Molecular Survey of Campylobacter jejuni in Broiler Chicken Farms in East Coast of Peninsular, Malaysia

Abdul Muhaimin Wahab1, Basit Zeshan2,*, Naveed Ahmed2, Muhammad Afzal2 and Muhammad Naveed2 1Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, (UMK), Kota Bharu, Kelantan, Malaysia 2Department of Microbiology, Faculty of Life Sciences, University of Central Punjab, Lahore, Pakistan Article Information Received 02 July 2018 Revised 23 June 2019 Accepted 06 August 2019 Available online 26 August 2020

Campylobacter spp. is responsible for food borne illness in humans while Campylobacter jejuni is the most common species for majority of human enteritis cases. The present study was conducted to determine the prevalence of C. jejuni, risk factors associated with the occurrence, identification control and preventive measure to reduce the prevalence in broiler chicken farms in Kelantan state located at east coast region of peninsula Malaysia. Eighty cloacal swab samples were collected from 4 different broiler chicken farms in district Tumpat, Machang and Bachok. The samples were processed for identification of C. jejuni followed by PCR to detect the presence of C. jejuni. Overall, 65% in total (80 samples) of cloacal swab samples showed positive reactions, where prevalence in farms A, B, C and D was 70%, 70%, 75% and 45%, respectively. Among, 2 of 5 identified risk factors through questionnaire showed significant difference which were open house system and untreated water source. Other risk factors which includes small scale, not using probiotic and poor biosecurity were not significant enough in association to occurrence of C. jejuni. Based on the risk factors that have been identified, control measure to reduce the occurrence was; by changing the open housing system into close housing system, using treated water source, use of probiotics, implementing strict biosecurity and good hygiene practices. In conclusion, the prevalence of C. jejuni in broiler chicken farms in Kelantan was high. Therefore, the farmer should always aware with this public health threat by avoiding those potential risk factors that are associated with the colonization of Campylobacter. C ampylobacter is one of the pathogen that causes most of the foodborne illnesses other than Salmonella species. It is a major contaminant in poultry meat, poultry products and the environment. A low infective dosage is enough to cause campylobacteriosis in human. According to the World Health Organisation (WHO), Campylobacter is 1 of 4 main global causes of diarrhoeal diseases and it is considered to be the most common bacterial cause for human gastroenteritis in the world. The high incidence of Campylobacter associated diarrhoea, its duration and possible complications makes it highly important from a socio-economic perspective. In developing countries, Campylobacter infections are frequently found in children under the age of 2 years, sometimes leads to death. The illness is characterized by acute watery or bloody diarrhea and abdominal pain. The food safety measures ensure that the food is safe to be consumed by humans without causing illness to them. Nevertheless, human illnesses associated with the consumption of food contaminated with pathogenic organisms or toxins are frequently reported. In humans, C. jejuni infection usually causes acute enteritis and abdominal pain which lasts for a week or more. Even though, it can be self-limiting, other complications may arise including bacteraemia, Guillain-Barre syndrome, reactive arthritis, and abortion (Skirrow, 2000).
Campylobacter species is prevalent in poultry farms and poultry product causing major sporadic gastroenteritis in humans. This happens due to handling and consumption of undercooked poultry meat. At low infective dosage which is 500 colony forming units (CFU), is enough to cause an infection to humans. Human campylobacteriosis is manifested by acute watery or bloody diarrhoea and abdominal pain. The great majority cases have been associated with C. jejuni followed by C. coli and C. lari. Among these, C. jejuni and C. coli are the most common pathogens responsible for the majority of human enteritis cases.

O n l i n e F i r s t A r t i c l e
The polymerase chain reaction (PCR) assays have been widely employed for identification of the pathogens owing to their sensitivity and cost effectiveness. A number of PCR assays have been described for detection of Campylobacters from food and fecal samples (Al Amri et al., 2007;Debruyne et al., 2008;Persson and Olsen, 2005;Sails et al., 2003). However, there are limited studies on occurrence of Campylobacter in broiler chicken farms in Malaysia. Moreover, there is limited data on the occurrence and risk factors of Campylobacter in broiler chicken farms in Kelantan. However, with development of technology and strategies implementation, transmission of those bacterial infections to humans can be prevented. The present study was conducted to assess prevalence of C. jejuni in faecal samples obtained from live chicken at various farms located within Kelantan state. The PCR assay was used to detect the C. jejuni in the collected faecal swabs and faeces samples. The results of our study could provide useful information on the future control and prevention strategies for this important food-born pathogen.

Materials and methods
Eighty cloacal swabs were collected from 4 different broiler chicken farms (20 samples from each) at District Tumpat, Bachok and Machang, Kota Bharu. Each cloacal swab was put into Amies transport medium and was labelled with farm identity number (ID) and sample ID. The samples were transported to laboratory within 2 h after collection in ice pack to maintain temperature at 2-8°C and to avoid them from sunlight.
Cloacal swabs were mixed with 50 ml of enrichment broth to make a homogenous suspension. The mixture was incubated under microaerophilic conditions at 37ºC for 3h and then further for 18h at 42ºC. Subsequently, it was centrifuged passively to remove debris. One ml of supernatant obtained was further centrifuged at 10000xg for 10min. The resulting pellet was re-suspended in 100µl of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), boiled for 10min followed by immediate cooling on ice. After centrifugation at 10000xg for 10min, a 5µl of supernatant was directly used as template in 25µl PCR reaction (Al Amri et al., 2007).
The reaction mixture comprised of 1x PCR buffer, 1.0mM MgCl 2 , 0.2mM dNTP mixture, 10pmol of each of the primers, 1µl of Taq DNA polymerase enzyme, 5µl of template DNA in 25µl of total reaction mixture. The primers used were based on mapA gene of C. jejuni (Denis et al., 1999). The sequences of forward and reverse oligonucleotide primers are as follows: Forward 5'-CTATTTTATTTTTGAGTGCTTGTG-3' and Reverse 5'-GCTTTATTTGCCATTTGTTTTATTA-3'.
The cyclic conditions for PCR were same as described by (Denis et al., 1999). In brief; the thermal cycler was set with following program: 1 cycle of 10 min at 95°C, 1 min and 30 sec at 59°C, 1 min at 72°C, and final extension steps of 10 min at 72°C will generate 589-bp DNA fragments corresponding to the C. jejuni. C. jejuni (ATCC 33560) was used as the positive controls and nucleasefree water was used as the negative control. PCR products were stained with 1% solution of GelRed stain and were visualised under UV light after gel electrophoresis using 1.2% agarose gel. Prevalence was calculated accordingly (Stevenson and EpiCentre, 2008).
Prevalence of chicken infected with Campylobacter = (No of chicken positive C. jejuni ÷ total number of samples taken) × 100 Risk factor that may associate with occurrence of C. jejuni were identified through questionnaire. After the risk factor identified, the association between risk factor and disease occurrence was determined by using 2×2 table where both risk factor and disease occurrence were binary variables (yes or no).
Chi-square was used to evaluate the significance difference between the risk factors that have been identified through questionnaire. A p-value of ˂0.05 is considered as statistically significant. Microsoft Excel and SPSS statistical software were used for data analysis. Results and discussion Table I shows prevalence of C. jejuni in different broiler chicken farms. From a total of 80 cloacal swab samples at 4 different broiler chicken farms, 52 (65%) were positive of C. jejuni through PCR in which out of 20 samples in each farm. But the total positive C. jejuni percentage showed high incidence of disease due to broiler consumption in north eastern Kelantan. And also these results showed the visible difference between the percentages of open house and close house farming of broiler and all other conditions which were responsible for colonization of C. jejuni in these farms. Out of all these four farms the highest prevalence was found in farm O n l i n e (2010 -2015) found 51% to 96% chickens were infected with Campylobacter (Saleha, 2004). Besides that, the neighboring country which is Thailand also recorded high prevalence of Campylobacter in chicken with 85% in farm (Padungtod et al., 2002). Supplementary Figure S1 shows the 500-600kb PCR product of mapA gene in different samples from different poultry farms.

F i r s t A r t i c l e C (75%) which is an open house farm and the lowest prevalence was in a close house farm D (45%). A number of recent studies in Malaysia
The odd occurrence C. jejuni was 3.09 times higher in open house system compared to close house system. There is significant difference between prevalence of Campylobacter in open house and close house system farms (p-value ˂ 0.05) (Table II). While the prevalence of C. jejuni is 1.95 times higher in small scale farm compared to medium scale farm. There is no significant difference between prevalence of Campylobacter in small scale farm and medium scale farm (p-value ˃ 0.05) ( Table II). The odd occurrence C. jejuni was 3.09 times higher in untreated water source compared to treated water source. There is significant difference between prevalence of Campylobacter in farm that practices untreated water and treated water (p-value ˂ 0.05) (Table III). While the C. jejuni was 1.95 times prevalent in higher poor biosecurity farm compared to fair biosecurity farm. There is no significant difference between prevalence of Campylobacter in poor biosecurity farm and fair biosecurity farm (p-value ˃ 0.05) (Table III).
Other risk factor such as small scale, not using probiotic and poor biosecurity were not significant to associate with occurrence of C. jejuni in farm even though that prevalence were recorded high in this study. Nevertheless, those risk factors should not be simply neglected such as using probiotic and practices good biosecurity. They are also a part of good management of farm not only to reduce C. jejuni but also other diseases and problems.
There are five risk factors that have been identified through the questionnaire which are open house system management, use of untreated water source, small scale farm, not using probiotic and poor biosecurity. Among five identified risk factors through questionnaire, two of them show significant value in term of risk factor that associate with occurrence of C. jejuni in broiler chicken farm. The two risks are significant (p-value ˂ 0.05); open house system farm (OR = 3.09) and untreated water source (OR = 3.09). This is supported by from study conducted by (Kapperud et al., 1993) in which the feeding broilers with untreated water was found to be independently associated with increased risk of Campylobacter colonization with OR = 3.42, p-value = 0.045 (Saleha, 2004) through her study found that 1.5% of untreated water supplies were positive for Campylobacter. Apart from that, chickens raised under closed-house system were not colonized by Campylobacter compared to those raised under open-sided house system. (Keener et al., 2004) in his comprehensive review article on Campylobacter suggesting chlorination of poultry drinking water as one of various on-farm strategies to reduce colonization of Campylobacter in poultry. Therefore, giving treated water instead of untreated water to poultry can reduce the incidence of Campylobacter.   Findings of present study showed that broiler chickens in farm were highly colonized by C. jejuni. Due to high prevalence in the farm level, it is possible to have contamination during slaughtering and processing plant. In order to prevent and reduce the contamination, it is advisable for farmer to adhere good animal husbandry practices (GAHP) and good biosecurity to ensure the level of colonization in the farm can be reduced therefore reduce the possible contamination that can occur during the post harvesting period.

Conclusion
As conclusion, broiler chickens are highly colonized with C. jejuni in the farm with prevalence ranging from 45% to 75%. Broiler chicken reared under open house system and given untreated water source tends to associate with high occurrence of C. jejuni in the farm with prevalence of 71.67% in each of both factors. Farm D was the lowest prevalence with 45% is because of the farm practices close house system and use treated water source.