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Research Article
 

Identification of Enteropathogenic Campylobacters in Poultries` Faeces by PCR and its Comparison with Culture in Zahedan (Iran)



Bokaeian Mohammad and Mohagheghi Fard Amir H.
 
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ABSTRACT

The present study was performed in order to identify the organisms in poultries by culture and the PCR. This is the first step in an effort to devise strategies for control of human Campylobacters infection in Zanhedan. One hundred and sixteen samples of rectal swabs from chickens of Zahedan (south-east Iran) poultry farms were collected and cultured on Campylobacter selective agar. Subsequently, identification was carried out by biochemical tests. DNA was isolated from suspensions, which were already prepared from stool samples and purified with specific kits. The PCR was done by application of species-specific primers of C. jejuni and C. coli. The results of culture in case of 11 samples were positive out of them 8 were as C. jejuni and 3 belonged to C. Coli, whereas in case of the PCR results 27 samples were positive out of them 18 isolates belonged to C. jejuni and 9 were found as C. coli. Hence, sensitivity and specificity of the PCR were found as 100 and 84.7%, respectively, therefore it is concluded that the PCR protocol applied in this study could identify all positive samples. Furthermore, detection of the species by the PCR was exactly identical with biochemical procedures, so taking into account accuracy and rapidity of the technique, it does seem that PCR is a reliable alternative for identification of Campylobacters in stool samples of the poultries.

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  How to cite this article:

Bokaeian Mohammad and Mohagheghi Fard Amir H. , 2006. Identification of Enteropathogenic Campylobacters in Poultries` Faeces by PCR and its Comparison with Culture in Zahedan (Iran). Journal of Medical Sciences, 6: 984-988.

DOI: 10.3923/jms.2006.984.988

URL: https://scialert.net/abstract/?doi=jms.2006.984.988

INTRODUCTION

Nowadays Campylobacter jejuni and Campylobacter coli are considered as the most common cause of bacterial diarrhea and in developing countries, they are associated mainly with illness in children (Coker et al., 2002; Oberhelman and Taylor, 2002).

Sporadic cases of human infection are generally attributed to the consumption of raw or undercooked poultry meat (Pearson et al., 2000; Deming et al., 1987) whereas outbreaks have been traced to contamination of the water supply (Pearson et al., 2000; Tokumaru et al., 1991; Sacks et al., 1986).

Natural reservoirs for Campylobacter jejuni include chicken and other poultries (Stern, 1992; Skirrow, 1991) and Campylobacter coli is usually isolated from intestines of pigs (Pearce et al., 2003).

The incidence of human Campylobacter enteritis in Iran has increased over past few years (Bokaeian and Dabirzaden, 1999; Bokaeian and Sultandallal, 1994). On the other hand, some difficulties in diagnosis of the species by culture (long incubation time, uncertainty of biochemical tests, live atypical strains incapable of growth on media) have induced attentions of researchers towards other alternative procedures such as the PCR (Magistrado et al., 2001; D'Sullivan et al., 2000).

Hence, as till now, there has been no investigation of the source(s) of human Campylobacter infections in Zahedan, the present study was performed in order to identify the organisms in poultries by culture and the PCR. This is the first step in an effort to devise strategies for control of human Campylobacter infection in Zahedan.

MATERIALS AND METHODS

A total of 116 samples in random from rectal swabs of chickens as of poultry farms in Zahedan were collected between January and June 2004. After enrichment of samples in Campy-Thio broth for 12 to 18 h, they were sub-cultured on Campylobacter selective agar (containing 5% defibrinated sheep blood in addition to vancomycin, polymyxin and trimethoprim). After 48 h incubation under microaerophilic condition at 42°C, colonies having Campylobacter characteristics (convex, gray, 2 to 4 mm in diameter) were gram stained. Furthermore, the species of Campylobacters were identified by application of catalase, oxidase and hyppurate hydrolysis tests plus antibiotic sensitivity tests applying 30 μg discs of cephalothin and nalidixic acid. Subsequently, for direct DNA isolation from the samples, High Pure Template preparation Kits (Roche, Germany) were employed and for this purpose enriched suspension of the samples in Campy-Thio was centrifuged (2000 rpm for 10 min) and the supernatant was then removed. Afterward, to the precipitant lysosyme and phosphate buffer were added and kept in water bath at 37°C for 30 min, subsequently to the mixture isopropanol was added and samples were transferred to filter tube and after centrifugation, inhibitor removal buffer was put in. The samples were then recentrifuged and wash buffer was added to them, once more the specimens were spun and this time elution buffer was put in. In the final step after centrifugation, DNA of the samples were collected and with the help of UV spectrophotometer, optical density of the specimens were assessed and DNA content of each sample was found out. Furthermore, two sets of primers were employed for the PCR amplification as: (JEJ1-JEJ2) Jejuni species specific and (COL1-COL2) coli species specific primers (Gonzalez et al., 1997). The sequence of primers were as following:

Image for - Identification of Enteropathogenic Campylobacters in Poultries` Faeces by PCR and its Comparison with Culture in Zahedan (Iran)

Concentrations of the reagents for the PCR cocktail (final volume 25 μL) were as: 100 ng of the purified DNA, KCL 50 mM, Tris-HCL (PH 8.3) 10 mM, Gelatine 0.001%, Mg Cl2 3 mM, dNTP 0.2 mM, primers 200 pM and Taq DNA polymerase 0.5 unit (Gonzalez et al., 1997). Total PCR cycles were 30 and each cycle was as following: denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec and finally extension at 72°C for 1 min (Gonzalez et al., 1997). The PCR product was then mixed with loading buffer and ethidium bromide before electrophoresis, which was carried out in presence of specific ladder. Moreover, in order to control PCR, DNA extraction and purification from control strains (Campylobacter jejuni Lior 1, Campylobacter jejuni Lior 2 and Campylobacter coli Lior 8) were also included exactly as for tests.

RESULTS

Out of 116 cultured samples, 11 were found positive by culture (9.4% of the total samples) having the morphology of campylobacters as: curved, gram-negative bacilli. Catalase and oxidase tests of all strains were established positive and all were resistant to cephalothin and sensitive to nalidixic acid. Furthermore, hyppurate hydrolysis test for 8 samples was positive and for the rest negative, therefore biochemical tests indicated that 72% of the strains belonged to C. jejuni species and remaining 28% to C. coli species.

Image for - Identification of Enteropathogenic Campylobacters in Poultries` Faeces by PCR and its Comparison with Culture in Zahedan (Iran)
Fig. 1: Electrophoresis of the PCR product of extracted and purified DNA from samples of poultries. M: marker for molecular weight, Lanes 3 and 5 samples of the poultries having C. coli, Lanes 1,2, 4 and 7: samples of the poultries having C. jejuni, lane 6: sample devoid of C. jejuni or C. coli

Table 1: The comparison of PCR and culture in identification of Campylobacters in poultries’ faeces
Image for - Identification of Enteropathogenic Campylobacters in Poultries` Faeces by PCR and its Comparison with Culture in Zahedan (Iran)

Out of 116 samples, which were subjected to DNA extraction and the PCR 27 were positive, that comprises 23.2% of the total specimens. Subsequently, 18 (66.7%) samples were amplified by C. jejuni specific primers and 9 (33.9%) with C. coli specific primers (Fig. 1).

All positive samples by culture (n = 11) were positive by the PCR too as well as other 16 specimens that were negative by culture found positive by the PCR. The remaining 89 samples were established negative by both culture and the PCR (Table 1).

DISCUSSION

Campylobacter is considered part of the normal flora of the gut of chickens but its isolation manifest massive differences. In some investigations 30 to 100% of the poultries have been regarded carriers of the bacteria as normal flora of intestinal tract (D'Sullivan et al., 2000). In the other hand, Magistrado et al. (2001) has reported only 5.9% isolation of Campylobacters and in our study, the isolation rate was 9.4% by culture method. There has been no evidence for these differences but age of chicken may be an important factor. In another study (Workman et al., 2005) all of the negative chicks were under the age of 3 weeks and in our study, 80% of examined samples were from chicks between 2 to 4 weeks and all of the positive chicks were above the age of 5 weeks.

Thus investigation of older chicks may yield a different prevalence rate and give a better indication of the overall prevalence of Campylobacter in chicks in Zahedan.

On the other hand, there are reports of seasonal variation in shedding by animals (Workman et al., 2005). Our study was done in winter and spring, thus frequent sampling throughout the year may give a better indication of the prevalence of Campylobacter in chickens. However in a study in Thailand the most Campylobacter isolated were collected during the rainy months of year (Meeyam et al., 2004).

In comparison to the culture, the PCR was capable of detecting more positive samples (27 versus 11) therefore it can be concluded that PCR is a suitable substitute for diagnosis of Campylobacters in chickens’ specimens. Other investigators have also reported that PCR detection of Campylobacter directly from feces may give better indication of the carriage rate in other animals (Inglis and Kalischuk, 2003)

The reasons for revealing culture negative samples in spite of being positive by the PCR in our study could be unsuitability of the used selective media for some strains, gap between sampling and culture and insufficient number of bacteria in some specimens. These factors have contributed to low Campylobacter isolation rates in chickens by others (Workman et al., 2005; Mahendru et al., 1997; Goossens et al., 1984).

In this study, 72 and 28% of the isolated species belonged to C. jejuni and C. coli, this is comparable with results of similar study in Thailand (Meeyam et al., 2004). Roughly in all other studies, C. coli has been the most common species isolated from pigs (Workman et al., 2005) but there are reports of isolation of this species from chicken meat (Workman et al., 2005; Meeyam et al., 2004).

Moreover the available data indicates enormous dissimilarities between species isolation from poultries for instance; in one study 99% of the infections in poultries were due to C. jejuni species (Ng et al., 1997) while Van Looveren et al., (2001) have attributed 79 and 21% of the isolations to C. jejuni and C. coli, respectively. In a similar study carried out by other researchers these figures were reported 67 and 33% correspondingly (Eyigor et al., 1999) and according to Magistrando and associates’ report out of 8 isolated strains of Campylobacters from poultries 3 belonged to C. jejuni and 5 to C. coli species (Magistrado et al., 2001).

Identification of strains by biochemical tests in this study was quite identical with that of PCR. Nonetheless, we recommend PCR as an efficient alternative for culture due to its sensitivity and speed in addition to intricacy of some strains’ cultivation in selective media (specially due to insufficient number of bacteria in poultries’ specimens) or at least for those samples whose results by culture are negative.

In our previous study in Zahedan (Bokaeian and Dabirzadeh, 1999) Campylobacters were identified in 5% of diarrheic children. As Campylobacter can transmit from chicken to human (Meeyam et al., 2004) and based on genotyping studies there are similarities between isolates from chicken and those isolated from human (Meeyam et al., 2004) health protection measures to control infectious disease in Zahedan should focus on preventing transmission from chicken meat to human.

We recommend of cooking chicken meat thoroughly and treatment with acidic juice and salt to prevent transmission if Campylobacters to human.

REFERENCES

1:  Bokaeian, M. and M.M. Sultandallal, 1995. Study of campylobacter diarrhea in Zahedan region. Iran. J. Public Health, 24: 45-54.
Direct Link  |  

2:  Bokaeian, M. and M. Dabirzadeh, 1999. Identification of campylobacter in stool of children with diarrhea referring to Ali Asghar Pediatric Clinic, Zahedan, Iran. Tabib-E-Shargh, 1: 25-30.

3:  Coker, A.O., R.D. Isokpehi, B.N. Thomas, K.O. Amisu and C.L. Obi, 2002. Human campylobacteriosis in developing countries. Emerg. Infect. Dis., 8: 237-244.
CrossRef  |  PubMed  |  Direct Link  |  

4:  Deming, M.S., R.V. Tauxe, P.A. Blake, S.E. Dixon and T.S. Jones et al., 1987. Campylobacter enteritis at a university transmission from eating chicken and from cats. Am. J. Epidemiol., 126: 526-534.

5:  D'Sullivan, N.A., R. Fallon, C. Carrol, T. Smith and M. Maher, 2000. Detection and differentiation of Campylobacter jejuni and Campylobacter coli in broiler chicken samples using a PCR/DNA probe membrane based colorimetric detection assay. Mol. Cell. Probes, 14: 7-16.
CrossRef  |  Direct Link  |  

6:  Eyigor, A., K.A. Dawson, B.E. Langlois and C.L. Pickett, 1999. Detection of cytolethal distending toxin activity and cdt genes in Campylobacter species isolated from chicken arcasses. Applied Environ. Microbiol., 65: 1501-1505.
PubMed  |  

7:  Gonzalez, I., K.A. Grant, P.T. Richardson, S.F. Park and M.D. Collins, 1997. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol., 35: 759-763.
PubMed  |  Direct Link  |  

8:  Goossens, H., M. Boeck, H. Coignau, L. Vlaes, C. Van den Borre and J.P. Butzler, 1984. Modified selective medium for isolation of Campylobacter spp. from feces: Comparison with Preston medium, a blood-free medium and a filtration system. J. Clin. Microbiol., 24: 840-843.

9:  Inglis, G.D. and L.D. Kalischuk, 2003. Use of PCR for direct detection of Campylobacter species in bovine feces. Applied Environ. Microbiol., 69: 3435-3447.
CrossRef  |  Direct Link  |  

10:  Magistrado, P.A., M.M. Garcia and A.K. Raymundo, 2001. Isolation and polymerase chain reaction-based detection of Campylobacter jejuni and Campylobacter coli from poultry in the Philippines. Int. J. Food Microbiol., 70: 197-206.
Direct Link  |  

11:  Mahendru, M., K.N. Prasad, T.N. Dhole and A. Ayyagari, 1997. Rapid identification of Campylobacter jejuni strains by polymerase chain reaction and their restriction fragment length polymorphism analysis. Indian J. Med. Res., 105: 9-14.

12:  Meeyam, T., P. Padungtod and J.B. Kaneene, 2004. Molecular characterization of Campylobacter isolated from chickens and humans in Northern Thailand. Southeast Asian J. Trop. Med. Public Health, 35: 670-675.

13:  Ng, L.K., C.I. Kingombe, W. Yan, D.E. Taylor, K. Hiratsuka and M.M. Garcia, 1997. Specific detection and confirmantion of Campylobacter jejuni by DNA hybridization and PCR. Applied Environ. Microbiol., 63: 4558-4563.
Direct Link  |  

14:  Oberhelman, R.A. and D.N. Taylor, 2000. Campylobacter Infections in Developing Countries. In: Campylobacter, Nachamkin, I. and M.J. Blaser (Eds.). 2nd Edn., American Society for Microbiology, Washington DC., pp: 139-153

15:  Pearce, R.A., F.M. Wallace, J.E. Call, R.L. Dudley and A. Oser et al., 2003. Prevalence of Campylobacter within a swine slaughter and processing facility. J. Food Prot., 66: 1550-1556.
Direct Link  |  

16:  Pearson, A.D., M.H. Greenwood, J. Donalson, T.D. Healing and D.M. Jones et al., 2000. Continuous source outbreak of campylobacteriosis traced to chicken. J. Food Prot., 63: 309-314.
PubMed  |  Direct Link  |  

17:  Sacks, J.J., S. Lieb, L.M. Baldy, S. Berta and C.M. Patton et al., 1986. Epidemic campylobacteriosis associated with a community water supply. Am. J. Public Health, 76: 424-428.
PubMed  |  Direct Link  |  

18:  Skirrow, M.B., 1991. Epidemiology of Campylobacter enteritis. Int. J. Food Microbiol., 12: 9-16.
CrossRef  |  PubMed  |  Direct Link  |  

19:  Stern, N.J., 1992. Reservoirs for Campylobacter jejuni and Approaches for Intervention in Poultry. In: Campylobacter jejuni: Current Status and Future Trends, Nachamkin, I., M.J. Blaser and L.S. Tompkins (Eds.). American Society for Microbiology, Washington, DC., pp: 49-60

20:  Tokumaru, M., H. Konuma, M. Umesako, S. Konno and K. Shinagawa, 1991. Rates of detection of Salmonella and Campylobacter in meats in response to the sample size and the infection level of each species. Int. J. Food Microbiol., 13: 41-46.
PubMed  |  Direct Link  |  

21:  Van Looveren, M., G. Daube, L. de Zutter, J.M. Dumont and C. Lammens et al., 2001. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J. Antimicrob. Chemother., 48: 235-240.
PubMed  |  Direct Link  |  

22:  Workman, S.N., G.E. Mathison and M.C. Lavoie, 2005. Pet dogs and chicken meat as reservoirs of Campylobacter species in Barbados. J. Clin. Microbiol., 43: 2642-2650.
CrossRef  |  Direct Link  |  

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