The current study was conducted to determine the prevalence of Campylobacter species, especially Campylobacter jejuni and Campylobacter coli in chicken, chicken products and associated personnel. A total of 120 samples were collected. The samples from chicken faeces 20, chicken products 80 and associated personnel 20 were used in the study. Among the chicken products samples of breasts 20, thighs 20, wings 20, and hearts 20 were respectively. All chicken samples were collected from local market along with faecal samples from the workers associated with chicken slaughtering (Butchers) around the different areas of Faisalabad district. Out of 120 samples examined 43 were found to be contaminated with Campylobacter. The overall prevalence of Campylobacter was found to be 35.83% out of which Campylobacter jejuni was 72.09 % and the remaining 27.91 % of the isolates were Campylobacter coli. Among the observed samples the highest prevalence 75% was recorded in chicken faeces followed by 45% in associated personnel while in chicken products the highest prevalence was recorded in breast 35% followed by 25% thighs, 20% wings and 15% in chicken hearts. The study reported that the prevalence of campylobacter species was significantly higher in the chicken faeces, raw chicken products and in butchers associated in chicken processing
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Campylobacters are among the chief organisms that cause diarrheal disease in human. The infection in most of the cases is from food and water that has an economic importance all over the world (Dingle et al., 2000; Yolanda et al., 2002). Mostly the cases gone undiagnosed or have not been reported affecting more that 2.4 million people each year. The distribution of disease is more frequent in summer that winter. According to CDC few numbers of Campylobacter organisms less than 500 could be infectious (CDC, 2005).
Campylobacteriosis is generally connected with handling of poultry, eating raw or under cooked meat of poultry or it may arise as cross contamination of raw meat with already cooked meat and other food items (Corry & Atabay, 2001). The important species of thermophilic Campylobacters are Campylobacter jejuni, campylobacter coli and Campylobacter lari, causing foremost cases of bacterial gastro enteritis and in 80 to 90 % of the cases food borne infections are due to Campylobacter jejuni (Frost et al. 1998; Park 2002).
The Campylobacter are dispersed into a wide range of species and is possible to isolate from animals and poultry kept for food production, wild animals, wild birds and from pet animals as commensal of their digestive system in conjunction to environment as well (Sandberg, 2002).
The increased consumption of chicken and chicken by products during current years led to increase in the out breaks of human enterocolitis spread around the world including developed and under developed countries and particularly in children, old people and patients with immunodeficiency(CDC, 2005; Corry & Atabay, 2001; NPHS, 2006).
Animal raised for food purpose could be carrier for Campylobacter and may be asymptomatic, during slaughtering and carcass dressing there is risk of contamination of the food products with this pathogen (Whyte et al., 2003).
The most important and widespread sporadic food borne and epidemic cases are due to the consumption of under cooked meat of poultry, unpasteurized milk and contaminated drinking water (Butzler and Oosterom 1991; Altekruse et al. 1999; Jorgensen et al., 2002).
The important Campylobacter specie that is the cause diarrhoea in children is Campylobacter jejuni and the frequency is increased when they have pet animals, drink untreated water and utilize chicken meat (Ali et al., 2003).
Campylobacter has been isolated from many food items like, raw milk, milk product, fresh salad and vegetables along with raw, chicken, beef and mutton meat from different areas of Pakistan. (Hussain et al., 2007; Mahmood et al., 2009)
Campylobacters are responsible for world wild infections in humans so there is a need to investigate this pathogen at retail level. The aim of this study was to determine the prevalence of thermophilic Campylobacter species especially Campylobacter jejuni and campylobacter coli in chicken meat, chicken by products obtained from retail shops in district Faisalabad. Additionally the prevalence of Campylobacter species in the workers involved in slaughtering and there processing. Subsequently, the aim was to determine the prevalence and Campylobacter species in the retail food products sold at the markets. The results from this study would be helpful in making assessment and finding the possible route of Campylobacter transmission from raw chicken meat and chicken products and finding the possible of Campylobacter transmission in workers during chicken slaughtering and carcass dressing.
2. Materials and methods
2.1. Sample collection
Always on Time
Marked to Standard
A total of 120 samples (20 samples of each category) were obtained over a period of 6 months from January 2008 to June 2008 on the day of slaughtering from various retail outlets located in Ghulam Muhammad Abad area Faisalabad. Samples include chicken faeces, chicken thighs, chicken wings, chicken hearts and stool of butchers. All samples were rapped with polyvinylidene films. The package permits anaerobic condition and chilling temperature (2-5 oC) was adopted with help of insulated bags containing ice packs immediately after collection samples were shifted to the department of Microbiology and analyzed within 24 hours to test their possible contamination with Campylobacters.
2.2. Sample preparation
A selective pre-enrichment of the samples was performed on selective enrichment broth and plating on selective media and The isolation media and conditions were adopted as described by Hussain et al. (2007), Petersen et al. (2001) and Nye et al. (2001).
The solid samples like chicken meat and by products including chicken breast, thighs, wings, and hearts. Using a sterile pair of scissor and forceps 10g from all samples were taken under aseptic conditions and put into 90ml of Campy CVA broth (Hardy Diagnostics, Santa Maria, USA) in sterile plastic beakers. Subsequently, they were homogenized for 1 min in an ultra homogenizer (Glas-ColÂÂ®, Terre Haute, Indiana, USA). Then homogenized material was transferred to sterile screw capped test tubes of 10 ml containing about 5 ml of homogenized sample. The headspace was minimized by adding some additional broth and the tubes were incubated at 42 oC for 24 hours. (Murray et al. 1999 and Robert et al. 1995)
Semi-solid samples, including chicken faeces and human stool were selectively enriched in Campy CVA broth in screw capped test tubes of 10 ml. 1g of chicken faeces and human stool samples were added to separate test tubes and the headspace was minimize by adding additional broth to create micro-aerophillic conditions inside the tubes and then incubated at 42 oC for 24 hours.
2.3. Microbiological analysis
After enrichment in broth, samples were inoculated into the Campy CVA Agar plates already prepared according to the recommendations of manufacturer (Hardy Diagnostics, Santa Maria, USA) along with provided antimicrobial agents and 5% lysed sheep blood and incubated at 42 oC for 24-48 hours placing into an anaerobic jar under micro-aerophilic conditions (CO2 10 %, O2 5 % and 85 % N2) The microaerophilic environment was adapted with the help of catalyst-free gas packs (Biomerieux, France) and examined for colony characteristic after the duration of 24 hours. (Murray et al. 1999)
Then the suspected colonies were sub-cultured onto modified charcoal cefoperazone deoxycolate agar (mCCDA-Preston) plates prepared according to the recommendation of manufacturer (Les Labs. QuÃÂ©-Lab Inc. Canada) and incubated at 42 oC for 24-48 hours placing into an anaerobic jar under micro-aerophilic conditions (CO2 10 %, O2 5 % and 85 % N2). Again the microaerophilic environment was adopted with the help of catalyst free gas packs (Biomerieux, France) and examined for colony characteristic after the duration of 24 hours. (Hutchinson and Bolton, 1984)
The isolates from mCCDA-preston agar were further streaked on MacConkey agar prepared according to Holt et al. (1994) and Cruickshank et al., (1975) to differentiate from other related organisms of the same family as a differential agar.
Suspected Campylobacter colonies were examined morphologically, and studied under microscope using Gram staining technique for presumptive identification as mentioned by Stern et al., (2001). The suspected positive samples for Campylobacter were finally confirmed and analyzed with help of analytical profile Index kit (APIÂÂ®, Biomerieux, France) as used by (Hussain et al., 2007 ; Stoyanchev et al., 2007). Along with Campylobacter identification this API is also helpful to identify Campylobacter at specie level and differentiation of Campylobacter jejuni and Campylobacter coli. The characteristic reactions for the isolates on API kits were based on indoxyl acetate hydrolysis, hippurate hydrolysis and on activity of urease.
2.4. Statistical analysis
The Campylobacter recovery rates from chicken, chicken products and associated personnel samples was compared statistically using computer tool Microsoft Excel and represented in graphically and tabulated form. The significance level of the data was distinct at P<0.05
Table 1: Distribution of collected samples on the basis of sources
Campylobacter positive samples
Table 2: Distribution of C. jejuni and C. coli among campylobacter positive samples
Campylobacter positive samples
3. Results and discussion
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The prevalence of Campylobacter in all the examined samples is summarized as graphical representation in Figure1. From 120 examined samples there were 43 found to be positive for Campylobacter. The overall prevalence found for Campylobacter in this study was 35.83% out of which 72.09% isolates were recognized as Campylobacter jejuni and 27.91% isolate were recognized as Campylobacter coli. Among the observed samples the highest incidence prevalence was recorded in chicken faeces 75% (15/20) followed by 45% (9/20) in associated personnel. While in chicken by-products the highest prevalence was recorded in chicken breast samples 35% (7/20) along with 25% (5/20) in chicken thighs, 20% (4/20) in chicken wings and 15% (3/20) in chicken heart. (Table1). Campylobacter jejuni is more prevalent in all the observed samples
Out of 120 samples examined 43 were found to be contaminated with campylobacter. The overall prevalence of campylobacter was found to be 35.83% out of which campylobacter jejuni was 72.09 % and the remaining 27.91 % of the isolates were identified as campylobacter coli. Among the observed samples the highest prevalence 75% was recorded in chicken faeces followed by 45% in associated personnel while in chicken by products the highest prevalence was recorded in chicken breast 35% followed by 25% in chicken thighs, 20% in chicken wings and 15% in chicken hearts.
For chicken and chicken by products a relatively low prevalence was noticed by Hussain et al. (2007) studied a total of 1636 food samples of meat, milk and other food commodities were procured Among meat samples, the highest prevalence (48%) of Campylobacter was recorded in raw chicken meat followed by raw beef (10.9%) and raw mutton (5.1%). Among other food commodities, the highest prevalence was observed in vegetable/fruit salad (40.9%), sandwiches (32%), cheese (11%) and raw bulk milk samples (10.2%). The overall prevalence of Campylobacter was found to be 21.5%, out of which 70.6% were identified as Campylobacter (C.) jejuni and 29.4% as C. coli.
In contrast of the above studies Sallam (2007) study the prevalence of Campylobacter in fresh chicken meat and chicken by-products and out of the 170 samples of chicken meat (breasts and thighs) and chicken by products (wings, livers, gizzards and hearts), 110 (64.7%) were contaminated with Campylobacter. Among the different products, chicken wings showed the highest contamination incidence (77.1%) followed by chicken thighs (70%), while chicken gizzards and hearts showed the lowest contamination incidence (45% and 40%, respectively). Of the 341 Campylobacter isolates, 278 (81.5%) were identified as Campylobacter jejuni and 63 (18.5%) isolates were identified as C. coli. These results are also similar to the results shown by Stoyanchev et al. (2007) who n = 210 of poultry carcasses and poultry products of these 35 frozen whole carcasses, 135 chilled poultry cuts (45 wing cuts, 45 thigh cuts and 45 fillet) and 40 thermally treated (ready-to-eat) poultry products. Although the results obtained showed that 35.2% of the frozen poultry carcasses for sale in the markets were Campylobacter contaminated is similar to the overall prevalence shown in my results but in chilled poultry cuts Campylobacter was isolated at the highest percentage in wing and thigh cuts, 91.1% and 88.9%, respectively. Vashin et al. (2005) reported the higher incidence of C. jejuni (89.7%), followed by lower incidence of C. coli (10.3%). Fernandez and Pison (1995) reported the higher incidence of campylobacter coli in chicken liver samples. From the 126 samples studied, 117 (92.9%).
And for the prevalence of chicken faeces samples a relatively high prevalence was recorded in a study from cloacal swabs of poultry by Kudirkiene et al. (2008) who reported that out of 40 broiler cloacal samples examined Campylobacter species were detected and confirmed in 37 samples (92.5 %) and C. jejuni was identified in 32 (86.5 %) and C. coli in 5 (13.5 %) out of 37 isolates.
Similarly a relatively higher incidence was found by Yildrim et al. (2005) who examined thermophilic Campylobacter species isolated from 393 (91.8%) of 428 samples. A total of 53 out of 57 rectal swab samples were positive for thermophilic Campylobacter species. Thermophilic Campylobacter species were isolated from 93.6%, 92.9% and 91.3% of broiler carcass, faecal and caecal samples, respectivelycies Overall, 92.2% and 7.8% of the isolates were identified as Campylobacter jejuni and C. coli
While for the chicken meat samples a relatively high prevalence of campylobacter was recorded from raw meat samples by Hong et al. (2007) who studied a total of 770 samples of retail raw meat and examined for the presence of Campylobacter species. The highest contamination rates were observed in chicken meat (220 181.4%] of 270 samples), whereas the rates of contamination in pork and beef were extremely low (1.6 and 1.2%, respectively. Similarly by Wong et al. (2007) who studied total of 1,011 uncooked retail meat samples (beef, un-weaned veal, chicken, lamb, mutton and pork) Prevalence of C. jejuni and C. coli were 89.1% in chicken, 9.1% in pork, 6.9% in lamb and mutton, 3.5% in beef, and 10% in unweaned veal and by Savas and Ozdemir 2006 who studied 127 samples of chicken meat for sale on the retail market Campylobacter species were isolated from 83.4% of the samples analyzed. Campylobacter jejuni was found in 74.8% of all samples. A total of 364 thermophilic Campylobacter strains were 70.1% C. jejuni, 21.1% Campylobacter coli and 8.6% Campylobacter lari.
Higher prevalence in the present study as compared to the previous work done by Hussain et al (2007) may be due to cross-contamination during manual skinning, evisceration and processing in the slaughter house/abattoir or in the butcher shops.
In Pakistan, the consumption of chicken and chicken by products could pose a potential risk to the public health. These foods are unlikely to be contaminated with Campylobacter unless they acquired the organism during preparation as a result of cross-contamination and the use of uncooked/undercooked component for chicken by products.
Further studies are required on the seasonal prevalence of Campylobacter in different food products. Surveillance data from the current study will be helpful to public health professionals to identify high risk food items sold in the supermarket/retail shops of Pakistan.