Isolation and molecular detection of enteric bacteria from neonatal calf in Chittagong

Abstract

Calf mortality due to neonatal calf diarrhea is a major problem in cattle farming in Bangladesh where certain groups of bacteria play crucial role. The aim of the present study was to carry out pathological and molecular investigation on enterotoxigenic Escherichia coli (ETEC) and Salmonella typhimurium in neonatal calves. During the study, fecal samples were collected from 200 calves (less than 40 days of age) from a total of 64 dairy and beef farms located in Chittagong Metropolitan Area and Patiya Upazila of Chittagong. The study was conducted during June, 2015 to March, 2016 when 5 dead calves with a history of diarrheal illness were examined through necropsy for gross and histopathological screening. Alongside classical culture technique molecular diagnostic tools like Polymerase chain reaction (PCR) was employed to identify the pathogens including E. coli and Salmonella spp. Two separate primer pairs namely ECO and K99 were used during this study where ECO is indicative of E. coli, and K99 is indicative for Enterotoxigenic E. coli (ETEC) K99. Besides, Salmonella spp. was confirmed by InvA gene in PCR. Typh and 104 primers were also used to identify S. typhimurium and multidrug resistant S. typhimurium DT104, respectively. Results indicated 19 (9.5%) cases of ETEC K99 out of 200 isolates, whereas S. typhimurium was recovered from only 8 calves (4%) and S. typhimurium DT104 was recorded from 2 calves. None of the dead animals was found positive for S. typhimurium through PCR. Gross pathological changes observed during necropsy include watery intestinal contents with bad odor, mucus and blood (in two cases) with dilatation of intestinal lumen. Histopathological investigation of intestine showed villous atrophy in all five cases of dead calves. Thickening of intestinal epithelium, sloughing off epithelium, congestion in lamina propria, infiltration of reactive cells were the findings of diarrhea caused by ETEC K99. Two out of five dead calves were found positive for ETEC K99 and one was isolated for mixed infection with E. coli and rotavirus in confirmatory molecular identification. Statistically significant difference (p<0.05) were observed in the prevalence of E. coli and S. typhimurium collected from different locations and herd size. Highest prevalence of ETEC K99 and S. typhimurium were found in Bakalia (30.43%) and Chawkbazar (27.27%), respectively. Compared to female animals, male calves were mostly affected by both ETEC K99 (13.27%) and S. typhimurium (27.27%). Small farms having <10heads had highest prevalence of ETEC K99 (42.86%). But S. typhimurium occurred highest in medium sized farms (5.81%) having 21-50 heads. Multilevel univariable and multivariable logistic regression was used to evaluate the risk factors associated with both infections. E. coli was significantly affected by farm size (OR = 3.54, 95% CI: 1.02, 12.78), population density (OR = 1.02, 95% CI: 1.00, 1.05), age (OR = 5.51, 95% CI: 1.59, 19.04), coexistence of other animal species in same pen (OR = 3.24, 95% CI: 1.12, 9.34), suckling as feeding regime (OR = 5.07, 95% CI: 1.43, 17.16). Interestingly, level of dehydration showed inverse association with prevalence of ETEC K99. Infection with Salmonella spp. was found to be significantly affected by the source of drinking water supply (OR = 32.28, 95% CI: 5.76-180.94), status of antibiotic therapy (OR = 7.03, 95% CI: 1.30, 37.81) and recent introduction of new calf in the farm (OR = 0.09, 95% CI: 0.01, 0.90). The results of the present study indicate the importance of PCR as rapid, effective and reliable tool alongside conventional culture techniques for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrheal illness may be helpful towards developing suitable prevention and control strategies.