Prevalence of Yersinia enterocolitica from food and pigs in selected states of Malaysia

Food Control 35 (2014) 94e100

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Prevalence of Yersinia enterocolitica from food and pigs in selected states of Malaysia Lai Kuan Tan a, b, Peck Toung Ooi c, Kwai Lin Thong a, b, * a

Microbiology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Laboratory of Biomedical Science and Molecular Microbiology, UMBIO Research Cluster, Institute of Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia c Clinical Veterinary Studies, Faculty of Veterinary Medicine, University Putra Malaysia, Malaysia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 April 2013 Received in revised form 18 June 2013 Accepted 25 June 2013

The aim of this study was to determine the prevalence of Yersinia enterocolitica and its bioserotypes from food and pigs in Malaysia. Fifty-eight raw porcine (raw pork meat, internal organs and other parts) and 48 non-porcine food (raw beef, poultry products, seafood, vegetables, tofu, and pasteurised milk) from wet markets located in Kuala Lumpur, Selangor, Perak, and Pahang were examined for the presence of Y. enterocolitica. Specimens (nasal, oral and rectal swabs) from 165 pigs (from nine farms) located at central and northern parts of Malaysia were also collected for Y. enterocolitica detection. Presumptive isolates were characterised biochemically and further confirmed by PCR. Out of 58 raw porcine food, Y. enterocolitica was detected in 7 (12.1%) samples in which raw pork meat (whole meat) had the highest prevalence 5/21 (23.8%), followed by raw pork liver 1/5 (20.0%) and raw pork intestine 1/8 (12.5%). No Y. enterocolitica was isolated from the 48 non-porcine foods. Overall, two pathogenic (bioserotypes 3 variant/O:3 and 1B/O:8) and one non-pathogenic (bioserotype 1A/O:5) Y. enterocolitica strains were isolated from food. Out of 165 pigs examined, 3 (1.8%) pigs were carriers for Y. enterocolitica. All 3 pigs were asymptomatic grower pigs from Penang, carried Y. enterocolitica bioserotype 3 variant/O:3. Postenrichment PCR approach gave a higher prevalence, 60.3%, 41.7% and 27.9% for porcine food, nonporcine food and pigs, respectively. Both pathogenic and non-pathogenic Y. enterocolitica were present in our domestic pigs and food. Improper food handling and processing may cause cross contamination of this pathogen to humans, affirms a potential risk for public health. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Food microbiology Pig Pork Yersinia enterocolitica

1. Introduction Yersinia enterocolitica is a bacterium which belongs to Enterobacteriaceae that is widely found in natural environments. It is psychrotrophic and has the capability to survive and multiply in cold (Annamalai & Venkitanarayanan, 2005; Neuhaus, Francis, Rapposch, Görg, & Scherer, 1999). It is enteropathogenic and causes gastrointestinal problems such as acute enteritis with fever, bloody diarrhoea and pseudo appendicitis, which frequently leads to unnecessary laparotomy in humans (Vlachaki, Tselios, Tsapas, & Klonizakis, 2007). Y. enterocolitica is notified as the third most important foodborne enteric pathogen in Europe after campylobacteriosis and salmonellosis (European Food Safety Authority & European Centre for Disease Prevention and Control, 2012).

* Corresponding author. Microbiology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: þ60 3 79675836; fax: þ60 3 79675908. E-mail address: [email protected] (K.L. Thong). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.06.053

Young children and infants are the most susceptible age group (Rosner, Stark, & Werber, 2010). In United States, it is estimated that Y. enterocolitica causes over 115,000 infections annually (Scallan et al., 2011). In Europe, there were 6776 reported yersiniosis cases in humans during 2012 (European Food Safety Authority & European Centre for Disease Prevention and Control, 2012). Reports on the incidence of yersiniosis in Southeast Asian countries are few. However, in the recent report of Ananchaipattana et al. (2012a, 2012b), Y. enterocolitica was reported present in some Thai food (beef, shrimp and tofu). This suggests a risk of human infection when such contaminated food is consumed in this area. Y. enterocolitica is ubiquitous in the nature and is routinely isolated from animals, food and environment (Fredriksson-Ahomaa & Korkeala, 2003). Among the sources, swine is reported as a major reservoir for Y. enterocolitica. The bacterium is often present in the oral cavity of pigs especially tonsils, intestinal content, faeces and lymph nodes (Fondrevez et al., 2010; Gutler, Alter, Kasimir, Linnebur, & Fehlhaber, 2005; Liang et al., 2012; Nesbakken,

L.K. Tan et al. / Food Control 35 (2014) 94e100

Eckner, Hridal, & Rrtterud, 2003). Besides the pigs, strains of Y. enterocolitica have been frequently isolated in raw pork too (European Food Safety Authority, & European Centre for Disease Prevention and Control, 2012). This is due to the cross contamination of the organism via oral cavity, faeces, and intestinal contents during slaughtering, cutting, further processing and distribution of fresh pork (Martínez et al., 2011; Terentjeva & Berzins, 2010). Other possible food vehicles of yersiniosis are ruminant and ruminant products (Fukushima, Hoshina, Itogawa, & Gomyoda, 1997), poultry (Dallal et al., 2010), vegetables (Xanthopoulos, Tzanetakis, & Litopoulou-Tzanetaki, 2010), milk and dairy products (Yucel & Ulusoy, 2006), ready-to-eat food (Xanthopoulos et al., 2010) and chitterlings (Lee et al., 1990). In Malaysia, there is limited study on Y. enterocolitica and the bacterium is not routinely isolated. The first case of human yersiniosis in Malaysia was reported by Jegathesan, Paramasivam, Rajagopalan, & Loo (1984) where Y. enterocolitica serotype O:3 was isolated from a 34-year-old female. The only food related prevalence report in Malaysia was from unpublished study of Dzomir (2005), Y. enterocolitica bioserotype 1A/O:52,53 and 1A/ O:41,42 were isolated from beef burger and chicken burger meats. Due to the limited study of this bacterium in Malaysia, the potential complications of yersiniosis in the country remain unknown. The actual incidence of this bacterium in various type of food is not well documented too. Therefore, the aims of this study were: (i) to determine the prevalence of Y. enterocolitica from porcine food, non-porcine food, and life pigs; and (ii) to determine the bioserotype of Y. enterocolitica that is currently present in Malaysia. 2. Materials and methods 2.1. Isolation of Y. enterocolitica from raw porcine food 2.1.1. Sampling Between June 2010 to March 2011, 58 raw porcine samples were sampled from wet markets at selected states in Peninsular Malaysia (Kuala Lumpur, Perak and Pahang). The raw porcine samples were further grouped into three categories as raw pork meats (n ¼ 25), raw pork internal organs (n ¼ 23) and other parts (n ¼ 10) (Table 1).

95

All raw food in this study were transferred in sterile plastic bags and transported in ice box to the laboratory. 2.1.2. Isolation and enumeration of Y. enterocolitica from raw porcine food Food samples were analysed for the presence of Y. enterocolitica by conventional culture methods and post-enrichment polymerase chain reaction (PCR) screening. Enumeration of Y. enterocolitica was performed using a 3  3 most probable number (MPN) method. Five g of raw food sample was cut into small pieces, added to 45 ml of selective enrichment broth in sterile plastic bag and homogenised manually by hand. Enrichment broths used were phosphate buffered saline (PBS, Sigma, Germany), Yersinia selective enrichment broth according to OSSMER (YSEO, Merck, Germany), and irgasan-ticarcillin-potassium chlorate (ITC) broth [ITC broth base (Fluka, Germany) supplemented with ticarcillin supplement (Fluka) and potassium chlorate supplement (Fluka)]. Food homogenates in ITC and PBS were incubated at 25  C for 2 days and 4  C for 3 weeks, respectively, and food homogenate in YSEO was used for MPN enrichment for food safety enumeration. Food particles in YSEO were left to settled down and the fluid was dispensed into a 3  3 MPN system consisting of 10 ml of undiluted fluid in each of three 10 ml test tubes (level A), 1 ml of fluid in 9 ml YSEO broth in each three 10 ml test tubes (a 1:10 dilution, level B), and 1 ml of a 1:10 dilution of the fluid in 9 ml YSEO broth in each three 10 ml test tubes (a 1:100 dilution, level C), incubated at 25  C for 18 h (Hudson et al., 2008). A loopful of each enriched samples was streaked onto cefsulodin-irgasan-novobiocin (CIN) agar [Yersinia Selective Agar Base supplemented with Yersinia Selective Supplement (Oxoid, UK)] and incubated at 25  C for 24e48 h. In parallel, sample was plated onto CIN agar immediately after alkaline treatment in which 0.5 ml of enriched culture was transferred into 4.5 ml of 0.25% potassium hydroxide (KOH): 0.50% sodium chloride (NaCl) solution (ISO standard 10273:2003; Hudson et al., 2008; Johnson, 1998). The alkaline treatment is recommended for samples which are normally highly contaminated with background microbiota. Y. enterocolitica can resist weak alkaline treatment, while background microbiota such as Pseudomonas, Proteus and Serratia can

Table 1 Prevalence of Y. enterocolitica from raw porcine food determined by cultural method and post-enrichment PCR screening. Food type

No. of samples

No of positives (%)a PCR

Cultural

Pathogenic

Non-pathogenic

Raw pork meat Whole meat Minced meat

25 21 3

20 (80.0) 18 (85.7) 2

5 (20.0) 5 (23.8) 0

4/5 (80.0) 4/5 (80.0) 0

1/5 (20.0) 1/5 (20.0) 0

Raw pork internal organs Liver Intestine Heart Kidney Throat

23 5 8 5 4 1

14 3 7 3 1 0

2 (8.7) 1 (20.0) 1 (12.5) 0 0 0

2/2 (100.0) 1/1 (100.0) 1/1 (100.0) 0 0 0

0 0 0 0 0 0

Other parts Skin Foot Fat tissue Ear Eye tissue Nose

10 4 2 1 1 1 1

1 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

Total

58

35 (60.3)

7 (12.1)

6/7 (85.7)

1/7 (14.3)

a b

(60.9) (60.0) (87.5) (60.0) (25.0)

1 (10.0)

Isolation rate by strain typeb (%)

The values are the number of positives detected in either of the enriched samples (YSEO, ITC, and PBS). The isolation rate refers to the number of positive samples determined by cultural methods.

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be suppressed (Schiemann, 1983). Typical Y. enterocolitica isolates (red bull’s eyes) were picked and tested with biochemical tests, i.e. oxidase test, Gram staining, and citrate test. All Gram negative, oxidase negative and citrate negative isolates were further characterised by using the API 20E strips (bio-MérieuxÒ SA, France). The strips were inoculated and read according to the recommendations of the manufacturer, with the exception of incubation at 28  C (Archer, Schell, Pennell, & Wick, 1987). Identity of Y. enterocolitica isolates was confirmed by using PCR targeting Y. enterocoliticaspecific 16S rRNA gene (Y1, 50 -AATACCGCATAACGTCTTCG-30 ; Y2, 50 CTTCTTCTGCGAGTAACGTC-30 ) (Neubauer, Hensel, Aleksic, & Meyer, 2000). The virulence of Y. enterocolitica was determined by PCR targeting yst gene (forward, 50 -GTTAATGCTGTCTTCATTTGGAGC-30 ; reverse, 50 -GACATCCCAATCACTACTGACTTC-30 ) (Gómez-Duarte, Bai, & Newell, 2009). Amplicons of selected PCR products were purified using MEGAquick-spinÔ PCR & agarose gel DNA extraction system (iNtRON Biotechnology, Korea) and sequenced to validate the identity. 2.1.3. Post-enrichment PCR screening from enriched food homogenates DNA templates were prepared from 1 ml of each enriched samples (YSEO, ITC, and PBS) using the cell lysate method. The presence of Y. enterocolitica was screened by PCR using the Y. enterocolitica-specific 16S rRNA gene primers (Neubauer et al., 2000).

(nasal, oral and rectal swabs) were collected from each pig and each maintained in Cary-Blair transport medium (Oxoid, UK) in ice box before being processed in laboratory. Specimens were processed by two methods, i.e. (i) direct streaking on selective agar plates and (ii) enrichment in ITC and PBS broths (as described in 2.1) followed by streaking on selective agar plates. Selective agars used were CIN or modified CIN. Modified CIN was made by adding 1% L-arginine (Sigma, Germany), 0.8 g/L ferric ammonium citrate (BDH Prolabo, UK), 6.8 g/L sodium thiosulphate (BDH Prolabo), and 2.0 g/L DL-phenylalanine (Sigma) at pH 7.4  0.02 into the CIN agar. The same procedures used for isolation, confirmation and post-enrichment PCR screening as stated in 2.1. except the MPN step. 2.4. Biogrouping and serotyping of Y. enterocolitica The biogroup of Y. enterocolitica was determined by using biochemical tests as described by Wauters, Kandolo, & Janssens (1987). The biochemical tests included lipase test, esculin hydrolysis, salicin utilisation, indole test, xylose utilisation, trehalose utilisation, nitrate reduction, pyrazinamidase test, b-D-glucosidase test, VogeseProkauer test, and DNase test. The serotype of Y. enterocolitica was determined by using the O-Antisera “SEIKEN” set purchased (DENKA SEIKEN Co., Ltd, Japan). 3. Results

2.1.4. MPN calculation The three digits for each level in the 3  3 MPN system were determined based on the post-enrichment PCR screening results (the YSEO enriched tubes). The MPN/g value was calculated using the Microsoft Excel spreadsheet provided by Institute of Environment Science and Research (ESR), New Zealand (Hudson et al., 2008). The range over which these nine tubes MPN system operates is between 0.30 MPN/g (LCI of 0.07 with one positive at level A) to 44.84 MPN/g (UCI of 198.70). 2.2. Isolation of Y. enterocolitica from non-porcine food Forty-eight non-porcine foods were purchased from wet markets (located in Kuala Lumpur, Selangor, and Pahang) and examined for the presence of Y. enterocolitica. Food types purchased included raw vegetables (n ¼ 19), raw seafood (n ¼ 11), raw poultry products (n ¼ 9), raw beef (n ¼ 6), tofu (n ¼ 2), and pasteurised milk (n ¼ 1). The same procedures used for isolation, confirmation and postenrichment PCR screening as stated in Section 2.1. were performed except the MPN step. 2.3. Isolation of Y. enterocolitica from pigs The presence of Y. enterocolitica from pigs in Malaysia was investigated during the period of October 2010 to September 2011. Random selection of participating pig farms was not possible in this observational study as selection was subjected to management and practices of the commercial pig farms. A total of nine pig farms located in three states in middle- and north- western part of Peninsular Malaysia, i.e., Selangor (Farms A, B, C), Perak (Farms D, E, F), and Penang (Farms G, H, I) were enrolled in this study. The three states are the top three largest pig-producing states in Malaysia (Department of Veterinary Services, Malaysia, 2011). A stratified random sampling was performed in categorising the pigs based on the health condition, i.e. healthy (pigs without disease symptoms) and unhealthy (sick, weak, and dead). A total of 165 pigs were selected (farms A, n ¼ 9; B, n ¼ 14; C, n ¼ 30; D, n ¼ 20; E, n ¼ 20; F, n ¼ 20; G, n ¼ 16; H, n ¼ 20; I, n ¼ 16) and three major specimens

3.1. Prevalence of Y. enterocolitica from raw porcine food The prevalence of Y. enterocolitica from raw porcine food was low by cultural method. Out of 58 food tested, 7 (12.1%) were naturally contaminated by Y. enterocolitica; i.e. raw pork meat (whole meat) 5/21 (23.8%), raw pork liver 1/5 (20.0%) and raw pork intestine 1/8 (12.5%) (Table 1). Twenty-six isolates of Y. enterocolitica (PCR confirmed) were isolated from the 7 positive samples. Twenty-three isolates were pathogenic (from 4 raw pork meats, 1 raw pork intestine and 1 raw pork liver) and 3 isolates were non-pathogenic (from 1 raw pork meat). Bioserotyping results revealed that the pathogenic Y. enterocolitica isolates were either bioserotyped as 1B/O:8 (n ¼ 3) or 3 variant/O:3 (VP negative variant strain, n ¼ 20), and all non-pathogenic Y. enterocolitica isolates were bioserotyped as 1A/O:5. All positive samples were from the same hawker stall. MPN/g of Y. enterocolitica in food was determined by using the YSEO enriched tubes and the results are tabulated in Table 3. Our results showed that the concentration of Y. enterocolitica in the positive samples ranged from <0.30 MPN/g to >43.84 MPN/g. Although there was no specific requirement for the levels of

Table 2 Prevalence of Y. enterocolitica from non-porcine food determined by cultural method and post-enrichment PCR screening. No of positivesa (%)

Food type

No. of samples

Raw beef Raw poultry products Raw seafood Raw vegetables Raw tofu Pasteurised milk

6 9 11 19 2 1

(66.7) (55.6) (45.5) (31.6)

0 0

0 0 0 0 0 0

Total

48

20 (41.7)

0

PCR

a

4 5 5 6

Cultural

The values are the number of positive detected in either of the enriched samples (YSEO, ITC, and PBS).

L.K. Tan et al. / Food Control 35 (2014) 94e100

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Table 3 The MPN/g values (calculated using the results of post-enrichment PCR) and the background information of food samples. Isolation time

Sample code

Food type

June 2010 June 2010 July 2010 Aug 2010 Sept 2010 Sept 2010 Sept 2010 Sept 2010 Jul 2010 Jul 2010 Jul 2010 Jul 2010 Jul 2010 Aug 2010 Sept 2010 Jan 2010 Jan 2011 Jan 2011 Jan 2011 Jan 2011 Jan 2011 Jan 2011 Jan 2011 Jan 2011 Mar 2011 Mar 2011

M1 M3 M5 M9 M12 M14 M13 M15 I2 I3 D1 D2 L1 D4 I5 I7 M16 M17 M18 M19 M20 YE032 YE036 YE037 K3 L3

Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw Raw

pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork pork

meat meat meat meat meat meat meat meat intestine intestine heart heart liver heart intestine intestine meat meat meat meat meat liver meat intestine kidney liver

Location

Bioserotype

MPNa/g

UCIb

LCIc

KLd KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL KL Taiping Taiping

3 variant/O:3 3 variant/O:3 e e e e 3 variant/O:3 e e e e e e e e e 1A/O:5 e e e e 1B/O:8 1B/O:8 3 variant/O:3 e e

<0.30 <0.30 0.30 0.30 1.58 7.60 1.90 0.62 0.30 0.36 0.30 0.60 3.12 18.98 0.30 1.90 >43.84 >43.84 >43.84 >43.84 2.71 0.36 0.36 0.36 0.73 4.57

ee e

0.07 0.07 0.07 0.07 0.35 1.68 0.42 0.14 0.07 0.08 0.07 0.13 0.69 4.19 0.07 0.42 e e e e 0.60 0.08 0.08 0.08 0.16 1.01

1.36 1.36 7.16 34.44 8.60 2.81 1.36 1.63 1.36 2.73 14.14 86.04 1.36 8.60 198.70 198.70 198.70 198.70 12.29 1.63 1.63 1.63 3.33 20.72

a

MPN, most probable number. UCI, upper confidence level. c LCI, lower confidence level. d KL, Kuala Lumpur. e Value cannot be calculated or identity cannot be determined. For the rest samples which were not mentioned in table, Y. enterocolitica was negative in both cultural and post-enrichment PCR method in all nine tubes of the 3  3 YSEO enrichment tubes. b

Y. enterocolitica in food under FDA Food Code (Lawley, Curtis, & Davis, 2012), majority of the positive samples had low MPN/g values (18.98 MPN/g), except for four samples (>43.84 MPN/g). A food sample was counted as PCR positive when either one of the 3 enriched cultures showed presence of amplicon. Postenrichment PCR detection showed a higher incidence of Y. enterocolitica (35/58) as compared to the cultural methods (raw pork meat, n ¼ 20; raw pork internal organs, n ¼ 14; skin, n ¼ 1). 3.2. Prevalence of Y. enterocolitica in non-porcine food Overall, no Y. enterocolitica was isolated via cultural methods from the 48 non-porcine food tested (Table 2). However, the postenrichment PCR screening indicated Y. enterocolitica was present in 20/48 (41.7%) of food samples; i.e. 4/6 (66.7%) raw beef, 5/9 (55.6%) raw poultry products, 5/11 (45.5%) raw seafood, and 6/19 (31.6%) raw vegetables. 3.3. Prevalence of Y. enterocolitica in live pigs Based on cultural methods, only 3 out of 165 pigs (1.8%) harboured Y. enterocolitica (Table 4). All the positive pigs were healthy grower pigs (asymptomatic) fed in the same pen from Farm I in Penang. All of them carried pathogenic Y. enterocolitica bioserotype 3 variant/O:3. These isolates were from all 3 swab types (nasal, oral, rectal) of 2 carrier pigs. For the other carrier pig, the bacterium was only identified in the nasal swab (total positive swabs ¼ 7). Overall, a total of 72 isolates were isolated (nasal swab, n ¼ 28; oral swab, n ¼ 20,; rectal swab, n ¼ 24). On the other hand, PCR detected a higher prevalence of Y. enterocolitica, in 46 pigs (27.9%), i.e. 11/22 (50.0%) healthy growers, 14/44 (31.8%) unhealthy weaners, 20/68 (29.4%) healthy weaners, and 1/11 (9.1%) healthy growers (Table 4). Our PCR results

indicated that Y. enterocolitica was most frequently found in nasal swabs, 29/165 (17.6%) followed by oral swabs, 25/165 (15.2%) and rectal swabs, 21/165 (12.7%) (Table 5). Overall, Y. enterocolitica was PCR detected in the swine hosts in the 3 states (Selangor, Perak, and Penang). 3.4. Confirmation of Y. enterocolitica by DNA sequencing DNA sequences of the 330 bp and 145 bp amplicons (Y. enterocolitica 16S rRNA and yst genes, respectively) were checked using the Basic Local Alignment Search Tool (http://blast. ncbi.nlm.nih.gov/) and the analyses confirmed the isolates were as Y. enterocolitica (99% and 96% homology, respectively). 4. Discussion The results showed that PCR-based detection method was more sensitive than the conventional cultural method, indicating that use of selective medium might underestimate the real prevalence of this pathogen in our local food and pigs. This result concurred with many other reports that the PCR detection was more sensitive than the cultural method (Bhaduri, Wesley, & Bush, 2005; Johannessen, Kapperud, & Kruse, 2000; Messelhäusser et al., 2011). For example, Messelhäusser et al. (2011) reported that 18% of the pork samples analysed was PCR- positive as compared to 10% positive by cultural method. However, these percentages are not necessarily comparable due to the different methods used in the detection of Y. enterocolitica. The primers used in the PCR assay are specific which amplified the targeted gene of Y. enterocolitica. In contrast, detection by cultural method is less sensitive as the method is based on the physiology and biochemical activities of bacteria. Besides, DNA templates for PCR assay were prepared from bacterial cells that were concentrated from 1 ml of enriched

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Table 4 Prevalence of Y. enterocolitica based on the age and health condition of pigs determined by cultural method and post-enrichment PCR screening. Health group

na

No of positives (%) Selangor (n ¼ 53)

Nj b c d e f g h i j

Penang (n ¼ 52)

Total positive

PCR

C

PCR

C

PCR

C

PCR

C

4 4 68 44 22 11 10 2

0 0 4 (5.9) 5 (11.3) 0 0 0 e

0 0 0 0 0 0 0 e

ee e 8 8 3 1 e 0

(11.8) (18.2) (13.6) (9.1)

e e 0 0 0 0 e 0

e e 8 (11.8) 1 (2.3) 8 (36.4) e e e

e e 0 0 3 (13.6) e e e

0 0 20 14 11 1 0 0

0 0 0 0 3 (13.6) 0 0 0

165

9 (5.5)

0

20 (12.1)

0

17 (10.3)

3 (1.8)

46 (27.88)

Hc pigletd UHf piglet H weanerg UH weaner H growerh UH grower H finisheri H sow

a

Perak (n ¼ 60) b

(29.4) (31.8) (50.0) (9.1)

3 (1.8)

n, number of pigs within each health group. C, cultural method. H, healthy. Piglet, <1 month old. e, sample not collected from this group. UH, unhealthy. Weaner, 1e2 months old. Grower, 2e4 months old. Finisher, 4e6 months old. N, number of pigs from each state.

homogenate, thus increasing the probability in getting more DNA of Y. enterocolitica. Moreover, the PCR can detect all kinds of cells in regardless of dead cells or viable including non-culturable cells which might not grow on artificial medium (Parker, 2002; Singh & McFeters, 1987). During the isolation of Y. enterocolitica from food and swine specimens, a high amount of natural background microbiota had been isolated as presumptive Y. enterocolitica from the CIN medium (colony appearance similar to Y. enterocolitica). Most of these bacteria were later identified as Citrobacter spp., Providencia rettgeri, Aeromonas hydrophila and Enterobacter cloacae by API 20E. The similarity in the colonies’ appearance caused difficulty in isolation of true Y. enterocolitica from these highly contaminated samples. In the later stage in our study, we modified the CIN medium by adding L-arginine, ferric ammonium citrate, sodium thiosulphate, and DLphenylalanine to improve the differentiation of Y. enterocolitica from these bacteria. Colony appearance of Y. enterocolitica on modified CIN remained unchanged (Y. enterocolitica utilise neither one of these substrates) as it was on CIN agar, but each of the microbiota (Citrobacter spp., P. rettgeri, A. hydrophila, and E. cloacae) appeared with a distinct colony appearance (utilised at least one of the substrates, and caused colour changes). We observed that lesser Yersinia-like bacteria (but not Y. enterocolitica) were miss-identified as presumptive Y. enterocolitica during the isolation since they were screened out on the modified CIN medium. This indirectly

decreased the workload for biochemical tests and the usage of API 20E kits. Further evaluation of this modified CIN needs to be carried out. We observed that the use of alkaline treatment prior to streaking onto the CIN or modified CIN agars eliminated a large amount of background microbiota on the agar plates during the isolation, such as that reported in Schiemann (1983). However, we observed no difference between both methods in terms of positive recovery according to the sample size (with alkaline treatment, n ¼ 12; without alkaline treatment, n ¼ 12). This concurred with study of Ratnam, Looi, & Patel (1983) who noted that alkaline treatment did not significantly enhance the isolation rates. In this study, Y. enterocolitica was isolated from both raw porcine food (12.7%) and pigs (1.8%). Three Y. enterocolitica bioserotypes were identified, 3 variant/O:3, 1B/O:8 and 1A/O:5. The results confirmed that Y. enterocolitica was present in our local porcine food and pigs. Interestingly, pathogenic Y. enterocolitica (3 variant/ O:3) was isolated from three healthy grower pigs. Similar finding was reported in Jos, Nigeria, where Y. enterocolitica was isolated from healthy pigs (Okwori et al., 2009). The infected pigs appeared asymptomatic due to the colonisation of Y. enterocolitica in the lymphoid tissue, particularly in tonsils (Horter, Yoon, & Zimmerman, 2003). The colonisation caused the identification of asymptomatic carrier animals difficult in disease control and/or pathogen elimination. These asymptomatic pigs serve as food for

Table 5 Distribution of the number of positive swab samples of pigs from Selangor, Perak and Penang using post-enrichment PCR screening and cultural methods. Swab type

na

No of positives (%) Selangor (n ¼ 53) PCR

Perak (n ¼ 60)

Penang (n ¼ 52)

Total positive

Cb

PCR

C

PCR

C

PCR

C

Nasal Rectal Oral

165 165 165

5 (9.4) 5 (9.4) 4 (7.5)

0 0 0

14 (23.3) 8 (13.3) 13 (21.7)

0 0 0

10 (19.2) 8 (15.4) 8 (15.4)

3 (5.8) 2 (3.8) 2 (3.8)

29 (17.6) 21 (12.7) 25 (15.2)

3 (1.2) 2 (1.2) 2 (1.2)

Nc

495

14 (2.8)

0

35 (7.1)

0

26 (5.3)

7 (1.4)

75 (9.9)

7 (1.4)

a b c

n, number of samples within each swab type. C, cultural method. N, number of pigs from each state.

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humans when they are matured to be sold. Cross-contamination of Y. enterocolitica from pigs’ oral cavity, intestine and faeces to meat is possible during the slaughtering and dressing operations through the slaughtering tools and containers (Gill & Jones, 1995; Nesbakken, 1988; Skjerve, Lium, Nielsen, & Nesbakken, 1998). Besides that, cross contamination may happen during food storage. For example, the interior surfaces of household refrigerators (Jackson, Blair, McDowell, Kennedy, & Bolton, 2007) or surfaces of storage containers. Y. enterocolitica may be transferred from the contaminated surfaces to other food items, especially the higher risk ready-to-eat foods. Improper food handling, processing and storing practices such as undercooked meats or cross contamination of contaminated meats or surfaces to other food or water are risk factors for yersiniosis in humans. Besides humans, companion animals such as dogs and cats are exposed to the risk of infection of Y. enterocolitica. This is because dogs and cats are more free-ranging and more likely to come in contact with the materials contaminated with Y. enterocolitica, especially stray dogs and cats in the vicinity of wet markets or pig’s abattoirs. Dogs and cats may be at risk if they were fed with contaminated meat. Several reports had indicated that companion animals are infected after the consumption of contaminated pork or by-products (Byun, Yoon, Lim, Lee, & Jung, 2011; FredrikssonAhomaa, Korte, & Korkeala, 2001; Greene, 2006). Besides consumption of contaminated food, humans might be infected through direct contact with sick animals. Farmers who work in pig farms or abattoirs may be infected through animal bits, saliva or faeces of infected pigs. Moreover, companion animals such as dogs and cats may also be another potential route for animal to human transmission. Infected dogs and cats can cause human yersiniosis when they are in contact with humans, i.e. through contact with animals’ excreta such as saliva and faeces (Fenwick, Madie, & Wilks, 1994; Stamm, Hailer, Depner, Kopp, & Rau, 2013; Wang et al., 2010). Human infection from companion animals is believed to be more serious than swine and consumption of contaminated food, especially people with poor hygiene practices, children, elderly, and immunocompromised patient (Plaut, Zimmerman, & Goldstein, 1996). More than 50 serotypes and 6 biogroups of Y. enterocolitica have been identified currently and their geographical distributions are diverse. In Europe, Y. enterocolitica particularly bioserotype 4/O:3 has been frequently isolated in humans, pig husbandry and food, followed by the less common bioserotype, 2/O:9 and 2/O:5,27 (European Food Safety Authority & European Centre for Disease Prevention and Control, 2012; Fondrevez et al., 2010; FredrikssonAhomaa, Gerhardt, & Stolle, 2009). In USA, O:8 is the primary infectious serotype, followed by O:5,27, O:13a, 13b, O:20, O:9 (Bottone, 1997; Kwaga, Iversen, & Misra, 1992). Y. enterocolitica also was frequently isolated in pigs from China in which the bioserotypes isolated were 2/O:9, 4/O:3, 3/O:3, 1A/O:5, 1A/O:8. Among the three bioserotypes identified in this study, Y. enterocolitica bioserotypes 3 variant/O:3 was the most common. In the past, the bioserotype 3 variant/O:3 had been reported in imported pork and chicken to Japan and food animals (pigs, rats and rabbits) in China (Fukushima, et al., 1997; Zheng & Xie, 1996). Y. enterocolitica bioserotype 3/O:3 was the major bioserotype in pigs, particularly from Jiangxi and Fujian Provinces with warmer climate (Liang et al., 2012; Wang et al., 2009). All these reports showed that this particular bioserotype 3/O:3 was frequently isolated from the regions with warmer climate. Therefore, Y. enterocolitica bioserotype 3/O:3 or 3 variant/O:3 strains might be the common strains in warm regions. Lim & Tay (1992) attempted to isolate Y. enterocolitica from over 6000 samples from patients with diarrhoea in Singapore, but no Y. enterocolitica was detected. They also suggested that

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Y. enterocolitica had no clinical importance in this region. However, this pathogen was recently isolated from unpacked tofu (Ananchaipattana et al., 2012a), beef and shrimp samples (Ananchaipattana et al., 2012b) in Thailand. These recent reports from Thailand indicated that there is a risk of human infection. In Malaysia, yersiniosis is rarely reported. The under-reporting in Malaysia could be due to several possibilities: (i) Malaysians prefer well cooked food to raw or undercooked meats (dietary preference), (ii) Y. enterocolitica is not the routine pathogen monitored in our diarrhoeal patients, or (iii) most of the food poisoning due to Y. enterocolitica cases are self-limiting. Although there is no other official report on yersiniosis in Malaysia since 1984, we should be cognisant that this bacterium could be another potential agent to contribute to the incidence of food poisoning cases in our country since this pathogen was confirmed present in our local food and pigs. Improper food handling and processing may cause cross contamination of this pathogen to humans and therefore affirms a potential risk for the consumers. 5. Conclusion This is the first report on the prevalence of Y. enterocolitica in pigs and food from Malaysia. Y. enterocolitica was isolated from raw porcine food and pigs. The prevalence of Y. enterocolitica in raw porcine food and pigs were 12.1% and 1.8%, respectively. The most common bioserotypes isolated were 3 variant/O:3, followed by 1B/ O:8 and 1A/O:5. The presence of pathogenic Y. enterocolitica in raw porcine food and pigs affirms a potential risk for the consumers. Improper food handling and processing may cause cross contamination of this pathogen to humans. Acknowledgements This work was supported by University of Malaya through UM PPP grant (PS316/2010B) and University of Malaya High Impact Research Grant (UM.C/625/1/HIR/MOHE/CHAN-02). Lai Kuan Tan is supported by a fellowship from University of Malaya. Special thanks to Dr. Hudson A. from Institute of Environment Science & Research (ESR) Limited, New Zealand in providing the Microsoft Excel spreadsheet for the MPN values calculation; and Dr. Gómez-Duarte O. G. in providing Y. enterocolitica positive control strain for our PCR test. References Ananchaipattana, C., Hosotani, Y., Kawasaki, S., Pongsawat, S., Latiful, B. M., Isobe, S., et al. (2012a). Bacterial contamination of soybean curd (tofu) sold in Thailand. Food Science and Techonology Research, 18(6), 843e848. Ananchaipattana, C., Hosotani, Y., Kawasaki, S., Pongsawat, S., Latiful, B. M., Isobe, S., et al. (2012b). Prevalence of foodborne pathogens in retailed foods in Thailand. Foodborne Pathogens and Disease, 9(9), 835e840. Annamalai, T., & Venkitanarayanan, K. (2005). Expression of major cold shock proteins and genes by Yersinia enterocolitica in synthetic medium and foods. Journal of Food ProtectionÒ, 68(11), 2454e2458. Archer, J. R., Schell, R. F., Pennell, D. R., & Wick, P. D. (1987). Identification of Yersinia spp. with the API 20E system. Journal of Clinical Microbiology, 25(12), 2398e2399. Bhaduri, S., Wesley, I. V., & Bush, E. J. (2005). Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States. Applied and Environmental Microbiology, 71(11), 7117e7121. Bottone, E. J. (1997). Yersinia enterocolitica: the charisma continues. Clinical Microbiology Reviews, 10(2), 257e276. Byun, J. W., Yoon, S. S., Lim, S. K., Lee, O. S., & Jung, B. Y. (2011). Hepatic yersiniosis caused by Yersinia enterocolitica 4:O3 in an adult dog. Journal of Veterinary Diagnostic Investigation, 23(2), 376e378. Dallal, M. M. S., Doyle, M. P., Rezadehbashi, M., Dabiri, H., Sanaei, M., Modarresi, S., et al. (2010). Prevalence and antimicrobial resistance profiles of Salmonella serotypes, Campylobacter and Yersinia spp. isolated from retail chicken and beef, Tehran, Iran. Food Control, 21(4), 388e392. Dzomir, M. A. Z. (2005). Isolation, identification and characterisation of Yersinia spp. from meat and meat products. Doctoral thesis of University Putra Malaysia. European Food Safety Authority, & European Centre for Disease Prevention and Control. (2012). The European Union summary report on trends and sources of

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