Prevalence of Campylobacter Contamination in Fresh Chicken Meat and Milk Obtained from Markets in the North-West Province, South Africa

Kamla-Raj 2011 J Hum Ecol, 36(1): 23-28 (2011) Prevalence of Campylobacter Contamination in Fresh Chicken Meat and Milk Obtained from Markets in the North-West Province, South Africa Keitumetse Idah Mabote 1,2*, Moses Mbewe 1 and Collins Njie Ateba 1,2 1 Dale Beighle Centre for Animal Health Studies, School of Agricultural Sciences, North-West University-Mafikeng Campus, P. Bag X 2046 Mmabatho 2735, South Africa 2 Department of Biological Sciences, School of Environmental and Health Sciences, Faculty of Agriculture, Science and Technology, North-West University Mafikeng Campus, Private Bag X2046, South Africa E-mail: ketumetsemabote@gmail.com KEYWORDS Campylobacter spp. Meat Contamination. Milk Contamination. Guillain-Barré Syndrome (GBS). Mast Colombia Agar (MCA) ABSTRACT Campylobacter species are food-borne enteropathogens that are usually transmitted to humans through the consumption of contaminated food products such as milk, beef and chicken. The aim of the study was to isolate and indentify Campylobacter species in chicken meat and milk obtained from some supermarkets in the North-west Province, South Africa. Sixteen chicken meat and milk samples were collected from seven regions in the Northwest Province. In chicken meat samples, the prevalence of C. coli contamination was 60 percent, 100 percent, 44.4 percent, 100 percent and 80 percent in Mafikeng, Rustenburg, Vryburg region 1, Vryburg region 2 and Dellareyville, respectively. On the other hand, C. jejuni was detected at 20 percent, 40 percent and 11.1 percent in Dellareyville, Mafikeng and Vryburg region 1, respectively. Only Vryburg region 1 had 44.4 percent prevalence of C. lari among chicken samples. In milk samples, the prevalence of C. coli contamination was 86 percent, 65 percent, 75 percent, 67 percent and 41 percent in Rustenburg, Vryburg region 1, Lichtenburg, Zeerust and Vryburg region 2, while 96 percent and 100 percent C. jejuni were obtained from Koster and Dellareyville, respectively. C. jejuni and C. coli were the most frequently (44.4 percent-100 percent) isolated contaminant among chicken samples when compared to C. lari (44.4 percent). The result of this study indicated a high prevalence of contamination with C. jejuni and C. coli, both of which are pathogens. This raises questions of food safety in South African markets.. INTRODUCTION Gastroenteritis (GE) in humans is a common problem in the developing and industrialized world (Bouknegt et al. 2004). The disease is caused by a variety of bacteria, but Campylobacter species account for most cases of human gastrointestinal infections worldwide (Khan et al., 2009). Currently, thermophilic Campylobacter species are recognized as food-borne enteropathogens of major public health significance (Whyte et al. 2003). Gastroenteritis caused by Campylobacter species may be self-limiting in some patients. However, in others the infections may progress to the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome that are usually more complicated (Rees et al. 1995; Winer 2001). Campylobacter species are widely distributed in the environment and the intestinal tract of both wild and domestic animals such as poultry, cattle, birds, sheep and pigs may act as reservoirs for Campylobacter species (Baylis et al. 2000; Sáenz et al. 2000; Hofshagen and Kruse 2005; Meldrum et al. 2005). Campylobacter species are transmitted to humans from the consumption of contaminated or improperly handled food products hence campylobacteriosis is considered a zoonotic disease in humans (Wheeler et al. 1999). Moreover, poultry processing plants may contaminate poultry carcasses with pathogens if proper hygiene practices are not implemented (Miettinen et al. 2002). Consequently, the occurrence of pathogenic microorganisms on meat and/or the level of contamination may depend on a number of factors that include contact with surfaces that have been contaminated with faecal matter and the conditions under which the carcass is dressed and butchered (Gill 2007). Campylobacter spp. are the major cause of bacterial gastrointestinal disease in the United Kingdom, and more than 60,000 cases of infection were reported in 1999 (McKay et al. 2001). Campylobacter jejuni and to a lesser extent by Campylobacter coli have been responsible for most sporadic cases and/or outbreaks of infections caused by these pathogens (Steinhauserová and Fortjikova 1999). Campylobacter species are

24 KEITUMETSE IDAH MABOTE, MOSES MBEWE AND COLLINS NJIE ATEBA susceptible to drying, high oxygen conditions and low ph (Bhavsar et al. 2004). Campylobacter jejuni can survive 2-4 weeks under moist, reduced oxygen conditions at 4 C, often outlasting the shelf life of the product, except in raw milk products. Although the prevalence of Campylobacter species in chickens have been well documented in countries that implement proper public health practices, in South Africa very little information currently exists (van Nierop et al. 2005). However, the detection of Campylobacter species depend upon the methods used to isolate and confirm their identities. In this study we utilized a number of preliminary identification tests coupled with the Camp Identification System as tools for determining the occurrence of Campylobacter species in chicken meat and milk in the Northwest province, South Africa. This was conducted to assess the health risks that these food products may pose on consumers in the Province. METHODOLOGY Collection of Meat and Milk Samples and Sampling Sites This research was conducted at the North-West University- Mafikeng Campus- North-West Province, South Africa. Chicken meat and milk samples were obtained from different regions in the Northwest Province. A sterile whirlpak bag was used to store chicken meat samples on ice while milk was collected into 100ml sterile bottles. The samples were transported to the laboratory and analysed immediately upon arrival. Sampling area, number, nature and source of samples are as shown in Table 1. Table 1: Area of collection, source, nature and number of samples collected. Sampling Sample Nature of Number of area source sample samples Rustenburg Chicken Meat 1 Koster Chicken Meat 1 Lichtenburg Chicken Meat 1 Zeerust Chicken Meat 1 Mafikeng Chicken Meat 1 Dellareyville Chicken Meat 1 Vryburg 1 Chicken Meat 1 Vryburg 2 Chicken Meat 1 Laboratory Analysis Media and Materials Used for Bacterial Isolation Meat samples were washed with 5ml of 2 percent peptone water (Biolab, Merck, S.A.) to remove microbes. The rinse fluid was used for isolation of Campylobacter species. The Mast Columbia agar (Mast Diagnostic, UK) media was autoclaved at 121 C for 15 minutes, cooled and held at 55 C. The agar was mixed with Campylobacter selective tablets and was also supplemented with 5 percent sheep blood. The media used for selective isolation of Campylobacter organisms. Plate count agar obtained from Merck, South Africa was used for bacterial counts. Nutrient agar media (Biolab, supplied by Merck, South Africa) was used to store isolates to be identified by biochemical tests. Sterile distilled water was used as a diluent for milk samples. Total Bacterial Isolation and Enumeration Bacterial isolation from chicken meat samples was carried out by suspending 1 g portion of each sample in 2 percent sterile peptone water. Serial dilutions were prepared using sterile distilled peptone water. Milk samples were also diluted serially from 10 0 to 10-6 using sterile distilled water. Aliquots of 100µl from each dilution for both meat and milk samples were spread-plated on Plate count agar and Mast Columbia agar. Plate count agar was used to determine total viable bacterial counts while Mast Columbia agar was used as a selective medium for the isolation of Campylobacter species. The addition of selective tablets was used to suppress the growth of background flora on the media. Plate count agar plates were incubated aerobically at 37ºC for 18 to 24 hours, while Mast Columbia agar plates were incubated aerobically at 42ºC for 24 to 48 hours. Isolates that were obtained on Mast Columbia agar plate were pure due to its high selective nature. These isolates were sub-cultured on nutrient agar and incubated at 37ºC for 18 to 24 hours. They were stored for further identification using biochemical tests. Preliminary Bacterial Identification In order to preliminary determine the identities of the isolates that grew on Mast Columbia agar, they were subjected to identification tests that included the wet mount to check darting motility. Gram staining was performed according to Cruikshank et al. (1975), in order to identify gram-negative rods. The catalase test and oxidase test were performed to identify

CAMPYLOBACTER CONTAMINATION IN FRESH CHICKEN MEAT AND MILK 25 isolates that were positive for these tests. The isolates that satisfied all the preliminary identification tests for Campylobacter were subjected to Camp identification system test. Confirmatory Biochemical Tests Camp Identification System Campylobacter identification system kit (Camp ID) obtained from Mast Diagnostics, UK that comprises a set of three biochemical tests were used to confirm the identities of thermophilic Campylobacter species. Only the isolates exhibiting characteristics of Campylobacter were selected. To perform the test, 1000µl of sterile distilled water was used to make a bacterial suspension. The isolates used were taken from fresh pure cultures. Aliquots of 500µl of the bacterial suspensions were added to the urease and hippurate test tubes, and the tubes were incubated at 37ºC for four hours. After incubation the results for the urease tests were read and recorded, while for the hippurate test, 2 drops of ninhydrin reagent were added into the tubes and left at room temperature for 10 to 15 minutes. The results were read and recorded thereafter. The swab from the indoxyl acetate tube was removed, dipped in sterile distilled water, and used to scrape several colonies from the plate that contained the test isolates, incubated at 37ºC for 30 minutes as instructed by the manufacturer. RESULTS Isolation of Campylobacter spp. Table 2 shows the number of bacteria isolated from plate count agar and mast Columbia agar. Contamination in chicken meat samples was observed in five regions, with Mafikeng having the highest level of contamination (3.7 percent), followed by Vryburg region 1 (1.81 percent) and the least obtained was Dellareyville (0.002 percent). There was no growth in MCA for chicken samples from Koster, Zeerust and Lichtenburg. The overall percentage of Campylobacter contamination observed in seven of the regions sampled was 62.5 percent. Contaminations with heterotrophic bacteria was observed in seven regions where milk samples were collected (Table 2). Dellareyville had the highest levels of contamination (4 percent). None of the milk samples obtained from Mafikeng was contaminated with Campylobacter species. The overall percentage of Campylobacter contamination observed in seven of the regions sampled was 87.5 percent. Table 2: Number of colony forming units (cfu) in plate count and mast Columbia agars and the percentages. Percentages were calculated by dividing the number of CFU in MCA by the CFU on PCA. Sampling Nature of CFU in CFU i Percent area sample PCA n MCA (MCA/ PCA) Pathogen contamination Mafikeng Meat 2.7x10 6 1 0 3.7 Milk 3.8x10 6 0 0 Rustenburg Meat 3.2x10 6 5x10 4 1.6 Milk 3.3x10 6 7 2.1 Koster Meat 5.3x10 4 0 0 Milk 3.3x10 4 2 3 0.06 Zeerust Meat 1.6x10 4 0 0 Milk 2.6x10 6 9 3.46 Vryburg 1 Meat 2.2x10 2 4x10 1.81 Milk 5x10 6 11x10 2 0.22 Vryburg 2 Meat 7x10 3 2 4 0.34 Milk 4x10 5 7x10 0.175 Lichtenburg Meat 1.3x10 6 0 0 Milk 2.5x10 6 1 0 0.0004 Dellareyville Meat 2.1x10 5 5 0.002 Milk 3x10 6 12x10 3 4 MCA-Mast Columbia agar; PCA-Plate Count agar; Numbers 1 and 2 indicate the number of samples collected from the same location Biochemical Test of Prospective Campylobacter Isolates Table 3 shows that all the isolates tested were motile. The results show that all the bacterial isolates were Gram negative rods and all were positive to catalase test and oxidase test. These isolates had a strong possibility of belonging to the genus Campylobacter. In order to establish the species composition, the Camp Identification Test Kit was used. Table 3: Biochemical tests performed for identification of Campylobacter spp. on meat and milk samples. Sampling Nature Moti- Gram Catala- Oxide area sample lity staining se test+ test + Mafikeng Meat 1 1 1 1 Milk 0 0 0 0 Rustenburg Meat 1 1 1 1 Koster Meat 0 0 0 0 Zeerust Meat 0 0 0 0 Vryburg 1 Meat 1 1 1 1 Vryburg 2 Meat 1 1 1 1 Lichtenburg Meat 0 0 0 0 Dellareyville Meat 1 1 1 1 -and + indicate the expected result for the test; 1 means all the isolates tested were 100 percent positive for the test.

26 KEITUMETSE IDAH MABOTE, MOSES MBEWE AND COLLINS NJIE ATEBA Identification of Campylobacter spp. using Camp Identification System Kit Tables 4 and 5 show the results of final identification tests that confirmed the identities of isolates as belonging to the genus Campylobacter. A camp Identification System test kit was used to identify Campylobacter species and during the interpretation of the test results, all the isolates were urease negative, which, is consistent with the identity of Campylobacter. The urease test results differentiate Campylobacter species from Helicobacter species, which are urease positive (Bhavsar et al., 2004). Table 4 shows that the most frequently isolated Campylobacter species in chicken meat samples included C. jejuni, C. coli and C. lari. C. coli was the most frequently isolated specie (Table 4). Samples obtained from Rustenburg, Vryburg region 2, Dellareyville and Mafikeng showed 100 percent, 80 percent and 60 percent contamination with C. coli, respectively. The lowest percentage of Campylobacter species was from Vryburg region 1 with only 11.1 percent of the isolates being C. jejuni and 44.4 percent C. lari and C. coli. No Campylobacter isolates were identified in samples obtained from Koster, Lichtenburg and Zeerust, indicating better hygiene practices were implemented when handling meat in these areas. An overall Campylobacter contamination of 62.5 percent of all sampled areas for chicken samples was observed. Table 4: Campylobacter spp. from chicken meat samples according to the Camp identification system kit. Sampling Percentage isolated area C. jejuni. C. coli C. lari. Mafikeng 2 0 6 0 2 0 Rustenburg 0 100 0 Koster 0 0 0 Zeerust 0 0 0 Vryburg 1 11.1 44.4 44.4 Vryburg 2 0 100 0 Lichtenburg 0 0 0 Dellareyville 0 8 0 2 0 The percentage proportions of Campylobacter species isolated from milk samples (Table 5). Similarly, three species of Campylobacter were isolated and these included C. jejuni, C. coli and C. lari. The results show that the samples from Mafikeng were not contaminated with Campylobacter species as none was isolated, indicating better hygiene practices in handling milk in the area. Table 5: Campylobacter spp. from milk samples according to the Camp identification system kit. Sampling area Percentage isolated C. jejuni C. coli C. lari Mafikeng 0 0 Rustenburg 1 4 8 6 0 Koster 9 6 0 4 Zeerust 3 3 6 7 0 Vryburg 1 3 5 6 5 0 Vryburg 2 5 9 4 1 0 Lichtenburg 2 5 7 5 0 Dellareyville 100 0 0 In Rustenburg and Dellareyville a large proportion of the isolates (96 percent to 100 percent) were contaminated with C. jejuni. Although C. lari was not isolated in most of the stations sampled, only a small proportion (4 percent) was positively identified in samples from Koster (Table 5). C. coli was isolated all the stations sampled except for Mafikeng, Koster and Dellareyville. Overall, the contamination with Campylobacter species in milk was 87.5 percent. C. jejuni and C. coli were the mostly isolated from milk samples in contrast to Hudson et al., (1999) who stated that Campylobacter jejuni and Campylobacter coli are the species of the pathogens that are the most frequently isolated from poultry. DISCUSSION Several studies have been conducted to evaluate the prevalence of Campylobacter contamination in chicken meat and milk samples (Jorgensen et al. 2002; Meldrum et al. 2005; Rosenquist et al. 2003; Rosenquist et al. 2006; Tereza et al. 2005; Thomas et al. 2005; van Nierop et al. 2005). Results obtained from some of these studies indicated a high prevalence of Campylobacter species in these food products (Meldrum et al. 2005) and were similar to those presented herein. The current study demonstrated a high prevalence of Campylobacter species in chicken meat and milk. This therefore indicates that chicken and bovine may serve as reservoirs of Campylobacter species. In some countries that have better public health monitoring systems, extensive work has been conducted to document the prevalence of food-borne pathogens and particularly Camplobacter species in chickens (Harrison et al. 2001; Refregier-Petton et al. 2001; Jorgensen

CAMPYLOBACTER CONTAMINATION IN FRESH CHICKEN MEAT AND MILK 27 et al. 2002; Miettinen et al. 2002). In Ireland and the United Kingdom (Fitzgerald et al. 2001), the two species that have been identified most often in diseased patients include Campylobacter jejuni, and to a lesser extent Campylobacter coli (Friedman et al. 2000). In France and UK, the percentage was found to be between 42.7 and 87 respectively, (Refregier-Petton et al. 2001; Jorgensen et al. 2002). However, in South Africa the occurrence of Campylobacter in poultry products have not been widely investigated (van Nierop et al. 2005). In the present study, the incidence of Campylobacter contaminations on chicken carcasses were comparable with or even higher than those reported in other studies (Refregier-Petton et al. 2001; Sackey et al. 2001; Zhao et al. 2001; Jorgensen et al. 2002). This explains the importance of such investigations. In the present study the prevalence of Campylobacter species was higher in milk than in chicken samples. This is contrary to a previous report (Whyte et al. 2004) in which the Campylobacter species were found to contaminate chicken meat (49.9 percent) than milk (1.6 percent). Contrary to the previous findings, in this study most of the isolates in chicken meat were C. coli than C. jejuni. Moreover, in milk samples a large proportion of the isolates were identified as C. jejuni than C. coli as was the case in the previously mentioned report (Whyte et al. 2004). It is therefore suggested that the prevalence of these species in food products greatly depends on the occurrence in the animal species. In this study, Campylobacter species were isolated from pasteurized milk. In this scenario, it suggested that the consumption of unpasteurized milk by farm families in the area might cause potential health risks to consumers (Whyte et al. 2004). Campylobacter species were not isolated from chicken meat in Koster, Zeerust and Lichtenburg. It is suggested that in Koster and Zeerust, these are small towns that have very few people visiting the shops at the same time. Moreover, the production rate is low in these cities and these minimizes cross contamination. However, the result obtained in Lichtenburg is not representative of observation made during the collection of samples. The results obtained for Rustenburg, Vryburg and Mafikeng may have resulted from the fact that these are highly populated cities which large number of individuals visiting the shops at the same time. This creates opportunities for cross contamination through the practice of poor hygiene at sale points. The detection of these pathogens in chicken meat and milk in the Northwest province of South Africa raises the question proper public health issues as they may cause severe health implications on those who consume these food products. CONCLUSION In conclusion, the presence of Campylobacter contamination in food products indicates the need to improve the hygiene practices that are utilized during production of these food products. Good hygiene must be practiced and necessary precautions should be taken when coming into contact with domestic animals since they are reservoirs for these organisms. Retail liquid milk must be pasteurised. In the study, it was found that milk samples as compared to chicken meat samples were often contaminated with Campylobacter species. Campylobacter are not only found on chicken meat and milk but also on various foods. Cross contamination of raw meat with cooked or ready to eat foods must be avoided. This may reduce the negative health implications of these pathogens to consumers and protect public health. ACKNOWLEDGEMENTS The work was funded by Centre for Animal Health, FAST, NWU - Mafikeng Campus. Thanks are also due to Mr. L. E. Motsei who assisted with the collection of samples and colleagues of the postgraduate microbiology group. REFERENCES Baserisalehi M, Al-Mahdi AY, Kapadnis BP 2005. Antimicrobial Susceptibility of thermophilic Campylobacter spp. isolated from environmental samples. Indian Journal of Medical Microbiology, 23(1): 48-51. Baylis CL, MacPhee S, Martin KW, Humphrey TJ, Betts RP 2000. Comparison of three enrichment media for the isolation of Campylobacter spp. from foods. Journal Applied Microbiology, 89: 884-891. Bhavsar SP, Baserisalehi M, Kapadnis BP 2004. Effect of gamma radiation on survival of Campylobacters in various food samples. Indian Journal of Medical Microbiology, 22(1): 39-43. Bouwknegt M, van de Giessen AW, Dam-Deeisz WDC, Havelaar A, Nagelkerk NJD, Henken AM 2004. Risk factors for the presence of Campylobacter spp. in Dutch broiler flocks. Prevention Veterinary Medicine, 62: 35-49.

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