MPN-PCR enumeration of Campylobacter spp. in raw chicken meats and by-products

Abstract

The goal of this study is to enumerate Campylobacter in chicken meats and by-products. In the current investigation, results showed that raw chicken meats and chicken by-products were contaminated with Campylobacter ranging from<3 to 4600 MPN·g^-1. Campylobacter jejuni showed a higher number compared to Campylobacter coli in the chicken samples. The current study showed that the percentage of chicken livers and gizzards harbored a higher number of Campylobacter (10³–10⁴ MPN·g^-1) than other chicken parts at 33.3% and 9.2%, respectively. The different concentrations of Campylobacter between chicken meats and chicken by-products reflect the differences in the contamination level. The data on Campylobacter concentration in chicken meats and by-products will be useful in risk analysis.

Keywords

MPN-PCR (most-probable-number PCR) / Campylobacter / chicken meats / chicken by-products

Introduction

Campylobacter spp. infection in humans has been increasing for the past 30 years and its infections on human had been reported to exceed the number of cases of Salmonella infections (Phillips, 1995). Campylobacter infections in humans are mainly food-borne. Campylobacter spp., particularly C. jejuni, C. coli and C. lari are carried in the intestinal tract of warm-blooded animals. Therefore, Campylobacter spp. is likely to contaminate food of animal origin and play an important role in human infections.

Consumption of chicken meats and products had been implicated with Campylobacter enteritis or campylobacteriosis in man (Tauxe, 1992) with possible complication such as Guillain Barré syndrome and neuropathy (Park et al., 1991). Black et al. (1988) reported that some highly infectious C. jejuni strains have an infective dose of as low as 500 cells. Chicken is recognized to be one of the major reservoirs for Campylobacter spp. Havellaar et al. (2007) reported that retail chicken meat free from Campylobacter is not realistic at this moment. Chicken is widely consumed worldwide as it supplies high quality protein that is rich in essential amino acids that cannot be found in plant protein. However, not only chicken meat is widely consumed but chicken by-products are also a very popular dish because of their low price, special textures and flavors.

There are numerous reports on the occurrence of Campylobacter in poultry farms (Ono and Yamamoto, 1999; Denis et al., 2001; Saleha, 2002; Tang et al., 2010) and chicken carcasses or meat (Ono and Yamamoto, 1999; Denis et al., 2001; Rodrigo et al., 2005; Han et al., 2007; Luber and Bartelt, 2007; Tang et al., 2010). Several studies have showed high prevalence of Campylobacter spp. in chicken samples at retail level (Denis et al., 2001; Sallam, 2007). Recently, much attention has been paid on the enumeration of Campylobacter in chicken samples in order to know the level of contamination that is required for the risk assessment studies (Rosenquist et al., 2003; Nauta et al., 2005; Uyttendaele et al., 2006). Previous studies showed high prevalence of pathogen in poultry did not necessarily harbor high concentration of pathogen (Campbell et al., 1983; Waldroup et al., 1992).

There are limited data available on Campylobacter concentration in chickens, especially chicken by-products. Our previous study found a high occurrence of Campylobacter in chickens from farm to retail level (Tang et al., 2010) and it would be interesting to know the concentration of Campylobacter in chicken samples. The present study aimed to enumerate thermophilic Campylobacter spp. in chicken meats and by-products from the wet market and hypermarket.

Materials and methods

Sampling

A total of 185 samples of chicken were purchased from 4 wet markets and 3 hypermarkets. Ninety-three chilled chicken parts were purchased from hypermarkets and 92 samples for fresh chicken parts were purchased from wet markets and transported to the laboratory within 2 h. Ten grams of each chicken sample (including skin in case of breasts, keels, drumsticks, wings and bishops) were cut into small pieces (<0.25 cm²) using a sterile scalpel blade. Each sample was added into a stomacher bag containing 90 mL of Bolton Selective Enrichment Broth (BEBB; Merck, Darmstadt, Germany) supplemented with Bolton antibiotic supplements (Merck, Darmstadt, Germany) and 5% lysed horse blood.

MPN-enrichment

The homogenates were transferred to three tubes MPN (most-probable-number) serial dilution and incubated in an anaerobic jar under microaerophilic condition produced using Anaerocult C (Merck, Darmstadt, Germany) at 42°C for 48 h.

DNA extraction

DNA extraction from enrichment samples were carried out in which portions of 1 mL of each positive sample were subjected to centrifugation at 15000 × g for 5 min to pellet the microorganisms. The supernatant was discarded and the pellet was washed once with 500 μL sterile distilled water. The pellet was then resuspended in 500 μL of sterile TE buffer (pH 8.0) by vigorous vortexing and boiled for 10 min to release the DNA from the microorganisms. The sample was later cooled at -20°C for 10 min. The cooled sample was again subjected to centrifugation at 15000 × g for 5 min. One hundred microlitre supernatant containing DNA was transferred to new sterile microcentrifuge tubes. These DNA samples were stored at -20°C until determination for the presence of Campylobacter spp., C. jejuni and C. coli using PCR assay.

MPN-PCR

MPN-PCR (Chai et al., 2007) was carried out with slight modification in which all MPN tubes were subjected to PCR assay. Boiled cell lysate containing DNA from each MPN tubes were examined for the presence of Campylobacter spp., C. jejuni and C. coli by optimized single PCR assay described in our previous study (Tang et al., 2009). Three Campylobacter primers were selected for the identification of Campylobacter spp., C. jejuni and C. coli using the 16S rRNA gene (Linton et al., 1996), the hip gene (Linton et al., 1997) and the ceuE gene (Gonzalez et al., 1997), respectively. The primer sequence used in this study for 16S rRNA gene (Linton et al., 1996) (primers: C412F 5′-GGA TGA CAC TTT TCG GAG C-3′; C1288R 5′-CAT TGT AGC ACG TGT GTC-3′), hip gene (Linton et al., 1997) (primers: HIP400F 5′-GAA GAG GGT TTG GGT GGT G-3′; HIP1134R 5′-AGC TAG CTT CGC ATA ATA ACT TG-3′), and ceuE gene (Gonzalez et al., 1997) (primer: F 5′-ATG AAA AAATAT TTA GTT TTT GCA-3′; R 5′-ATT TTA TTA TTTGTA GCA GCG-3′) were used in the PCR identification of the pathogens found in the samples. DNA from reference cultures, C. jejuni (ATCC 33560) and C. coli (ATCC 43478), were included as a positive control in every PCR assay.

Optimized PCR amplification (Tang et al., 2009) was performed in 25 μL reaction mixture as shown in Table 1. PCR reaction mixtures were heated at 95°C for 2 min as an initial denaturation step followed by 30 cycles of denaturation at 95°C (30 s), annealing (60 s) and extension 72°C (40 s). Annealing temperature for Campylobacter spp., C. jejuni and C. coli were 55°C, 59°C and 55°C, respectively. All PCR assays were terminated with a 3 min extension at 72°C and were performed with Veriti^TM 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA).

For visualization of PCR products, 5 μL of PCR products were run on 1.0% agarose gel at 90 V for 40 min. The gel was then stained with ethidium bromide and viewed under ultraviolet (UV) light. A DNA-molecular ladder (100-bp ladder) (Vivantis Technologies, Selangor, Malaysia) was included in each gel.

Results

Representative gel of PCR amplification for Campylobacter spp., C. jejuni, and C. coli from samples and reference strain C. jejuni ATCC 33560 and C. coli ATCC 43478 is shown in Fig. 1.

The concentration of Campylobacter spp., C. jejuni and C. coli in chicken meats and chicken by-products was summarized in Table 2. The present study showed that concentration of Campylobacter spp. contamination on chicken meats and by-products at retail level were less than 100 MPN·g^-1 (Table 3) in most samples. Chicken livers and gizzards harbored higher concentration (10³-10⁴ MPN·g^-1) of Campylobacter with percentages of 33.3% and 9.2%, respectively (Table 3). All chicken meats showed Campylobacter spp. concentration less than 10² MPN·g^-1 except chicken keels. A small percentage (4.3%) of chicken keels harbored 10³-10⁴ MPN·g^-1 of Campylobacter spp. (Table 3).

Discussion

Poultry products contaminated with Campylobacter in poultry has been recognized worldwide (Son et al., 1996; Denis et al., 2001; Saleha, 2004; Havelaar et al., 2007; Sallam, 2007). Such high prevalence of Campylobacter might be due to improper handling, contaminated water and cross-contamination in various stages of chicken processing as well as packaging. Most of the studies reported on the prevalence of Campylobacter in chickens (Harrison et al., 2001; Lindblad et al., 2006; Arsenault et al., 2007; Meldrum and Wilson, 2007; Luber and Bartelt, 2007; Pointon et al., 2008). There are limited studies reported on Campylobacter numbers in retail chicken: chicken carcasses (Manfreda et al., 2006; Klein et al., 2007) and chicken breast fillets (Luber and Bartelt, 2007). The reasons for these might be due to the difficulty in culturing Campylobacter spp. (Solomon and Hoover, 1999); and they are generally inactive in many conventional biochemical tests (Stern et al., 2001). The amount of Campylobacter on retail chicken products will be useful in risk assessment studies. In addition, Black et al., (1988) indicated 500 cells (10²-10³ cells) of highly infectious Campylobacter strain are capable of causing symptoms in humans. Low concentrations of Campylobacter spp. in raw chicken products will reduce the risk of ingestion of Campylobacter through proper cooking of chicken products.

Campylobacter concentration in chicken samples from both hypermarkets and wet markets showed higher number of C. jejuni compared to C. coli. This was in line with reports from different studies (Denis et al., 2001; Whyte et al., 2004; Sallam, 2007) showing C. jejuni is predominant in chicken samples while C. coli is less frequently encountered.

Higher amount of Campylobacter found in chicken livers and gizzards might be due to the cross-contamination from fecal material during evisceration. Because chicken livers and gizzards are removed together along with the intestinal tract of the chicken during evisceration before being separated, this may suggest greater risk of cross-contamination from fecal material that may harbor Campylobacter. The range of Campylobacter colonization in the small intestine was reported to be from log 5 to over log 9 CFU·g^-1 (Berndtson et al., 1992; Rosenquist et al., 2006). Any improper handling of viscera content after evisceration poses high chances of cross-contamination of chicken livers and gizzards with Campylobacter from intestinal content. Insufficient cooking of chicken livers and gizzards are common especially for steamboat to retain the soft texture as overcooking will cause the chicken livers and gizzards to become hard. This will lead to possible ingestion of Campylobacter through undercooked chicken livers and gizzards.

High concentration of Campylobacter will pose risks for human infection through undercooked meat or cross contamination. Fischer et al. (2007) reported that insufficient cooking and cross-contamination are the most important factors that determine food safety during the preparation of poultry meat. Undercooking of chicken is very likely to occur when whole chicken is cooked before cutting into pieces, e.g., salads, sandwiches, etc. A high load of Campylobacter also pose a higher risk of cross-contamination of kitchen utensils used to cut raw chicken and later used to cut other food stuff.

Conclusions

The present study showed that Campylobacter concentration found in most raw chicken meats and chicken by-products examined were below 100 MPN·g^-1. The presence of Campylobacter in chicken samples poses a health risk to consumers and a monitoring of chicken products from farm to retail would be desirable.

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