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BRIEF COMMUNICATION
Year : 2012  |  Volume : 30  |  Issue : 1  |  Page : 85-88
 

Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, south India


Department of Gastrointestinal Sciences, Christian Medical College, Vellore - 632 004, Tamil Nadu, India

Date of Submission02-Sep-2011
Date of Acceptance19-Oct-2011
Date of Web Publication22-Feb-2012

Correspondence Address:
S S Ajjampur
Department of Gastrointestinal Sciences, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.93049

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 ~ Abstract 

Campylobacter spp. are an important cause of bacterial gastroenteritis frequently isolated from animal, poultry and environmental samples. In this study, we investigated the zoonotic potential of Campylobacter spp. by comparing prevalence rates and species in 394 children with diarrhoea and 652 animals in Vellore using PCR-based tools. Eighteen children (4.5%) had campylobacteriosis, a majority of whom had co-pathogens (15/18) and most were infected with Campylobacter jejuni (16/18). A few C. coli and mixed infections with both species were also seen. Among the animal samples, 16/25 chicken samples (64%) were positive and all were found to be C. jejuni.


Keywords: Campylobacter, chicken, gastroenteritis, zoonotic


How to cite this article:
Rajendran P, Babji S, George A T, Rajan D P, Kang G, Ajjampur S S. Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, south India. Indian J Med Microbiol 2012;30:85-8

How to cite this URL:
Rajendran P, Babji S, George A T, Rajan D P, Kang G, Ajjampur S S. Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, south India. Indian J Med Microbiol [serial online] 2012 [cited 2019 Sep 22];30:85-8. Available from: http://www.ijmm.org/text.asp?2012/30/1/85/93049



 ~ Introduction Top
Campylobacter spp. are one of the most common causes of bacterial gastroenteritis with human campylobacteriosis caused principally by Campylobacter jejuni and C. coli and occasionally by C. lari. Campylobacter gastroenteritis is especially common in children during the first 5 years of life with reported isolation rates of up to 46%. [1] This pathogen is also associated with post-infectious auto-immune sequelae including Guillain-Barrι syndrome. In developed countries, consumption of contaminated chicken, red meat, water, milk, and contact with pets and farm animals have been implicated as potential sources of Campylobacter infection. The precise extent of the risk posed by campylobacteriosis to human health in developing countries where the practices for food handling and hygiene are different from industrialised countries is not clear. In the present study, we aimed to identify the potential zoonotic sources of Campylobacter associated diarrhoea in South India and if there was any species-specific risk.


 ~ Materials and Methods Top


Stool samples were collected from children with diarrhoea aged less than 5 years, admitted to the institution between January 2003 and May 2006 for studies on diarrhoeal etiology. Written informed consent was obtained prior to enrolment from parents or guardians of the children. The study was approved by the institutional review board. Diarrhoea was defined as the passage of three watery stools in a 24-h period. Diarrheal samples from animals were collected from a veterinary clinic and several dairy farms near Vellore between February 2007 and May 2008. Faecal samples from 25 chicken from a poultry farm in Vellore were also collected in 2009.

Animal and poultry stool samples were treated with proteinase K (2 μg/ml in 20 mM Tris, pH 7.5, 10 mM EDTA, and 0.1% SDS) followed by a previously validated extraction protocol with alkaline (1M KOH and dithithreitol), acid (25% HCl and 2M Tris HCl) and phenol chloroform treatment and Qiamp DNA stool minikit extraction (Qiagen, Valencia, CA, USA). DNA extraction of stool samples from children was carried out with the same kit but without pre-treatment of samples. Campylobacter spp. PCRs targeting the 16S rRNA gene [2] [Figure 1]a for screening C. jejuni and C. coli were carried out on all samples. Species identification was carried out on positive samples with hippuricase PCR [Figure 1]b (hippuricase gene seen exclusively in C. jejuni) [3] and asp PCR [Figure 1]c (aspartokinase gene seen in C.coli but not in C. jejuni and C. lari) [4] using previously published primers. Diarrhoeal samples from children were also screened for rotavirus by ELISA (Rotavirus IDEIA, UK), Cryptosporidium spp. by microscopy (modified acid fast stain) followed by SSU rRNA PCR-RFLP for species determination and diarrhoeagenic E. coli by multiplex PCR using previously described methods. [5]
Figure 1: Agarose gel electrophoresis of PCR products for identification of (a) Campylobacter spp. (16S rRNA PCR, 820 bp) (b) C. jejuni (hippuricase PCR, ~180 bp) (c) C. coli (asp PCR, 500bp). Lane 1 in all gels is a 100 bp molecular weight marker and NC is the negative control

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 ~ Results Top


A total of 394 stool samples from children aged less than five years presenting with diarrhoea were screened. The median (inter-quartile range, IQR) age of the children enrolled in the study was 10 months (7) and the mean duration of diarrhoea was 2.8 days. Campylobacter spp. were detected in 18 (4.5%) children, a majority of whom (15/18) were co-infected with other pathogens [Table 1]. There were no significant differences in features of diarrhoea between children with Campylobacter-associated mixed infections and other children enrolled in this study (data not shown). Of the 18 Campylobacter spp. positive samples, 13 children were infected with C. jejuni alone, two with C. coli alone and three with both C. jejuni and C. coli.
Table 1: Campylobacter species and co-pathogens identified in children with diarrhoea


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A total of 627 samples from animals with diarrhoea were collected, including 589 cows (25 were calves), 2 buffaloes, 11 bullocks and 25 goats (11 were kids). The mean duration of diarrhoea was 4.5 days for adult animals, 4 days for calves and 3 days for goat kids. None of the animals were found to be infected with Campylobacter spp. When stool from 25 chickens at a poultry farm in Vellore were screened, 16 chickens were found to be infected with Campylobacter spp. all of which were characterised as C. jejuni.


 ~ Discussion Top


Previous studies from India using culture as a screening tool have documented a wide variation of prevalence rates in children with diarrhoea ranging from 3.2% in Karnataka, [6] 8% from Chennai [7] to 13% from Lucknow [8] and up to 11.1% in asymptomatic controls. [9] In studies using PCR-based methods, Campylobacter spp. was found to be associated with 5.7% of diarrhoea in children in South India [5] and 5.1% in North Indian children. [10] In this study, a comparable prevalence rate of 4.5% was seen but Campylobacter strains were rarely identified as single pathogens indicating that the role of this organism as the etiological agent of diarrhoea may be questionable. Studies from India and other developing countries have also identified high rates of mixed or polymicrobial infections involving Campylobacter.[11],[12],[13] However, a recent study in north Indian children documented a higher risk of GBS among children with a history of Campylobacter-related diarrhoea [14] demonstrating the importance of detection and prevention of these infections.

Our data indicated that poultry was the major reservoir of infection in this region. Data on exposure to animals or poultry was not available for the children enrolled in the study; therefore, further risk assessment could not be carried out. Previous studies from India have, however, documented poultry as a reservoir of Campylobacter spp. with reported prevalence rates of 39.3% and 48%, respectively. [15],[16] Our findings of only C. jejuni in poultry is also in agreement with data from other parts of India which showed that C. jejuni were more frequent than C. coli in poultry. [16]

Although the current study did not detect Campylobacter spp. in cattle and other animals, studies from India have identified cattle and other livestock as reservoirs for this bacterium. [16] The lack of detection of Campylobacter isolates in cattle in our study could either be due to a low burden among these animals or due to the presence of inhibitors affecting PCR on animal faeces. [17],[18] Additional techniques, including culture on enrichment and/or semi-selective media or on template DNA obtained from enrichment medium may have improved detection rates.

In conclusion, the present study has shown that Campylobacter associated with diarrhoea in south Indian children usually occurs as a polymicrobial infection. Future community-based studies are required for insights into the role of Campylobacter in diarrhoea in Indian children. While other studies have reported cattle as a major reservoir of Campylobacter, we were only able to identify poultry as a potential source of infection. In addition, the observation that chicken-isolated species were common among human isolates is consistent with other studies where typing has indicated that strains from chickens are often linked to human campylobacteriosis and is an important first step in planning preventive strategies.

 
 ~ References Top

1.Yang JR, Wu HS, Chiang CS, Mu JJ. Pediatric campylobacteriosis in northern Taiwan from 2003 to 2005. BMC Infect Dis 2008;8:151.  Back to cited text no. 1
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2.Linton D, Lawson AJ, Owen RJ, Stanley J. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. J Clin Microbiol 1997;35:2568-72.  Back to cited text no. 2
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3.Marshall SM, Melito PL, Woodward DL, Johnson WM, Rodgers FG, Mulvey MR. Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 1999;37:4158-60.  Back to cited text no. 3
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4.Persson S, Olsen KE. Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples. J Med Microbiol 2005;54:1043-7.  Back to cited text no. 4
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5.Ajjampur SS, Rajendran P, Ramani S, Banerjee I, Monica B, Sankaran P, et al. Closing the diarrhoea diagnostic gap in Indian children by the application of molecular techniques. J Med Microbiol 2008;57:1364-8.  Back to cited text no. 5
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6.Naik DG, Jayaraj YM. Campylobacter jejuni diarrhea in north Karnataka. Indian Pediatr 1998;35:768-70.  Back to cited text no. 6
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7.Ananthan S, Swarna SR, Alavandi SV. Isolation of nalidixic acid resistant Campylobacters from cases of paediatric diarrhoea in Chennai. J Commun Dis 1998;30:159-62.  Back to cited text no. 7
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8.Jain D, Sinha S, Prasad KN, Pandey CM. Campylobacter species and drug resistance in a north Indian rural community. Trans R Soc Trop Med Hyg 2005;99:207-14.  Back to cited text no. 8
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9.Sen Gupta PG, Nair GB, Mondal S, Gupta DN, Sen D, Sikdar SN, et al. Epidemiology of campylobacteriosis in a cohort of rural population near Calcutta. Epidemiol Infect 1991;106:507-12.  Back to cited text no. 9
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10.Sinha S, Prasad KN, Pradhan S, Jain D, Jha S. Detection of preceding Campylobacter jejuni infection by polymerase chain reaction in patients with Guillain-Barre syndrome. Trans R Soc Trop Med Hyg 2004;98:342-6.  Back to cited text no. 10
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11.Albert MJ, Faruque AS, Faruque SM, Sack RB, Mahalanabis D. Case-control study of enteropathogens associated with childhood diarrhea in Dhaka, Bangladesh. J Clin Microbiol 1999;37:3458-64.  Back to cited text no. 11
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12.Glass RI, Stoll BJ, Huq MI, Struelens MJ, Blaser M, Kibriya AK. Epidemiologic and clinical features of endemic Campylobacter jejuni infection in Bangladesh. J Infect Dis 1983;148:292-6.  Back to cited text no. 12
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13.Bichile LS, Saraswati K, Popat UR, Nanivadekar SA, Deodhar LP. Acute Campylobacter jejuni enteritis in 385 hospitalised patients. J Assoc Physicians India 1992;40:164-6.  Back to cited text no. 13
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14.Kalra V, Chaudhry R, Dua T, Dhawan B, Sahu JK, Mridula B. Association of Campylobacter jejuni infection with childhood Guillain-Barre syndrome: A case-control study. J Child Neurol 2009;24:664-8.  Back to cited text no. 14
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15.Chattopadhyay UK, Rashid M. Rapid screening of chicken intestinal contents for Campylobacter jejuni using coagglutination. Vet Rec 2003;152:654-5.  Back to cited text no. 15
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16.Chattopadhyay UK, Rashid M, Sur SK, Pal D. The occurrence of campylobacteriosis in domestic animals and their handlers in and around Calcutta. J Med Microbiol 2001;50:933-4.  Back to cited text no. 16
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17.Wani SA, Bhat MA, Ishaq SM, Ashrafi MA. Determination of bovine rotavirus G genotypes in Kashmir, India. Rev Sci Tech 2004;23:931-6.  Back to cited text no. 17
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18.Wilde J, Eiden J, Yolken R. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol 1990;28:1300-7.  Back to cited text no. 18
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    Tables

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