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Pathological Study on Colibacillosis in Chickens and Detection of Escherichia Coli by Pcr

N.S. Tonu
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. A. Sufian
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

S. Sarker
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M.M. Kamal
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M.H. Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M.M. Hossain
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

The aim of the present study was to detect the pathogenic Escherichia coli (E. coli) through pathological study of the colibacillosis affected birds. These isolated E. coli were further confirmed by PCR using specific primer. For this purpose, a total of 20 swabs (10 from lung and 10 from intestine of 10 dead birds) were collected in sterile nutrient broth. The histopathological samples were collected in 10% buffered neutral formalin. The used methods were histopathology, isolation and identification of E. coli by conventional methods and as well as by PCR method. A total of 10 isolates of E. coli from 20 swabs of lung and intestine was characterized by conventional routine methods of bacteriology. Gross pathological lesions of all lungs in the present investigation were congested and consolidated. Duodenum showed congestion and hemorrhages with excess mucus in the luminal surface of it. Microscopically, all the lungs showed severe congestion, infiltration of heterophils, macrophages and lymphocytes in the wall of bronchus as well as in the peribronchial alveoli. E. coli infected all the duodenum showed severe infiltration of leukocytes mainly heterophils, lymphocytes and macrophages in the submucosa of the duodenal wall. In this study, DNA of 8 isolates out of 10 isolated E. coli organisms was amplified by PCR using ECO-f and ECO-r primer targeting 16S ribosomal DNA and found 585 bp amplicon which is specific for E. coli with enteroinvasive type confirmed by histopathological lesions in duodenum. Further investigation should be focused on serotyping and detection of genes of E. coli which are responsible for pathogenicity of the organism.

Keywords: Colibacillosis, histopathologcal, chickens, PCR

Location: Mymensingh

Start Date: 01-06-2009

End Date: 31-01-2010

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

The present research work was performed with these objectives (a) pathological studies of colibacillosis affected dead birds. (b) isolation and identification of E. coli from the collected sample of dead birds. (c) detection of pathogenic E. coli using specific primer.

Methodology:

A total of 10 dead birds from Krishibid Poultry Farm, Bhaluka (8 birds) and S.K. Diagnostic Center (SKDC), Mymensingh (2 birds) and a total of 20 swabs samples (10 from lung and 10 from intestine) were collected from necropsy cases during the study period from June 2009 to January 2010.All lung and intestinal swabs were placed in nutrient agar plate and incubated for overnight at 37°C for the growth of the organisms. After primary culture of the organisms, a small amount of inoculum from nutrient agar were subcultured in the nutrient agar and MacConkey agar to observe the colony morphology. Characteristic colony morphology of the organisms indicating the features of E. coli was selected for subculture on EMB agar (Carter, 1986). Morphological characteristics (shape, size, surface texture, edge and elevation, color, opacity etc. of the suspected colonies on different agar media developed within 18 to 24 hours of incubation were carefully studied and recorded.For PCR analysis of locally isolated E. coli, oligonucleotide primers targeting 16S rDNA gene (Candrian et al., 1991; Amit-Romach et al., 2004) (Science Park Rd. #01-23, The Gemini, S’pore) ECO-f and ECO-r were used. DNA was extracted from E. coli isolate using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) and extracted DNA was quantified by using spectrophotometer (DU-640, Beckman, Germany) on the basis of optical density ratio at 260: 280 nm.PCR amplification was performed in a final volume of 20μl containing 4μl (50ng/μl) of DNA template, Taq buffer- B 2μl, dNTPs 2μl (250mM), primer-F 1μl (100mM), primer-R 1μl (100mM), Taq DNA polymerase 0.4μl (3U/μl) and 8.4μl nuclease free water (Science Park Rd.#01-23 The Gemini, S’pore). Three independent reactions, with the primers, were made for DNA template. Amplification was carried out in Gene amplification PCR system 9600 Thermocycler (Master cycler eppendorf, Germany). Initial denaturation was at 94⁰C for 3 min., followed by 35 cycles for 94⁰C for 30 sec., annealing at 60⁰C for 1 min and extension at 68⁰C for 2 min, with a final extension at 68⁰C for 7 minutes and hold for 40C. At the end of the study period all findings were compiled, scrutinized and analyzed for comprehensive interpretation

Source of Information: Bangl. J. Vet. Med. (2011). 9(1): 17 – 25

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Locally isolated 8 isolates (out of 10) of E. coli were tested by ECO-f and ECO-r primer and showed 585-bp products from each field samples after 1% agarose gel electrophoresis (Fig. 8). Traditionally, diagnosis of colibacillosis was carried out by isolation and identification of E. coli. The isolation and identification depend upon the culture of the organism using different selective media and biochemical tests. Instead of biochemical and ELISA tests, PCR and its related method have been reported to identify E. coli. The PCR is used as a highly sensitive and specific test for the presence of pathogenic bacteria in clinical specimens (Cohen et al., 1993). PCR is also more rapid, reliable than traditionally culture methods. In this study, the isolated E. coli organisms from all collected birds were grown in nutrient broth, extracted DNA and amplified by PCR using ECO-f and ECO-r primer targeting E. coli 16S ribosomal DNA and found 585 bp amplicon after 1% agarose gel electrophoresis. The similar result also found other authors (Amith-Romach et al., 2004. This base pair is specific for E. coli not for others.

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