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Investigation of Pathogenic Escherichia Coli from Diarrheic Calves in Selective Area of Bangladesh

A. K. M. A. Islam
Department of Medicine, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

A. Nahar
Department of Medicine, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

A. Khair
Department of Medicine, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. M. Alam
Department of Medicine, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Molecular technique was used to investigate the prevalence of virulent diarrheic genes in pathogenic Escherichia coli and their antibiotic sensitivity patterns. A hundred samples from 100 different diarrheic calves from mid-north-western part of Bangladesh were screened for the presence of virulence factors associated with diarrhea. Following isolation and identification on the basis of cultural, morphological and biochemical properties, the presence of the virulence genes such as eaeA, bfpA, elt, est, stx1 and stx2 were examined using PCR. Antimicrobial susceptibility of 57 E. coli was determined by agar disk diffusion method for 8 antimicrobial agents. Out of 100 samples 57 (57%) were found to be positive for E. coli and their distribution rates according to their age, breed and sex were 66.7% ( 6 days old ), 85.7% (Sahiwal breed) and in 64.2 % (female calves) respectively. Among 57 E. coli isolates, only 16 isolates were analyzed for the detection of the said genes. Among them, only eaeA gene was detected in 2 E. coli isolates (12.5 %). Antibiotic resistance patterns revealed that Oxacillin, Rifampicin and Penicillin were 100% resistant followed by Erythromycin which was more than 80% resistant. In case of Amoxicillin and Tetracycline, about 59.65% and 61.40% were found to be resistant respectively whereas all 57 E. coli isolates showed moderately susceptible (30%) to Cefuroxime, a second generation Cephalosporin. Therefore, none of the eight antimicrobials studied can not be recommended as single best therapeutic agent for the treatment of neonatal calf diarrhea. In addition, this study indicated that diarrhea in calves in these locations can be ascribed to mainly Enteropathogenic E. coli (EPEC) which was atypical (only contained the eaeA genes but not bfpA). However, further studies are necessary to characterize the isolated eaeA gene positive E. coli by serotyping, tissue culture assay and other molecular techniques to find out the potentiality of those virulent genes contributing pathogenicity of E. coli causing diarrhea in calves.

Keywords: Prevalence, Polymerase chain reaction, Virulence genes, Antibiotic sensitivity, Atypical EPEC

Location: Bathan, Sirajgonj

Start Date: 00-01-2014

End Date: 00-06-2014

Program Area: Animal Health and Management

Commodity: Calf

Objectives:

To investigate the presence of E. coli causing diarrhea in calves
To detect their virulence factors as well as the study of their antibiotic sensitivity patterns.

Methodology:

The study was conducted from January 2014 to June 2014 on the calves of Bathan regions in Sirajgonj district. Diarrheic samples from this region were brought to the Laboratory of Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh as soon as possible after collection for further examinations. Bathans are considered the most important production zone of dairy cattle in Bangladesh where calves are reared in clusters. A hundred diarrheic faecal samples were collected from the Sirajgonj district of Bangladesh. Each sample was aseptically collected in a sterile stool pot and transported to the laboratory of department of Medicine as soon as possible for further bacteriological examinations. Samples were processed within 24–48 h after reception. For the isolation and identification of E. coli standard methods. Nutrient broth (NB) was used for primary culturing of E. coli organisms that were present in the collected faecal samples. For the differentiation of the bacteria MacConkey (MC) agar medium was used followed by Eosin Methylene blue (EMB) agar medium which was used as a selective medium and these are used. The isolated bacteria was confirmed with their distinctive cultural characteristics, morphology with Gram’s staining and biochemical tests. Among 57 E. coli isolates, only 16 E. coli isolates were subjected to PCR for detection of six diarrheic pathogenic genes (eaeA, bfpA, stx1, stx2, est and elt). Bacterial DNA extraction was done through boiling methods. For the extraction of genomic DNA of E. coli, a single colony of E. coli was taken in 1000 µl of TE buffer in Eppendorf tube. The mixture was then vortexed and boiled at 100°C for 10 minutes. After boiling the tubes were immediately placed on ice for 5 minutes followed by centrifugation at 14,000 rpm for 5 minutes. The supernatant was collected and stored at -20°C which was used as template DNA. The base sequences, PCR conditions and predicted sizes of the amplified products for the specific oligonucleotide primers used in the study. Analysis of the PCR products was then carried out by agar gel electrophoresis method at 50 Volt for 60 minutes using 1% agarose gel stained with ethidium bromide. Finally the PCR products were visualized under UV transluminator. Susceptibility to different classes of antimicrobial agents was evaluated for E. coli using standard disc-diffusion method in Mueller Hinton agar using the inhibition-zone patterns. Antibiotic susceptibility and resistance patterns of E. coli against different antibiotics were measured. The zone diameter interpretative criteria of E. coli were used to classify isolates as susceptible, intermediate or resistant based on the standard interpretation table updated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2007). The discs containing the amount of antibiotics used: Penicillin-G 10 µg, Amoxycillin 30 µg , Rifampicin 5 µg, Gentamicin 10 µg, Cefuroxime 30 µg, tetracycline 30 µg, Erythromycin 15 µg, and Oxacillin 1 µg.

Source of Information: Bangl. J. Vet. Med. (2015). 13 (1): 45-51, ISSN: 1729-7893, 2308-0922

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study indicated that diarrhea in calves in these locations can be ascribed to mainly Enteropathogenic E. coli (EPEC) which was atypical (only contained the eaeA genes but not bfpA). However, further studies are necessary to characterize the isolated eaeA gene positive E. coli by serotyping, tissue culture assay and other molecular techniques to find out the potentiality of those virulent genes contributing pathogenicity of E. coli causing diarrhea in calves.

Knowledge Management: Journal