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Diagnosis of chronic enteritis in goats by polymerase chain reaction.

Introduction

Enteritis is the most obvious outward sign of a gut abnormality and should always be treated as potentially serious. In North East India, goat population reared primarily for meat apart from skin and to some extent milk account for a total of 45 lakhs, distributed in varied agro-climatic zones in the regions. Amongst various diseases affecting the goats in the region, gastrointestinal (GI) tract infections with mild or chronic form of enteritis remains a major cause of concern from economic point of view. Both aerobic and anaerobic bacteria can cause GI tract infections leading to the symptoms of chronic enteritis.

Amongst aerobic bacteria, shiga toxin producing Escherichia coli (STEC) are able to cause severe enteritis in humans when being transmitted through the food chain from their animal reservoirs (Wasteson, 2001). The major virulence factor of STEC is shiga toxin production (stx), formerly known as verotoxins/shigalike toxins (Paton and Paton, 1998). The STEC found in the faecal flora of wide variety of animals is the important source of infection for human. The shiga toxin (encoded by stx1 and stx2 gene), enterohemolysin (encoded by hlyA gene), intimin (encoded by eaeA) and lipopolysaccharide (encoded by rfbO157 gene) are the most common virulence factors produce by the STEC strain found associated with gastroenteritis in animal and human (Paton and Paton, 1998).

Amongst anaerobic bacteria, Clostridium perfringens types (type A to E) are known to cause enteritis or enterotoxaemia in goats (Songer, 1996). C. perfringens are toxin typed (A to E) by the presence of four major toxins- alpha, beta, iota and epsilon (Songer and Meer, 1996). Along with four major toxins, enterotoxin and beta2 toxins produced by types of C. perfringens are considered as important toxins for enteric diseases of both animal and human (Smedley III et al., 2004). Polymerase chain reaction nowadays has been used a rapid diagnostic techniques to detect the presence of toxin genes and to identify the specific strains of both E. coli and C. perfringens (Yamagishi et al., 1997; Paton and Paton, 1998). The present study reports the diagnosis of goats suffering from the chronic enteritis by PCR.

Materials and Methods

History of the animals and sample collection

Sixteen goats of age 11 to 24 months old were suffering from chronic enteritis maintained in a semi intensive rearing system of an organized goat farm in Meghalaya, India were investigated. Infected animals showed rough hair coat, poor growth, anorexia, pyrexia and prolonged episodes of diarrhoea. The animals were unresponsive to the treatment with 5mg tetracycline (Himedia, Mumbai) per kg of body weight parentarily. The atmospheric temperature and humidity recorded were between 30-35[degrees]C and 85-90% respectively with heavy rainfall during the month of July and August. Diarrhoeic faecal samples from all the ailing goats were collected aseptically for the isolation of bacteria.

Isolation and identification of bacteria

Both aerobic and anaerobic bacterial isolations were attempted. For aerobic isolation, collected samples were first inoculated in peptone water broth with novobiocin and incubated at 37[degrees]C for 4hr. Soon after, inoculum from each tube were transferred to freshly prepared sterile MacConkey lactose agar (MLA) and incubated at 370C for 24hr. Bacterial colonies were purified and identified as per standard bacteriological procedures (Holt et al., 1994) and got serotyped at National Salmonella and Escherichia Center, Central Research Institute, Kasauli, Himachal Pradesh, India.

For anaerobic isolation, samples were inoculated in Robertson's cooked meat (RCM) medium broth supplemented with glucose, hemin and vitamin K (Himedia, Mumbai) with neutral oil overlay and incubated at 37[degrees]C for 48hr. The inoculums from the RCM media were seeded onto 10% goat blood agar and incubated anaerobically with an anaerobic gas-pack system (BBL Microbiology Systems Cockeysville, Md. [Div. Becton Dickinson and Co.]) for 24hr at 37[degrees]C. Bacterial colonies were purified based on the size, shape, color and patterns of haemolysis on blood agar and were subjected to Gram's and malachite spore staining. The isolates were identified based on the litmus milk test, gelatinase, deoxyribonuclease (DNase), lecithinase and fermentation of glucose and lactose (Holt et al., 1994).

Detection of toxin genes from E. coli isolates by PCR

Polymerase chain reaction assay was employed to detect the virulence toxin genes like stx1 and stx2 genes, hlyA gene and rfbO157 gene of E. coli. A single colony from nutrient agar was suspended in 100[micro]l of sterile distilled water and boiled at 100[degrees]C for 10 min. After boiling, the cell suspensions were cooled on ice bath, centrifuged and the top supernatant was used as a source of template DNA.

The amplification was carried out in 25[micro]l reaction volume containing 12.5[micro]l of 2x PCR master mix (Promega, USA) containing 4mM magnesium chloride, 0.4mM of deoxynucleotide triphosphates (dNTPs), 0.5U of Taq DNA polymerase, 150mM trishydrochlroric acid, pH 8.5 (Promega, USA), 0.5[micro]M of each forward and reverse primer primers and 2.5[micro]l of template DNA. The PCR reactions were performed in iCycler (BioRad, USA). After initial denaturation at 94[degrees]C for 4 min, the amplification cycle had denaturation, annealing and extension at 94[degrees]C, 55[degrees]C and 72[degrees]C for 1 min each respectively. Final extension was done at 72[degrees]C for 10 min. The specific forward and reverse primer pairs for stx1 gene of 348bp were [5.sup./]-cagttaatgtggtggcgaagg-[3.sup./] and [5.sup./]-caccagacaatgtaaccgctg-[3.sup./] (Cebula et al., 1995), stx2 gene of 584bp were [5.sup./]-atcctattcccgggagtttacg-[3.sup./]

and [5.sup./]-gcgtcatcgtatacacaggagc-[3.sup./] (Cebula et al., 1995), hlyA gene of 1551bp were [5.sup./]-ggtgcagcagaaaaagttgtag-[3.sup./] and [5.sup./]-tctcgcctgatagtgtttggta-[3.sup./] (Schmidt et al., 1995), eaeA gene of 384bp were [5.sup./]-gacccggcacaagcataagc-[3.sup./] [5.sup./]-ccacctgcagcaacaagagg-[3.sup./] (Paton and Paton, 1998), rfbO157 gene of 497bp were [5.sup./]aagattgcgctgaagcctttg-[3.sup./] and [5.sup./]-cattggcatcgtgtggacag-[3.sup./] (Desmarchelier et al., 1998) were commercially synthesized (GENSET, USA). Standard E. coli H 3159, E. coli ED933 and O157 (E771/01) strains were used as positive controls for both stx1 and 2, hlyA and rfbO157 genes, respectively and C. perfringens isolate used as negative control. The PCR amplicons (5[micro]l) were electrophoresed in 1.5% agarose (Promega) gel prepared in 1x TAE (tris-acetate-EDTA; pH 8.0) buffer and stained with 0.4 [micro]g/ml ethidium bromide and observed in gel documentation system (Image Master[R] VDS, Pharmacia Biotech, Sweden).

Detection of toxin genes from C. perfringens isolates by polymerase chain reaction

Polymerase chain reaction assay was employed to detect the virulence toxin genes like alpha toxin gene (cpa), beta toxin gene (cpb), epsilon toxin gene (etx), iota toxin gene (iA), enterotoxin gene (cpe) and beta2 toxin gene (cpb2) of C. perfringens according to Songer and Meer (1996). The amplification reaction in PCR was carried out according to the method described above. The specific forward and reverse primer pairs for cpa gene of 324bp were [5.sup./]-gctaatgttactgccgttga-[3.sup./] and [5.sup./]cctctgatacatcgtgtaag-[3.sup./] (Titbal et al., 1989), cpb gene of 180bp were [5.sup./]gcgaatatgctgaatcatcta-[3.sup./] and [5.sup./]-gcaggaacattagtatatcttc-[3.sup./] (Hunter et al., 1993), etx gene of 655bp were [5.sup./]-gcggtgatatccatctattc-[3.sup./] and [5.sup./]-ccacttacttgtcctactaac-[3.sup./] (Hunter et al., 1992), iA gene of 446bp were [5.sup./]-actactctcagacaagacag- [3.sup./] and [5.sup./]-ctttccttctattactatacg[3.sup./] (Perelle et al., 1993), cpb2 gene of 567bp were [5.sup./]-agattttaaatatgatcctaacc-[3.sup./] and [5.sup./]caatacccttcaccaaatactc-[3.sup./] (Gibert et al., 1997) and cpe gene of 233bp were [5.sup./]ggagatggttggatattagg-[3.sup./] and [5.sup./]-ggaccagcagttgtagata-[3.sup./] (Czeczulin et al., 1996) were commercially synthesized (GENSET, USA). C. perfringens type A positive for cpa genes isolated from atypical blackleg in cattle was used as positive control (Shome et al., 2006) and E. coli strain used as negative control. The PCR amplicons (5[micro]l) were electrophoresed in 1.5% agarose gel in TAE buffer, stained with ethidium bromide and observed in gel doc system.

Result and Discussion

Isolation, identification and detection of toxin genes from E. coli

On aerobic isolation, 16 bacterial isolates characterized by Gram negative, rod shaped positive for indole and methyl red and negative for citrate were identified as E. coli. The serogroups were identified as O5 (2), O8 (2), O15 (1), O26 (1), O63 (1), O78 (2), O128 (3), O157 (2) and UT (2). In PCR screening, 11 (68.75%) isolates were identified as shiga toxin producing E. coli (STEC) distributed among the serogroups of O5, O15, O26, O78, O128 and O157 (Table 1). The PCR assay revealed both stx1 and 2 genes, stx1 gene, hlyA gene and rfbO157 gene in 6 (37.5%), 5 (31.2%), 2 (12.5%), 2 (12.5%) cases respectively (Figure 1 and 2). None of the E. coli isolates was positive eaeA genes. In congruence, Zschock et al., (2000) reported the detection of both stx1 & 2, stx1 alone, stx2 alone and hlyA toxin gene in 70 E. coli isolated from goats by multiplex PCR. In the present study, high prevalence (68.75%) of STEC, positive for both hlyA and rfbO157 toxin genes from the faecal samples of goats were also reported earlier (Shome et al., 2004; Wani et al., 2006).

Isolation, identification and detection of toxin genes from C. perfringens

On anaerobic isolation, a total of 13 bacterial isolates characterized by Gram positive, rod shaped bearing sub-terminal oval endospore, showed double hemolysis on blood agar with DNase and lecithinase activities were identified as C. perfringens. In PCR, both cpa and cpb2 toxin genes were detected from all the isolates (Table 1; Figure 3).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

None of the C. perfringens isolates was positive for cpb, etx, iA and cpe genes. The PCR assay thus revealed that all isolates were C. perfringens type A. The detection of both alpha and beta2 toxin genes of C. perfringens type A from 5 week old goat died in enterotoxaemia by PCR was earlier reported (Dray, 2004). C. perfringens type A, producing alpha toxin, is a common isolate from cases of caprine enterotoxemia (Daube et al., 1996), but its pathologic role is equivocal, as it is a normal commensal of the gastrointestinal tract (Songer, 1996). Similarly, the cpb2 gene has been identified in a number of enteritis cases from a variety of species but the exact role of beta2 toxin in pathogenesis of chronic enteritis is not straight forward. Both the E. coli and C. perfringens type A are opportunistic bacteria can cause chronic enteritis in goats by releasing different toxins. As both the organisms are zoonotically important, surveillance for the dissemination of the toxin genes by molecular methods will be crucial.

[FIGURE 3 OMITTED]

Conclusion

Present study revealed that the chronic enteritis in goats was multifactorial. Both shiga toxigenic E. coli and beta2 toxigenic C. perfringens type A have appeared to play crucial dual role in chronic enteritis in goats in Meghalaya. As classical identification methods are expensive, time consuming and also gives low sensitivity results, PCR can be very useful tool to determine the presence of toxin genes and identification of E. coli and C. perfringens from the clinical samples. Since, both the bacteria can cause disease in human by entering into the food chain, therefore consumption of diseased goat meat for human has a crucial impact on public health.

As the disease is having public health importance, good management practices, awareness regarding the disease is in first priority. Further, molecular analysis of the pathogens and the role of toxin genes in association with the disease is required to be understood for undertaking the development of control measures, especially for the formulation of cost effecting vaccine.

References

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[18] Titball, R. W., Hunter, S. E. C., Martin, K. L., Morris, A. D., Shuttleworth, A. D., Rubdige, T., Anderson, D. W., and Kelly, D. C., 1989, "Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens," Infect. Immun., 57(2), pp. 367-376.

[19] Wani, S. A., Samanta, I., Munshi, Z. H., Bhat, M. A., and Nishikawa, Y, 2006, "Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties," J. Appl. Microbiol., 100(1), pp. 108-113.

[20] Wasteson, Y., 2001, "Zoonotic Escherichia coli." Acta Vet. Scand., 95, pp. 79-84.

[21] Yamagishi, T., Sugitani, K., Tanishima, K., and Nakamura, S., 1997, "Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens," Microbiol. Immunol., 41(4), pp. 295-299.

[22] Zschock, M., Hamann, H. P., Kloppert, B., and Wolter, W., 2000, "Shigatoxin-producing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties," Lett. Appl. Microbiol., 31(3), pp. 203-208.

Arunava Das *, Yahya Mazumder and Merlin Rajesh Lal L. P.

(1) Department of Biotechnology, Bannari Amman Institute Technology, Sathyamangalam-638401, Erode District, Tamil Nadu, India.

* Corresponding Author: Dr. Arunava Das, Faculty of Department of Biotechnology, Bannari Amman Institute Technology, Sathyamangalam-638401, Erode District, Tamil Nadu, India, E-mail: drarunavadas@rediffmail.com
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Author:Das, Arunava; Mazumder, Yahya; Lal, Merlin Rajesh L.P.
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2009
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