PREVALENCE OF CAMPYLOBACTER SPP. AND ITS PATHOGENIC GENES IN POULTRY MEAT, HUMAN AND ENVIRONMENT IN ASWAN, UPPER EGYPT

Assiut University web-site: www.aun.edu.eg

PREVALENCE OF CAMPYLOBACTER SPP. AND ITS PATHOGENIC GENES IN POULTRY MEAT, HUMAN AND ENVIRONMENT IN ASWAN, UPPER EGYPT 

MOHAMED KARMI

Department of Food Hygiene, Faculty of Veterinary Medicine, Aswan

University, 81528 Aswan, Egypt

Received: 12 March 2019;     Accepted: 9 April 2019

INTRODUCTION

Campylobacter is the most common cause of gastroenteritis worldwide especially in children (Ruiz-Palacios, 2007, Kaakoush et al., 2015), C. jejuni infection frequently causes an acute enteritis with diarrhea, malaise, fever and abdominal pain, vomiting and/or bloody diarrhea specific for the more sever end of the disease spectrum and may affected by the host susceptibility and/or infective dose (Gillespie et al., 2006). Campylobacter enteritis signs can lasts for more than a year and may contribute to post-dysenteric irritable bowel syndrome (PD-IBS) (Spiller et al., 2000) and may causes Guillain-Barre Syndrome (GBS) during the subsequent 2-month period post infection (Tam et al., 2006). Guillain-Barre Syndrome (GBS) and Miller-Fisher Syndrome (MFS) are related to prior infection by C. jejuni in up to 40% of cases (Dingle et al., 2001). 

The majority of outbreaks of acute campylobacteriosis in human worldwide were associated with consumption of chicken and chicken meat products (Corry  and   Atabay, 2001,   Friedman

Corresponding author: Dr. MOHAMED KARMI

E-mail address: karmy99@yahoo.com

Present address: Department of Food Hygiene, Faculty of Veterinary Medicine, Aswan University, 81528 Aswan, Egypt

et al., 2004) also, contact with cattle, consumption of beef and milk were responsible for more than 90% of all sporadic human cases. Other sources such as sheep, contact with wild birds, contaminated water and pet animals were contribute for human infection but in much lesser extent (Wilson et al., 2008). The use of the same cutting board for chicken meat and salad without intermediate cleaning, spreading of pathogens via the kitchen environment and contaminated tools and equipments may causes cross-contamination more than the risk associated with undercooking of poultry meat (Luber, 2009). Drinking water may conform the common reservoir linking infection between humans and animals, including poultry and wild birds (Kapperud et al., 2003). Poultry meat and its products are the most implicated food and the most significant risk factor for acquiring infection in human campylobacteriosis. Genotyping with multilocus sequence typing (MLST) and relatedness study of large number samples of C. jejuni isolated from humans and broilers was carried out, all results of source attribution analysis confirmed the strong linkage between broiler C. jejuni and human cases (Griekspoor et al., 2015).

About 35 virulence genes were discovered in campylobacter isolates using PCR testing, include those involved in motility, chemotaxis, cell adhesion, invasion, cytotoxin production, capsule, multidrug and bile resistance, stress response/survival and the iron uptake system, there was no discernible difference in the virulence profiles of human and poultry isolates (Koolman et al., 2015). Several authors reported that some bacterial factors are more essential for the pathogenesis of campylobacter, including the motility and adherence of bacteria to intestinal mucosa, capability to invade enterocytes and toxin production (Datta et al., 2003). One of these virulence markers is the cadF gene, which encodes a 37 KDa protein belonging to the group of outer membrane proteins (OMPs) that functions as an adhesion protein responsible for certain steps of invasion (Konkel et al., 1999). Another interesting region, designated an invasion-associated marker (iam), has been identified in some C. jejuni and C. coli strains (Carvalho et al., 2001). CDT is composed of three subunits designated CDTs A, B and C. The B subunit targets the eukaryotic DNA and triggers a signaling pathway involving different protein kinases which results in a cell block before entering into mitosis. Until now, the individual role of the A and C subunits has not been totally elucidated. Its exact role in pathogenesis is not yet clear, but possible actions include inhibition of epithelial cell proliferation, apoptosis of immune cells and inhibition of a fibrotic response (Ceelen et al., 2006). Different distribution of genetic markers between human and chicken isolates indicates that some campylobacter infections in children may have additional sources other than contaminated chicken meat (Rozynek et al., 2005).

MATERIALS AND METHODS

Samples

A total of 250 samples including 100 samples from chicken meat (fresh chicken, frozen chicken and chicken meat products which include chicken luncheon, nuggets, pane, frozen liver and giblets), 75 samples human diarrhea (from hospitals and medical laboratories), 75 samples from environment (water, poultry slaughter house, meat shops, supermarkets, hospital`s kitchens and restaurants kitchens) from Aswan, Egypt were collected. Sampling box containing ice pads was used for carrying the samples maintaining low temperature from market to laboratory in the faculty of veterinary medicine, Aswan University. Samples were preserved in sterile polyethylene bags in the refrigerator. Twenty five grams of each chicken meat sample were aseptically transferred to sterile stomacher bag containing 225 ml Bolton broth with 5% lysed horse blood and antibiotic supplement. The bag content was homogenized using a Stomacher® 400 Circulator (Seward Ltd., UK) for 1 minute (FDA et al., 1998). Human diarrheal samples were collected in clean sterile containers. Environmental samples were collected by swabbing and the water samples collected in clean sterile bottles then concentrated by centrifugation at 20000 rpm for 10 minutes, the pellet was re-suspended in Bolton broth (Fricker and Park, 1989).

Isolation and Identification

Aseptically transfer 25 grams of each chicken sample to a sterile 250 ml flask and incubated for 4 hours at 37°C, followed by further incubation at 42°C/48 hours under micro-aerophilic condition (5% oxygen, 10% carbon dioxide and 85% nitrogen). Also, each environmental swab was immersed into sterile flasks containing 10 ml of Bolton broth (FDA et al., 1998). Loopful (10 µl) was taken from each Bolton broth enrichment culture after 48 hours and streaked on Charcoal Cefoperazone Deoxycholate Modified Agar Base (mCCDA) (Oxoid, Code: CM0739) selective solid medium with selective supplement, the culture plates were incubated micro-aerobically in a micro-aerobic atmosphere using anaerobic jar and campylobacter gas generating kits at 42°C for 48 hours (Bolton et al., 1984). Diarrheal and water samples were inoculated directly on mCCDA (Maher et al., 2003). Positive colonies are grayish, flat and moistened with tendency to spread and may have a metal sheen. Positive strains were confirmed with Gram's staining, oxidase, catalase, hippurate hydrolysis, Analytical Profile Index (API) Campy and other biochemical tests.

Genetic Characterization

DNA extraction was carried out by using boiling method; DNA was prepared by the whole-cell procedure. Each DNA template was prepared by using approximately half a loopful of culture transferred to 1 ml of brain heart infusion broth (Oxoid, Code: CM1135). The optimized whole-cell DNA preparations from all campylobacter species were further diluted 1:500 in distilled water and were heated at 100°C for 10 minutes. A five µl aliquot was directly used as a template for PCR amplification (Shah et al., 2009). Primer sequences were used as forward and reverse for 23S rRNA, hipO, glyA, cadF, cdtB and iam genes used for identification of Campylobacter species and for pathogenic genes (Table). Multiplex PCR amplification of Campylobacter species genes (23S rRNA, hipO, glyA) and the second multiplex PCR for amplification of pathogenic genes (cadF, cdtB, iam). Each reaction consists of 2.5 µl of 10x buffer, 2.5 µl master mix, 2.5 µl Taq polymerase, 1 µl each primer, 0.5 µl DNA template and nuclease free water till 25 µl volume. Thermacycler (Eppendorf, Germany) was used with initial denaturation step at 95 for 6 minutes followed by 30 cycles (denaturation at 95°C for 0.5 minute, annealing at 59°C for 0.5 minute and extension at 72°C for 0.5 minute for species genes) (Wang et al., 2002), (denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 1 minute for pathogenic genes) ending with final extension at 72°C for 7 minute. Amplified products were analyzed by 1.5% agarose gel electrophoresis stained with ethidium bromide and visualized on UV transilluminator (Andrzejewska et al., 2011).

RESULTS

Prevalence of campylobacter in chicken meat, human diarrhea and environmental samples were 32%, 14.7% and 13.3%, respectively. Results showed that prevalence rates were 50% for fresh chicken carcasses and 12.5% for frozen chicken carcasses. Prevalence rates of human diarrheal were 17.5% in children and 11.4% in adults while these rates were 18.75% in males and 11.62% in females. Prevalence rates in environmental samples were 44.4% in slaughter houses, 40% in restaurant`s kitchens while poultry meat shops, supermarkets, hospital`s kitchens and tape water were free from campylobacter. Multiplex PCR analysis of 23S rRNA gene to identify campylobacter species of positive isolates revealed lower prevalence rates which were 6% for chicken meat, 5.3% for human diarrhea and 4% for environmental samples. Genetic analysis of positive isolates for differentiation of campylobacter species through detection of hipO and glyA genes revealed that 64.3% were Campylobacter jejuni, 21.4% were Campylobacter coli and 14.3% were mixed culture. Prevalence of Campylobacter jejuni were 83.3%, 50% and 66.6% and of Campylobacter coli were 16.7%, 50% and 0% in chicken meat, human diarrhea and environmental isolates, respectively. Prevalence of virulence genes, Iam, cdtB and cadF genes in all examined samples were 61.5%, 69.2% and 84.6%, respectively. Prevalence of cadF, cdtB and Iam genes presence in Campylobacter jejuni isolates were 100%, 77.8% and 55.5%, respectively, while in Campylobacter coli were 33.3%, 33.3% and 66.6%, respectively.

Table 1: Prevalence of campylobacterspecies in chicken meat, human diarrhea and environmental samples.

Table 2: Prevalence of campylobactervirulence genes in examined samples.

Table 3: Primer sequences of campylobacterspecies genes and pathogenic genes.

DISCUSSION

In this study, the prevalence of campylobacter in chicken meat samples was 32% which is similar to that found in Gauteng, South Africa, 32.3% (Van Nierop et al., 2005) and lower than rates in Spain in which campylobacter isolated with percentage of 49.5% from chicken meat samples (Dominguez et al., 2002). Poultry can be contaminated from a variety of sources on farms and the contaminants are spread during processing, scalding, defeathering, evisceration and giblet operations are major points of spread, further spread can occur during handling in markets and kitchens. Insufficient thermal processing or cooking allows survival, Improper handling of cooked chicken frequently results in cross contamination from previously handled raw carcasses and parts (Bryan and Doyle, 1995). Prevalence of campylobacter in fresh chicken carcasses and in frozen carcasses was 50% and 12.5%, respectively. These rates are lower than results of fresh chickens sold in Ontario and Ohio which are 62% and 54%, respectively, (Park et al., 1981) and 64% in Sapporo, Hokkaido, Japan (Sallam, 2007), lower rates, 39.2% was recorded (Sproston et al., 2014), also, it is noticed that prevalence of campylobacter in fresh chickens was higher than that of frozen ones, reduction in positive samples in frozen chickens may be due to killing of campylobacter spp. or sub-lethal injury with or without reduction in viable counts under investigated storage temperatures which may indicates the ability of campylobacter to survive in chicken meat stored under refrigerated and frozen conditions (Eideh and Al-Qadiri, 2011), also, may be due to oxidative stress contribute to the freeze-thaw induced killing of campylobacter (Stead and Park, 2000). Prevalence of campylobacter in human diarrheal samples was 14.7% which are higher than results of Bangkok and its suburb in Thailand, 3% (Samosornsuk et al., 2015), and results of northern region of India, 2.6% (Vaishnavi et al., 2015) and lower than results obtained in Indonesia which was 79.5% (Pagaya et al., 2015) and that obtained in Pakistan which was 30% (Guhar et al., 2015), variations of rates in various parts of the world may be due to the varying standards and styles of living conditions, water supply and feeding habits. Rate of campylobacter in children was 17.5% which was lower than results in Egypt which was 35% (Barakat et al., 2015), higher than results of India which was 10% (Salim et al., 2014) and similar to results of northern Thailand which was 18% (Padungtod and Kaneene, 2005). Rate in adults was 11.4% which was higher than in northern Thailand which was 5% (Padungtod and Kaneene, 2005), prevalence in children was higher than that in adults may be due to lower immunity in children and lower standard of personal hygiene in contrast to adults, in older ages, most infections by campylobacter are mild or asymptomatic, probably because of immunity that may follow frequent exposure to contaminated food or water (Havelaar et al., 2009). Campylobacter infections were more common in males, 18.7%, than in females, 11.6%, and this may be due to that males are more exposed to infection than females, also, males eating food outside home more than females (Sadkowska-Todys and Hucharczyk, 2012). Prevalence of campylobacter in environmental samples was 13.3% which are lower than results of Israel which was 38.7% from environmental swabs and 100% from washing water (Rogol et al., 1985) and higher than results of Turin, northern Italy which was 0% for swabs (Bellio et al., 2014) and in Sao Paulo, Brazil which was 4.9% in abattoirs (Cortez et al., 2006). Prevalence rates in environmental samples were 44.4% in slaughter houses, 40% in restaurant`s kitchens while poultry meat shops, supermarkets, hospital`s kitchens and tape water were free from campylobacter. These results indicate higher rates of contamination in slaughter houses and restaurants that may attributed to low level of hygiene and cleanliness, high incidence of cross contamination from other birds and from use of equipments and utensils, low personal hygiene of workers (Tang et al., 2011). Transfer of campylobacter from naturally contaminated raw chicken products to cooked chicken products via cutting board occurred and that both C. jejuni and C. coli are able to transfer (Guyard-Nicodeme et al., 2013). There is no campylobacter found in tape water which is similar to results obtained in Finland (Hänninen et al., 2003), this may be due to well chlorination of water (Moore et al., 1996). Results showed high proportion of C. jejuni relative to C. coli in all tested samples. Prevalence of  campylobacter species in chicken meat  was 83.3% C. jejuni and 16.7% C. coli, these findings were differ than results obtained in Yangzhou, China which was 45.5% C. jejuni and 30.9% C. coli (Huang et al., 2016) and closely similar to results of Sao Paulo, Brazil which was 91.6% C. jejuni and 8.3% C. coli (Carvalho et al., 2013). Prevalence of campylobacter species in human samples was 50% C. jejuni and 50% C. coli which are closely similar to results of Thailand which was 58% C. jejuni and 40% C. coli (Samosornuk et al., 2015) and differs than results of Kayseri, Turkey which was 84% C. jejuni and 13% C. coli and 5% other species (Kayman et al., 2013) and fifers than results of north Lebanon which was 10% C. jejuni, 10% C. coli, 20% mixed culture and 60% other species (Dabboussi et al., 2012). Prevalence of campylobacter species in environmental samples was 66.7% C. jejuni and 33.3% mixed culture, this finding was differs than results of Brazil which was 93.3% C. jejuni and 6.7% C. coli from abattoir samples (Cortez et al., 2006), while in Italy was found 0% for any of Campylobacter spp. in environmental samples (Bellio et al., 2014). Genetic characterization of pathogenic genes results showed that in chicken meat samples, prevalence of Iam gene was 60% in C. jejuni and 100% C. coli, cdtB gene was 80% C. jejuni and 0% C. coli and cadF gene found in 100% C. jejuni and 0% C. coli. In human samples, prevalence of Iam, cdtB and cadF genes were 100 % C. jejuni and 50% C. coli in all samples. In environmental samples, prevalence of cdtB gene was 50% in C. jejuni and 100% in mixed culture, cadF gene was found in 100% of samples while Iam gene was not found in   C. jejuni isolates and found in 100% in mixed culture. Iam gene is responsible for invasiveness of Campylobacter species and marker potentially associated with the severity of campylobacter-induced enteritis (Carvalho et al., 2001) and this gene was prominent in C. coli compared to C. jejuni (Andrzejewska et al., 2015). cadF gene is gene of adhesion of campylobacter to fibronectin, an outer membrane protein (Monteville et al., 2003), cadF gene was found nearly in all campylobacter isolates (Rozynek et al., 2005). The cdtB gene is the toxin-forming gene of campylobacter jejuni, seems to be important for cell cycle control ond induction of host cell apoptosis and recognized as a major pathogenicity-associated factor (Dasti et al., 2010), the possible actions of this toxin are firstly inhibition of epithelial cell proliferation and apoptosis allowing bacterial invasion, secondly cell cycle arrest of immune cells ensuing local immune suppression and finally inhibition of fibrotic response (Ceelen et al., 2006).CDT activity requires the function of three genes: cdtA, cdtB and cdtC (Lara-Tejero and Galan, 2001).

CONCLUSION

Poultry meat and poultry meat products were an important source of campylobacter infections, also, campylobacter contamination can occur through environmental sources and from human itself. Campylobacter food poisoning caused mainly by Campylobacter jejuni and to lesser extent by Campylobacter coli and other species and most of the isolates was Campylobacter jejuni. Most campylobacter isolates have pathogenic genes responsible for adhesion, invasion and cytolethal toxins production which increase the virulence and pathogenicity of the microorganism.

AUTHORS’ CONTRIBUTIONS

Author performs collection, preparation, processing, and analysis of samples, isolation of bacteria, data acquisition, writing, preparation and revision of manuscript.

ACKNOWLEDGEMENTS

I thank Faculty of Veterinary Medicine, Aswan University for Financial support and Veterinarian, for his technical support and help in isolation of bacteria.

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مدى انتشار ميکروب الکامبيلوباکتر و جيناته الممرضة فى لحوم الدواجن و الأنسان و البيئة فى أسوان – مصر

محمد کرمى

E-mail:karmy99@yahoo.com           Assiut University web-site: www.aun.edu.eg

تم تجميع عدد 250 عينة عبارة عن 100 عينة من لحوم الدواجن و 75 عينة من اسهالات الأنسان و 75 عينة من البيئة المحيطة. تم عمل الفحص الباکتيريولوجى والبيوکيميائى لجميع العينات وذلک لعمل عزل وتعرف على الأنواع المختلفة من ميکروب الکامبيلوباکتر. کما تم عمل أختبار تفاعل البلمرة المتسلسل المتعدد للکشف على بعض جينات التعرف مثل 23S rRNA, hipO, glyA genes وکذلک الجينات الممرضة مثل  Iam, cdtB and cadF genes. أظهرت النتائج أن النسبة المئوية لمدى انتشار ميکروب الکامبيلوباکتر فى لحوم الدواجن ومنتجاتها وفى عينات الأسهال فى الأنسان وفى عينات البيئة المحيطة بالأغذية هى 32% و 14.7% و13.3% على التوالى وذلک باستخدام الطرق التقليدية للعزل بينما تغيرت هذه النسبة بشکل کبير باستخدام أختبار تفاعل البلمرة المتسلسل المتعدد فکانت 6% و 5.33% و 4% على التوالى. کما أظهرت النتائج أن 64.33% من العينات کانت ايجابية لميکروب الکامبيلوباکتر جيجوناى وأن 21.4% من العينات کانت ايجابية لميکروب الکامبيلوباکتر کولاى وأن 14.3% من العينات کانت ايجابية لأنواع مختلفة من ميکروب الکامبيلوباکتر کذلک أظهرت النتائج أن معظم العينات کانت ايجابية لوجود الجينات الممرضة بها. نستخلص من هذه الدراسة أن لحوم الدواجن ومنتجاتها والبيئة المحيطة بالأغذية تشکل مصدرا خطيرا لميکروب الکامبيلوباکتر الممرض للأنسان وأن معظم هذه الميکروبات تحتوى على جينات الضراوة الخطيرة.