MOLECULAR CHARACTERIZATION OF CAMPYLOBACTER SPECIES FROM TURKEYS FLOCKS IN DELTA GOVERNMENTS

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

MOLECULAR CHARACTERIZATION OF CAMPYLOBACTER SPECIES FROM TURKEYS FLOCKS IN DELTA GOVERNMENTS

KHALIL, M.R. ¹; AMIRA, A. MOAWAD ^2; KAFAFY, M.H. ³; HANAN, A. FAHMY ⁴ and

MONA, M. SOBHY ⁵

¹ Poultry Dis. Animal Health Research Inst. (AHRI), Mansoura Branch, ARC.

²Microbiology, AHRI. Mansoura Branch, ARC.

³ Pox Vaccines Dept. Vaccines and Serum Veterinary Res. Inst., Abassia, ARC, Egypt.

⁴ Biotechnology Dept. AHRI, Dokki, ARC. Egypt.

⁵ Reproductive Diseases Dept. ARRI. ARC, Giza, Egypt.

Received: 31 December 2019;     Accepted: 28 January 2020

INTRODUCTION

Campylobacteriosis is considered as the major important zoonotic gastrointestinal disease around the world caused mainly by C. jejuni (Gormley et al., 2008). Campylobacter species mainly C. jejuni and C. coli cause acute human gastroenteritis (Friedman et al., 2000). C. jejuni is part of normal intestinal flora of poultry and is present within the first few days post-hatch. The avian intestine seems to be a favourable environment for proliferation of C. jejuni (Lee and Newell 2006).

Campylobacter species are recognized as the most common cause of foodborne bacterial gastroenteritis in human (Nguyen  et  al., 2016). Campylobacteriosis

Corresponding author: Dr. MONA, M. SOBHY

E-mail address: monagabr17@yahoo.com

Present address: Reproductive Diseases Dept. ARRI. ARC, Giza, Egypt.

are predominantly caused by the Thermophilic Campylobacter including C. jejuni and C. coli (Griffiths and Park, 1990). The main route of infection is ingestion of food of animal origin and particularly consumption of poultry meat (Butzler and Oosterom 1991).

Campylobacter has been found to contaminate 81% of fresh, whole broiler chicken carcasses and a third of turkey carcasses (Anonymous, 2007). Campylobacter is present in the crop and ceca contents; thus, a single rupture early in processing can contaminate carcasses from several flocks (Musgrove et al., 2001).

Thermophilic Campylobacter species have Gram negative cell wall with capsule and flagella. The bacteria are slender, curved rod to small spiral shape with 0.2-0.5mm width and 0.5-5.0 mm length. They need microaerophilic atmosphere at 37-42°C for 48 ± 4 hours for optimal growth (Shane and Harrington, 1998).

Campylobacter can colonize normally in the intestine, so it can directly contaminate the meat product during evisceration inside the slaughter houses and is the major source of transmission of disease to human (Rahimi and Tajbakhsh, 2008). Meat of broilers, turkeys, and ducks are major sources of C. jejuni for humans (Friesema et al., 2012). Turkey products are an important commercial poultry commodity and limited information is available on colonization of turkeys with Campylobacter.

Campylobacters increased from day l to day 14 in the carriage rate and diarrhea. By day 39, the excretion rate had reached 6 x 10 (7) campylobacters in fresh faeces, peaks in Campylobacter numbers on days 19 and 75, corresponding to peaks in diarrheic samples (Wallace et al., 1998).  The introduction of new birds resulted in an increase in Campylobacter in birds with diarrhea. Turkeys at slaughter showed increased of Campylobacter from beak and highest in caeca (Zhao et al., 2001). Up to 34.9% of examined turkey carcasses were positive for Campylobacter after chilling (Logue et al., 2003).

Birds infected with Campylobacter will contaminate the food processing environment. The concentration of Campylobacter on turkey carcasses and in caeca is positively correlated to human campylobacteriosis cases (Rosenquist et al., 2003 and Anonymous, 2007). Campylobacter will have a positive impact on consumers’ perceptions related to food safety, the food industry and public health agencies (Wassenar   et al., 2007).

When considering the overall burden of campylobacteriosis, in addition to the corresponding acute morbidity and mortality described above as well as the long-term post-infectious squealed a consideration must also be made regarding anti-microbial resistance (AMR) (Poly et al., 2019).

 In USA, over two million cases of Campylobacter related illness are reported annually.  Campylobacter infections being responsible for 5% of food-related deaths (Mead et al., 1999). In 2004, the incidence of Campylobacter infection in the European Union exceeded than of Salmonella infection for the first time (Zoonotic Agents, 2005). Chicken meat has been reported to be contaminated with Campylobacters up to 100% the major sources of infection of C. jejuni (Borck and Pedersen, 2005).

Turkey meat is increasingly being chosen by consumers because the adherence to low-fat diets. The consumption of turkey meat has increased in recent times. It is of great importance to identify and assess the potential risks arising from turkey products. The aim of this study was to examine turkey flocks and turkey meat retail products in Delta Governorates for the prevalence of Campylobacter by convential cultural method and molecular characterization.

MATERIALS AND METHODS

1- Sampling:

Two hundred and forty samples were collected from turkeys (150 cloacal swabs from turkey farms and 90 samples from freshly slaughtered turkeys from various markets) in Delta Governorates from May 2018 till June 2019 for Campylobacter spp. isolation (Table, I). The samples were cultured onto thioglycollate media within four hours (Smibert, 1974).

Table I: Number and types of examined samples of turkeys for Campylobacter Spp.

2- Isolation and identification of Campylobacter isolates:

A loop full from each sample were cultured directly onto thioglycollate medium for 24-72 hours in sterile tubes, then a loop full from each tube was cultured on modified Campylobacter blood free selective medium with antibiotics and 10% sheep blood. All inoculated plates were incubated in microaerophilic condition contain CO₂ (10%), O₂ (5%) and N₂ (85%) in 37ºC for 48 hours and were demonstrated for characteristics colonies. The suspected colonies will be identified by biochemical test described by El-Gohary (1998).

3-Multiplex PCR assay:

Extraction of bacterial DNA from Campylobacter species was performed in a 0.5 ml Eppendorf tube containing PBS by heating at 100°C for 10 min in a heat block according to OIE (2008). After centrifugation DNA was collected and stored till used. Quantification of genomic DNA extracted from Campylobacter isolates by using UV Visible Spectrophotometer.

Oligonucleotides primers used were supplied from Metabion (Germany) are listed in Table (2), according to Wang et al. (2002).

DNA amplification of Campylobacter: The amplification of DNA in a 50 µl reaction containing thermoscientific Taq for Multiplex PCR Master Mix (2X) was carried out in Bio-RAD thermo cycler with positive and negative controls and cycling condition was illustrated in Table (3).

Detection of PCR products using agarose gel electrophoresis:

The amplified PCR products were electrophosed on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer with ethidium bromide. For gel analysis, 10 µl of the products was loaded in each gel slot. Thermoscientific, Gene Ruler 100 bp ladder was used to determine the expected fragment sizes (Sambrook et al., 1989). The gel was photographed by a gel documentation system (Alpha Innotech, Biometra).

Table 2: Oligonucleotides primers used for detection of Campylobacter strains in Turkey by Multiplex PCR.

Table 3: Cycling conditions of the primers during PCR according to (Wang et al., 2002).

RESULTS

Table 4: Detection of C. jejuni and C. coli in turkey samples by conventional cultural method.

Table 5: Occurrence of Campylobacter jejuni and Campylobacter coli in different turkey samples.

Table 6: Biochemical tests to differentiate between C. jejuni and C. coli.

Table 7: Incidence of Campylobacter jejuni and Campylobacter coli by multiplex PCR.

Fig. (1): Multiplex PCR of C. jejuni hipO and C. coli were detected in turkey samples in 1.5% agarose gels electrophoresis. All the 47 Campylobacter isolates yielded the genus specific as 31 isolates of C. jejuni at 323 bp while 14 produced the C. coli at 126 bp. Lane 1 and 6 samples positive of C. coli. Lane: 5,10,11,12,13,15 and 16 samples positive of C. jejuni. M: 100-600 bp ladders; Lane 8: Positive controls of C. jejuni and C. coli. Lane 7: Negative control.

Fig. (2): Agarose gel electrophoresis of multiplex PCR for 23S rRNA at 650 bp and glyA at 126 bp from turkeys. Lane 1: Thermoscientific, GeneRuler 100bp DNA Ladder, (#SM0243). Lane 2: control Negative. Lane 3: Positive control for C. jejuni and C. coli. Lanes 4-12: samples positive C. jejuni for 23S rRNA in all Campylobacter spp. with C. coli at 126 bp, except 7- 12 lane has no C. coli.

DISCUSSION

The prevalence of Campylobacter in turkeys (22.5%) observed in this study as shown in table (4) is with values reported by Noormohamed and Fakhr (2014) and Logue et al. (2003). The highest incidence was in Dakahlia and Kafr El-Sheikh Governorates 24% followed by Gharbia 21.5% and finally in Sharkia 20% (Table, 4). The prevalence of Campylobacter jejuni in turkeys was 15.4% which is near from results obtained by Perko-Makela et al. (2009). Campylobacter coli percentage in turkeys was detected as 7% which was agreement with Atanassova et al. (2007) and Gahlan et al. (2017). The prevalence rate of C. jejuni was higher than       C. coli in this study which agree with Weber et al. (2014). The difference was notably due to a positive hippurate test identified as C.jejuni but absence for   C. coli (Table, 6) (Sincinschi, 1995).

 Table (6) revealed the high incidence of Campylobacter in liver was (30%) followed by skin (26.7%), cloacal swabs (21.3%) and finally intestinal content (16.7%). The high incidence of Campylobacter due to contamination rate attributed to that damage to intestinal tract during evisceration can lead to direct contamination of the carcasses. Contamination can also occur indirectly through the hands of the processors and material or instrument used in processing (Salihu et al., 2009).

The Polymerase Chain Reaction (PCR) technique is the most widely used of all molecular techniques as it is highly sensitive, specific and rapid for the detection of food-borne pathogens (Samosornsuk et al., 2007). The specificity of this assay to detect C. coli or         C. jejuni was 97% (Nayak et al., 2005). Table (7) showed the amplification of the DNA belonging to Campylobacter isolates obtained in the present study. This amplification was performed strictly according to the Multiplex PCR methods and amplification parameters as specified by Wang et al. (2002). All samples yielded the genus specific (23S rRNA) with percentage (19%); (13.8%) C. jejuni specific at 650 bp and (5.8%) C. coli specific at 126 bp (Fig. 1 and Fig. 2). These results detected by Waller and Ogata (2000) and Rajagunalan et al. (2014).

PCR analysis detection of Campylobacter can be utilized as a simple and rapid tool to discriminate stains recovered from different sources (French, 2008 and El-Adawy et al., 2012). Finally, we concluded that prevention and control of C. jejuni in turkey farms would reduce the risk of human exposure to Campylobacter and is an important food safety issue. Multiplex PCR was found to be more reliable than the conventional cultural methods in species level for identification and differentiation of Campylobacter isolates. Further epidemiological studies at different geographical areas, carried to discover the prevalence, magnitude and importance of Campylobacter infection in turkey farms.

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