300 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XVII, Issue 3 - 2018, pag. 300 - 308 THE POTENTIAL ROLE OF CHICKEN MEAT IN TRANSMISSION OF CAMPYLOBACTER JEJUNI TO CONSUMERS *Nagwa Thabet ELSHRAWAY 1 , Ahmed Ibrahim YOUSSEF 2 Mohamed Shaker ABDEL HAFEZ 3 1Food Hygiene Department, 3Poultry Diseases Department, 1,3Faculty of Veterinary Medicine, Assuit University, New Valley Branch, Egypt, 1dr.nagwa2004@yahoo.com 2Division of Zoonoses, Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. *Corresponding author Received 6th January 2018, accepted 24th September 2018 Abstract: Food-borne zoonotic diseases are a significant and widespread global public health threat. Campylobacter jejuni is a most common contaminant of chicken meat. This study was conducted to investigate the presence of C. jejuni and some of its virulence genes in chicken meat in New Valley, Egypt and determine their zoonotic impacts. A total of 300 chicken meat specimens were collected from 100 freshly slaughtered from breast (pectoral), thigh, and liver (100 of each) from (healthy and diarrheic) live bird markets and analyzed for C. jejuni load using specific and selective nutrient media and molecular diagnosis. The identified strains were screened for virulence factors (VirB11, fla A and Iam) genes. The results revealed that throughout 300 meat samples, C. jejuni was detected in 74 (24.67%). The highest microbial load was in liver samples (26%) followed by pectoral (25%) then thigh muscle samples (23%). The virulence gene markers of C. jejuni was detected in chicken meat and liver samples as ViroB11 (2.27%), fla A gene (3.34%) and Iam gene (26.66%). Out of 50 diarrheic patients with food-poising signs 18 (36%) was positive for C. jejuni. The virulence markers genes in the human isolates showed that the prevalence of VirB11 gene, fla A, and Iam genes was 14.29 %, 71.43%, and 35.71%, respectively. In conclusion, this survey revealed that raw poultry meat available for consumers in ElKharga, Egypt was contaminated with zoonotic C. jejuni that might represent a threat to public health. Keywords: chicken, Campylobacter jejuni, enterotoxine, food poisoning 1. Introduction Foodborne diseases especially food poisoning is going up worldwide and becomes an important issue in public health impact by its fast contagiousness high morbidity and lethality. Food-borne zoonotic pathogens caused by consuming undercooked food or drinking water contaminated by pathogenic micro- organisms. Many of Food-borne zoonotic diseases are commonly found in the intestines of healthy food-producing animals and birds. Chicken is one of the major food-borne pathogens source by contaminated raw or undercooked chicken meat [1]. Live bird markets are places where customers can buy domestic bird where either slaughter birds in the retail shops or at homes where minimal, if any, food safety standards or veterinary inspection are applied [2]. These markets related to traditional preference for consumption of freshly slaughtered poultry. Live poultry markets are an important part of the poultry supply chains in different parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonosis, In Egypt, the poultry meat trade depends mainly on that types markets that characterized by unhygienic slaughtering process, lack of marketing infrastructures. http://www.fia.usv.ro/fiajournal mailto:dr.nagwa2004@yahoo.com Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 301 C. jejuni is the major pathogen causing foodborne diseases worldwide [3]. The incident of C. jejuni is one of the most common causes of bacterial enteritis in human. Poultry products play an important role in transmission of Campylobacter bacteria to humans. C. jejuni contamination of poultry meat during processing has been reported [4]. Campylobacter is most often detected in fresh broiler meat and in the EU the prevalence of these bacteria in broiler carcasses identified at the retail level varied from 3.1% to 58.8%, depending on the Member State [5]. Several studies showed that certain bacterial factors are essential for the pathogenesis of C. jejuni including the motility and adherence to intestinal mucosa, capability of the bacteria to invade enterocytes as well as toxin production [6]. Recently, some genes have been recognized as responsible for the expression of pathogenicity such as flagellin gene fla A, virB11 and invasion associated markers (Iam) genes which are genetic markers for C. jejuni [7]. It is necessary to know the extent to which the public is exposed to zoonotic C. jejuni infections via poultry meat as observed in slaughtered markets. The main objectives of this study were to assess the role of fresh chicken meat sold on the Egyptian live bird markets in the transmission of C. Jejuni entero-pathogene, detection of these genes have been recognized as responsible for the expression of pathogenicity and identifying its zoonotic implications. 2. Material and methods Ethical approval: This study has been approved by the animal rights and ethical use committee of Suez Canal and Assiut Universities. The study area: This study was conducted in Elkharga which, is the capital of New Valley Governorate which is a part of the oasis which is located to the west of the Nile Valley, 232 kilometers to the South of Assuit Governorate. The New Valley Governorate represents about 45% from the total Egypt area. Samples Collection: From farm birds, a total of 300 meat specimens were collected from 100 freshly slaughtered from 3 sites which were breast (pectoral) meat, thigh meat, and liver (100 of each) from New Valley public from (healthy and diarrheic) live bird markets. All data was recorded and samples were transported with minimal delay in an ice-box to the laboratory for microbiological and molecular examinations. Samples preparation: Samples were prepared following the protocols of APHA, [8] as following: Ten grams from each sample were weighted under complete aseptic conditions, and transferred into sterile polyethylene bag containing 90 ml of sterile 0.1% peptone water (Oxoid). Samples were blended in a stomacher (Lab-blender, 400) for one minute to provide 10-1 dilution. Human samples: to identify the occurrence of C. jejuni food poisoning bacteria among human cases, 50 stool swabs were collected from the outpatients suffering from diarrhea and fever of Elkharga general hospital. Stool swab samples were examined for C. jejuni infections. Patients were interviewed and filled out a standardized questionnaire addressing the family’s chicken consumption and purchasing and preparation conditions for chicken and their contacts with people having presented with an episode of diarrhea within 2-5 days after ingestion. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 302 Microbiological examination: Total bacterial isolation was performed by adding 0.1ml of each dilution to the following media in duplicate: Standard plate count agar (Oxoid) using drop technique. The plates were incubated at 35oC/24hr. Plates with distinct colonies counted between 30-300 colony were enumerated as Colony Forming Units (CFU) and in Preston enrichment broth were incubated at 37ºC for 24 hours. After enrichment, 0.1 ml of the broth was streaked onto modified Campylobacter selective agar base Cefoperazone Charcoal Desoxycolate Agar (Oxoid) containing antibiotic supplement (Oxoid). The plates were then incubated at 42ºC for 48 hours under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) using Campylobacter gas generating kits (Oxoid) [9]. All the isolates were picked up and preserved on nutrient agar (Oxoid) and Preston broth (Oxoid) examined microscopically by Gram's stain to observe the morphological arrangement and staining reaction and pure cultures of the isolates were biochemically identified using catalase test, oxidase test, urea hydrolysis test, hydrogen sulphide (H2S) production, citrate utilization test and rapid hippurate hydrolysis test according to Quinn et al. [10]. Ten pure positive isolates for C. jejuni further enterotoxins and pathogenicity gene identification by PCR. PCR detection: genomic DNA was extracted from the selected C. jejuni isolates from chicken and human samples using the QIA amp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis at 56○C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Primers used were supplied from (Biobasic, Canada) as listed in table (1). Primers were utilized in a 25µl PCR reaction containing 12.5 µl of 2X DreamTaq Green Mastermix kit (Fermentas, Germany), 1 µl of each primer of 10 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reactions were performed in applied biosystem 2720 thermal cycler. The thermal cycle condition was used as initial denaturation at 94○C for 4 min, followed by 35 cycles of 94○C denaturation for 30 sec, annealing (temperature differed as mentioned in table 1), for 30 sec, and extension at 72○C for 45 sec. followed by on cycle of final extension at 72○C for 5 min. Table 1 Primers sequences, target genes, amplicon sizes and annealing temperature of PCR reactions. Target gene Primers sequences (5` to 3`) Amplified segment (bp) Annealing Reference flaA AATAAAAATGCTGATAAAACAGGTG 855 53˚C Datta et al., 2003 TACCGAACCAATGTCTGCTCTGATT VirB11 TCTTGTGAGTTGCCTTACCCCTTTT 494 53˚C CCTGCGTGTCCTGTGTTATTTACCC iam GCGCAAATATTATCACCC TTCACGACTACTACTATGCGG 518 46˚C Wieczorek et al., 2012 The PCR products were separated by electrophoresis on 1.2% agarose gel (Applichem, Germany, GmbH) in 1X TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 10 µl of PCR products were loaded in each gel slot and 100 bp plus DNA Ladders (Qiagen, Germany, GmbH) were used to determine the fragment sizes. Control positive and control negative were included in each reaction. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 303 Table 2 Total prevalence of C. jejuni among chicken meat and stool samples from diarrheic human Samples examined Number of the examined samples No (%) Chicken Pectoral meat 100 25 (25%) Chicken Thigh meat 100 23 (23%) Chicken liver 100 26 (26%) Total 300 74 (24.67%) Human samples 50 14 (28%) 3. Results 1- Total prevalence of C. jejuni in chicken meat and stool samples from diarrheic human As listed in the table (2) and figure (1), the results revealed that the total prevalence of C. jejuni in chicken samples was of 24.67% whereas the total prevalence in diarrheic human stool samples was 28%. The higher C. jejuni prevalence was in liver samples 26 (26%) followed by pectoral muscles 25 (25%) and thigh muscle 23 (23%). All these percentage of chicken meat and liver exceeded the Egyptian Organization Specification (EOS) levels of C. Jejuni: considered unfit for human consumption because of high microbial load of C. jejuni exceeding the permissible limits. Fig. 1 . The percentage of chicken meat and liver samples exceeding the EOS standards of C. Jejuni. 2- Prevalence of VirB11, fla A, and iam virulence genes in C. jejuni isolates from chicken: The prevalence of three main virulence genes in ten C. jejuni from chicken meat and liver samples was illustrated in table (3) and figure (2) as following: the total prevalence of VirB11, fla A, and iam genes was 6.67%, 3.34%, and 26.66%, respectively. VirB11 gene was detected in 2 (20%) in pectoral samples. fla A gene was detected in 1(10%) only in thigh meat. The highest rate was detected in iam gene by a percentage of 3 (30%) in pectoral and liver samples and 2 (20%) of the thigh muscles. 25% 23% 26% 21% 22% 23% 24% 25% 26% 27% Pectoral meat Thigh meat Liver Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 304 PCR amplification of the enterotoxins (VirB11) 494 bp products of DNA extracted from C. jejuni, enterotoxins (iam): 518 bp molecular size ladder. Enterotoxins (flaA): 855bp of C. jejuni isolates from chicken samples respectively. Fig. 2 Agarose gel electrophoresis of specific dose-dependent amplification of C. jejuni pathogenic genes (VirB11,Iam, flaA). Table 3 Prevalence of three virulence genes of in C. jejuni isolates from chicken meat and human stool samples Samples Virulence C. Jejuni genes VirB11 No (%) Fla A No (%) iam No (%) Chicken Pectoral meat 2 (20%) *ND 3 (30%) Chicken Thigh meat *ND 1 (10%) 2 (20%) Chicken liver *ND *ND 3 (30%) Total No=30 2 (6.67%) 1 (3.34%) 8 (26.66%) Human samples 2/50 (14.29%) 10 (71.43%) 5 (35.71%) *ND: Not Detected 3- Prevalence of VirB11, Fla A, and iam virulence genes in human samples As illustrated in the table (3) and figure (2), out of 50 diarrheic patients with food poising signs, 18 (36%) for C. jejuni, Among C. jejuni isolates, the virulence gene markers were detected as following VirB11, 2 (14.29%), fla A 10 (71.43%), Iam 5 (35.71%). All human subjects were living in rural areas and all were buying chicken from public live bird markers. 4. Discussion Chicken are susceptible to many bacterial diseases which may be caused by a lack of hygiene, extensive exposure to the microbial contaminants and being carriers of these diseases due to their genetic immunity. Many chicken contaminating pathogens have potential risk for consumers directly from eating contaminated food by the microorganisms or through their enterotoxins. C. jejuni are associated in a wide range of food sources such as poultry. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 305 Symptoms of food poisoning are ranged from mild to severe include stomach pain, abdominal cramps, nausea and vomiting, diarrhea, fever, and dehydration [11]. Egyptians have been costumed to buy live chicken from public live bird markets and slaughter immediately after selection in the markets using the same primitive manual equipment in slaughtering that considered excellent sources to spreading microbial contamination to the slaughtered bird. That ‘on-the-spot’ slaughter, plucking and evisceration in the live bird markets lead to carcass contamination and poses hazards of food borne diseases to the consumers [1, 2]. Meat handlers could be sources of C. jejuni contamination particularly in case of following improper personal hygienic precautions. Moreover, shutting down live poultry markets is extremely effective in preventing human cases. Egyptian Organization Specification, [12] and European regulations, (European Commission, [13]) prohibit consumption of any food containing any amount of C. jejuni. In the present study, the detection of C. jejuni with zoonotic potentials that exceed the standards indicate public health hazards to the consumers and raise the needs for proper implementation of preventative programs and regular surveillance. C. jejuni infect intestinal tract of several domestic poultry types frequently which be more sever in chicks which known as enter-invasive transient diarrhea resulting as watery droppings, hepatic focal necrosis, focal hemorrhage, jejunum distention or the absence of clinical signs. Infected bird shed C. jejuni after 2-3 weeks of infection [14]. However, it was illustrated that 100% of chickens infected asymptomatically by C. jejuni in their intestinal tracts and shading the organism through feces [15]. In this study, the total prevalence of C. jejuni among chicken meat and liver was 24.67% which was consistent with other studies that recorded 22.6% in Egypt [2] and 25% in England [16]. Moreover, a higher, this higher percentage could be attributed to the sampling of stool samples from human in contact with food animals [17]. However, lower C. jejuni isolation rate of 16.7% was reported in chicken meat of Giza, Egypt by Hassanien et, al., [17], 21.5% in Bin-suef, Egypt chickens meat by El Fadaly, et. al., [18]. Other studies reported a very high prevalence of C. jejuni reached 40.4 % and 37.5 % in chicken liver and meat in Egypt [19], 51.06% in Malaysian [20], 60% in Portugal [21]and even over 90% [22]. In 2008, an extensive survey on the prevalence of Campylobacter spp. in broiler carcasses from slaughterhouses in the European Union showed that 75.8% of samples were contaminated with these bacteria with 100% records in some countries [23]. The differences in the prevalence of C. jejuni could be related to differences in geographical area and population. This study was conducted in a desert area and very hot and dry weather might be a determinant of the prevalence rate. Regarding the site of contamination of the examined bacteria, pectoral muscle the total contamination rate of C. jejuni was highest in liver samples 26 (26%), followed by breast muscles 25 (25%) and thigh muscles 23 (23%). Ilida and Faridah, [20] reported that poultry samples contaminated by C. jejuni were (66.67%) of breast & liver and (75%) of thigh. These results concluded that all poultry meat are subjected to contamination by C. jejuni from intestinal contents that might be occur during improper slaughtering, buckling and evisceration processes. Compared with the numbers found on poultry meat and liver surfaces, bacterial numbers inside tissues are low but nonetheless may be significant when undercooking occurs [20]. Regarding human samples, the prevalence rate of C. jejuni among diarrheic human samples was Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 306 28%. C. jejuni was detected in 35% children stool in 3 governorates of Egypt [19]. Campylobacter organism is one of the most common causes of human bacterial gastroenteritis. About 15 of every 100,000 people are diagnosed with Campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor Campylobacter in their gut. All this emphasizes the importance of chickens as a potential reservoir and source of C. jejuni infection in human. Some potential genetic markers of bacterial virulence have been identified. Fla A gene involved in adhesion and colonization, virB11gene as pathogenic genes responsible for the expression of invasion and iam gene is associated with invasiveness and play a role in the transmission of Campylobacter and/or its adaptation to different hosts [6, 7, 24, 25]. In this study, the prevalence of VirB11 gene, fla A, and iam genes was 2.27%, 3.34%, and 26.66%, respectively in chicken meat and liver. Results of VirB11 gene in chicken was lower than that reported by many authors; 6.1% [26], 7.3% [27] and 9.5% [5]. The fla A gene was detected in 3.34% of chicken meat, which was much lower than previous reports, which reached even 100% Wiseczorek et al., [26]. The iam gene was detected in 26.66% of chicken meat which was higher than the previous study by Gonzalez-Hein et al, [27] (6.2%), whereas it was much lower than that detected by Wiseczorek et al., [26] who recorded 53.8%. The virulence markers genes in the human isolates showed that the prevalence of VirB11 gene, fla A, and iam genes was 14.29 %, 71.43%, and 35.71%, respectively. Results of VirB11 gene was lower than that reported by Datta et al., [5] who recorded 10·7%. Another study showed that the virB11 gene was detected in 3.6% isolates from human [27]. The fla A was detected in a high percentage of the isolates tested (71.34%). These results were much lower than the data previously reported by other authors [5, 28]. The iam gene of Campylobacter was another virulence marker detected in this study. Differences in the prevalence of the iam factor were found by other authors [24, 28, 29]. The iam gene has been detected in the majority of invasive C. jejuni retrieved from humans. Furthermore, the detection of iam in C. jejuni isolated from two important hosts, humans and chickens, suggested a role for this marker in C. jejuni’s colonization of multiple hosts. Moreover, it was proposed that the iam gene is not only essential for the colonization of the chicken gut but is also responsible for the induction of diarrhea in humans [28, 29]. The detection of high rates of virulence genes among chicken and human samples referred to the potential of zoonotic infection of human by eating undercooked or improperly cooked chicken meat and liver. 5. Conclusion In conclusion, this survey revealed that raw poultry meat available for consumers in Egypt was often contaminated with zoonotic C. jejuni that might represent a threat to public health. Furthermore, several strains were positive for the several putative virulence marker genes. The consumption of undercooked meat cross- contaminated with C. jejuni may pose a serious threat to consumer health. Therefore, adequate cooking of chicken meat, the staff’s and equipment’s cleanliness and chemical disinfectants, hygienic handling, storage and effective processing of chicken meat are warranted. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Nagwa Thabet ELSHRAWAY, Ahmed Ibrahim YOUSSEF, Mohamed Shaker ABDEL HAFEZ, The potential role of chicken meat in transmission of campylobacter jejuni to consumers, Food and Environment Safety, Volume XVII Issue 3 – 2018, pag. 300 – 308 307 6. Acknowledgments This study was funded by the corresponding author. 7. References [1] GWIDA M, EL-GOHARY A., Prevalence and Characterization of Antibiotic Resistance Food Borne Pathogens Isolated from Locally Produced Chicken Raw Meat and their Handlers. Journal of Dairy, Veterinary & Animal Research. 3(1): 00062, (2015). [2] KHALAFALLA FA, ABDEL-ATTY NS, SOAD A ABDEL-WANIS, ADEL S HANAFY, Food Poisoning Microorganisms in Chicken Broiler Meat. Global Veterinaria 14(2): 211-218, (2015). 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