficacy of NAC for uterine infections.