EPIDEMIOLOGICAL PATTERN AND DETECTION OF YERSINIA ENTEROCOLITICA IN FOODS OF ANIMAL ORIGIN: A REVIEW

Yersinia enterocolitica is an important organism causing yersiniosis in animals and human beings. It has an immense zoonotic importance. The most common mode of transmission is through faecal oral route. The detection of Y. enterocolitica in foods of animal origin like meat and milk is gaining importance from health and trade point of view. Many detection techniques like culture methods on selective and differential media have been used earlier but novel methods like serological and molecular techniques are now widely being used because of authentic and rapid screening. Key words: Yersinia enterocolitica.






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Publication: Journal of Cell and Tissue Research
Author: Arora, D
Date published: January 1, 2012

INTRODUCTION

The organism now recognized as Yersinia enterocolitica was first described by Schleifstein and Coleman in 1939 and included in the family Enterobacteriaceae [1]. The nameYersinia was given in the honour of a French bacteriologist, Yersin who first isolated the causal organism of the plague [2]. Yersinia enterocolitica is characterized by Gram negative rods (0.99-3.54 m x 0.52-1.27 m ), arranged singly or in chains or heaps. Coccoid forms predominate in young cultures grown at 22 to 25C on selective differential media [3]. Cultures grown at 25C showmotility due to presence of peritrichous flagella while cultures grown at 37C are non-motile. These organisms are usually methyl red positive, Voges-Proskauer and citrate test negative at 25C but variable at 37C [4]. The pH range for survival and growth of Y. enterocolitica reported to be 4.6 - 9.0 with the optimum range of 7.0-8.0 [5].

Y. enterocolitica strains and related species can be serogrouped on the basis of their heat-stable somatic antigens. 54 serogroups for Y. enterocolitica and related species have been described [6] and 18 serogroups containing 20 O-antigens within the Y. enterocolitica have been proposed [7]. Presently, pathogenic strains of Y. enterocolitica belong to serogroups include O:1, 2a, 3;O:2a, 3;O:3; O:8;O:9; O:4, 32; O:5, 27; O:12, 25; O:13a, 13b; O:19, O:20 and O:21. Serogroups that predominate in human beings are O:3, O:8, O:9, O:13 and O:5, 25 [8].

These organisms are causing yersiniosis in wild and domestic animals as well as humans. Yersiniosis being a zoonotic disease warrants detection and epidemiological studies in man and animals. It has been reported that pigs are infected early in life and maintain infection in the tonsils for longer periods, perhaps for life [9]. Y. enterocolitica is an important emerging food and water-borne enteric pathogen that causes acute gastroenteritis, enterocolitis and mesenteric lymphadenitis as well as a variety of extra intestinal disorders [10,11]. The most prevalent clinical signs in human appear within 2-3 days with abdominal pain, fever, diarrhea and nausea. Sometimes vomiting may also occur. Diarrhoea may be watery lasting for 3-14 days and 5% cases may have blood in their stool. The disease can range in severity from a self-limiting gastroenteritis to potentially fetal septicemia. The most susceptible population is either the very young or the old and persons undergoing immunosuppressive therapy.

Acute yersiniosis in animals more frequently caused by Yersinia enterocolitica is characterized by enteritis and enlargement of lymph nodes and spleen, whereas, chronic infections may cause granulomatous nodules and localized abscesses affecting various organs, typically liver and lungs [12, 13].

Epidemiology of Yersinia enterocolitica in humans and animals: Yersinia infections in man and animals have been reported as a result of the frequent isolation froman increasing number of countries from all parts of the world. Epidemiological evidence has demonstrated that pigs and pork are important sources of yersiniosis in humans. Yersinia enterocolitica and Yersinia pseudotuberculosis were recovered from diverse animal sources ranging from farm animals and domestic pets to free living and captured wild animals [11,14]. However, human pathogenic strains of Yersinia enterocolitica have frequently been isolated only fromasymptomatic pigs at slaughter [15]. In Switzerland, the prevalence of pathogenic Yersinia enterocolitica has shown to be high in the tonsils of pigs at slaughter, 85 and 34% with PCR and culturing, respectively [16].

It was reported that Yersinia enterocolitica is an important gastro-intestinal pathogen, which has been isolated from3%of the 1189 stored samples collected from pediatric diarrhoeic patients and 32.9% of the 492 throat swabs collected from swine in Delhi. Yersinia enterocolitica was also isolated fromground water, waste water and river Yamuna. In addition, Yersinia intermedia and Yersinia frederiksonii were also isolated from human stool and swine throat samples. All the Yersinia enterocolitica strains belong to biotypes IA. This study represented first isolation of Yersinia enterocolitica fromswine throat swabs, ground water and surfacewater in India [17].

The most common mode of transmission of Y. enterocolitica is thought to be the faecal-oral route via contaminated food. Direct person to person transmission has not been demonstrated but the infection of Y. enterocolitica 0:3 in infants probably on exposure to their carriers has been reported [18]. The main sources of human infection are assumed to be pork and pork products that have been undercooked or improperly handled. Pathogenic Y. enterocolitica can be transmitted from slaughter house to meat processing plants and then to retail level [19]. Transmission probablymore often occurs via pork and other food products.

Human Outbreaks: Y. enterocolitica was first isolated inAsia fromJapan in 1971 [20] and presumed that this pathogenic strains have been introduced through imported pork, beef and fowl [21]. Yersinia food borne infections in humans have also been reported in Japan [22], Czechoslovakia [23], Canada and United States [24]. In several tropical countries such as Iran, Israel, South Africa and Brazil, Y. enterocolitica has been observed to be a significant cause of human infections. The isolation of this organismwith high frequency fromman and animals, some foods of animal origin and also from a large number of different food-stuffs as well as from nonchlorinated well water used for drinking has also been reported [25].

Y. enterocolitica was first isolated in Czechoslovakia in 1963 in humans. By the end of 1971, 845 cases of human Y. enterocolitica infections had been diagnosed. During 1969-1974, 1355 strains of Y. enterocolitica were reported from 1096 persons in Hungary. Over 99% of the isolates were characterized as serotype O:3. The frequency of isolation of Y. enterocolitica from persons suffering fromenteritis in the southern region ofWest Germany was 1.5%. Y. enterocolitica 4/O:3 was the most frequent cause of sporadic human yersiniosis in Finland and Germany [15].

In New York State in 1976, there has been a clearly documented food borne outbreak in which serotype (O:8) was isolated from patients as well as from the left over food. In Belgium, Canada, Japan and other countries where Y. enterocolitica rivals strains of Salmonella and Campylobacter and serotype O:3 is the predominant serotype [26]. In various studies conducted worldwide, Yersinia enterocolitica has been isolated from 5.9% of 1635 patient stool, 3.4% of 206 appendices and 4%of 555 control stools [27]. Also, in patients suffering fromgastroenteritis, it was reported in only three (0.2%) out of 1470 faecal specimens tested [28]. However, the isolation rate of Yersinia enterocolitica from diarrhoea cases in several other countries ranged from 2 to 5%.

In India there was an outbreak of food poisoning associated with Y. enterocolitica in Tamil Nadu that affected 25 of 48 individuals who participated in a feast and the risk of developing illness was associated with consumption of buttermilk (relative risk 3.8). This is the first report from India of a food poisoning outbreak associated with this organism [29]. On testing the faecal samples in Ludhiana, it was found that three hundred and seventy-four (18.7%) faecal samples were positive for bacterial pathogens, of which 41 (2.05%) had Y. enterocolitica infection. Y. enterocolitica was more common in patients above 16 years of age (23/41, 56%). Higher rates of isolation were recorded during winter months [30].

The first reported Norwegian outbreak of Y. enterocolitica O:9 disease took place in January 2006 involving 11 persons [31] where in later a ready to eat pork product was considered as the probable source [32].

Prevalence in Animals: Y. enterocolitica has been detected from many domestic and wild mammals, as well as some birds. Most important are swine, dogs, and cats from which serotypes O:3 and O:9 have been reported. In addition, serotype O:5 was found in swinewhich is common in people of Japan.Among wild animals in New York State, serotype O:8 has been isolated from a gray fox (Urocyon cinereoargenteus) and from a porcupine (Erethizon dorsatum); serotype O:3 has been isolated from another gray fox. Both serotypes are reported to be pathogenic for man [33]. The incidence of Y. enterocolitica has been reported from swine and a group of buffaloes in India [34]. Four isolates of Y. enterocolitica were recovered from 127 pig rectal swab samples in Uttar Pradesh [35].

Y. enterocolitica serotype O:3 has been exclusively isolated fromswine inDenmark, Belgiumand Sweden [36-38] which reveals that in some European countries Y. enterocolitica O:3 is a normal inhabitant of the oral cavity of swine that may play important role [37,38]. It was reported that more than half of containers of chitterlings sampled, originating from pigs slaughtered in different regions of the United States, were positive for isolation of Y. enterocolitica serotype O:3 [18].

The studies on prevalence of Y. enterocolitica in different production phases viz: gestating, farrowing, suckling, nursery and finishing on swine farms was conducted. On all farms, there was a trend of increasing prevalence as pigs mature [39]. Y. enterocolitica has been isolated from young sheep with enterocolitis inNewZealand and also in Southern Australia [40]. Abortions have been described in association with Y. enterocolitica in cattle in the former Soviet Union and Great Britain. In India, serotype O:9 was isolated from nine buffaloes that aborted; this serotype shares common antigens with Brucella and gives serologic cross reactions [34].

The tonsils of 630 pigs from English farms were examined using three different rearing methods (Assured British Pigs, Open Management and Organic) in order to investigate if the low incidence of human yersiniosis could be attributed to a low prevalence of enteropathogenic Yersinia among English pigs [41]. They reported high prevalence and a wide diversity of bioserotypes of enteropathogenic Yersinia as compared to other European countries.

Prevalence of Yersinia enterocolitica in food: Y. enterocolitica have also been isolated fromvacuumpackaged meats [42] and can survive for extended periods in frozen food, even with repeated freezing and thawing [43]. The association of human illness with consumption of Y. enterocolitica contaminated food and unchlorinated water is well documented [44,45]. Contamination with this organismis possible either at the manufacturing site [44] or in the home [45]. Refrigerated foods are potential vehicles for infection because this organism may survive and grow during refrigerated storage. The prevalence of Yersinia enterocolitica was investigated on chicken egg shell surfaces in San Luis, Argentina eight Y. enterocolitica O:9 strains were recovered after enrichment with a prevalence of 2.27% [46].

As far as milk and milk products are concerned, Yersinia spp. have been reported from raw milk in Australia [47], UK[48-50], Canada [51], France [52], Italy [53], Scandinavia [54,55] and USA [56]. In another study, Y. enterocolitica was isolated from 7% of cottage cheese [48] and 9.2% of cheese curd samples [51]. Contamination of butter, pasteurized cream, ice cream and yoghurt with Y. enterocolitica has also been reported. Yersinia spp. has been isolated fromcaprine milk inAustralia [57], England andWales [58] and Northern Ireland [50]. The strains obtained were similar to those occurring in bovine milk.

Techniques for Detection of Yersinia enterocolitca: The source of Y. enterocolitica may markedly affect the methods of isolation. During phases of acute gastroenteritis or with organ abscess, Y. enterocolitica is often dominant [59] and so may be isolated by direct plating on conventional enteric media [3,60]. In other clinical specimens and food samples however, Y. enterocolitica may be present in low numbers and so require enrichment prior to inoculation on solid media. Isolation of Y. enterocolitica fromfood is more difficult than faeces taken from patients with active infections where the numbers of Yersinia spp. may be smaller and the background flora likely to be greater in both number and variety [61].

The addition of sorbitol (1%) and bile salts (0.15%) to Phosphate Buffer Saline (PBS) is claimed to reduce the incubation time for detection of Y. enterocolitica and increase the sensitivity of the medium [62] and so has been frequently added in enrichment medium methods [63]. Alkali treatment has been used for the isolation of Yersinia spp. from foods and environmental samples [64-66] and may recover these microorganisms after 1-3 day enrichment in PBS incubated at 25C [67].

1. Culture Methods: Cefsulodin-irgasannovobiocin (CIN) is a highly selective and differential medium that provides recovery of all types of Y. enterocolitica on 18 to 20 h incubation at 32 C [68] and has been well accepted. Y. enterocolitica appears as 0.5- to 1.0- mmcolonies with a red "bull's eye" and a clear border. Use of this medium allows differentiation between Y. enterocolitica and Y. enterocolitica- like isolates.

CIN agar is found to be a highly specific medium for isolation of Y. enterocolitica, requiringKligler's Iron agar (KIA) slants and urease broth for confirmation of presumptive colonies [69]. Modified Rappaport broth and Bile-oxalate-sorbose (BOS) broth work well as a secondary enrichment at 22 to 25C for 4 days following cold enrichment [61]. Potassium chlorate was also used as a selective agent in a solid plating medium incubated anaerobically [2]. Better results were achieved by direct plating than by cold enrichment, as only one 0:3 strain recovered after enrichment versus five recovered by direct plating [67].

A new culture method was developed employing a potassium hydroxide treatment and compared with the conventional cold enrichment method for efficacy in recovering Yersinia spp. from naturally and artificially contaminated food [70]. This newmethod increased the yield of Yersinia spp. four-fold and the sensitivity 100-fold, shortened the incubation period and appreciably decreased the growth of non-Yersinia bacteria.

The psychrotrophic nature of Y. enterocolitica is unusual amongst enteric micro-organism and consequently cold enrichment in phosphate-buffered solution (PBS) at 4C for 3 weeks has been successfully used for isolation fromfaecal specimens [71, 72]. Isolation through enrichment in coldmannitol broth or a bile medium at 4C for 10-60 days was equally effective. Cold enrichment of stored samples at 4C for two weeks before incubation yielded better results. Y. enterocolitica was diagnosed from 6 of 67 samples of pig tongue and 4 out of 34 samples of pig tonsils and 1 out of 23 poultrymeat samples [73].

A shortened enrichment procedure (25C for 24 h) was compared with cold enrichment procedures (4C for 1 to 3 weeks) and direct plating for isolation of Yersinia enterocolitica. The organism was isolated from 34% of the ground beef samples. The highest isolation rate was 32% for the 4C/3 week enrichment, followed by 28% for the 4C/2 week enrichment, 26%for the 25C/24-h enrichment, 22% for the 4C/1-week enrichment, and 10% for direct plating [74].

On examination of 139 slaughtered pigs by enrichment and direct plating procedures, 52 porcine tonsils (37.4%) were found positive for the presence of pathogenic Y. enterocoilitica belonging to bioserotype 4/0:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating [75].

2. Serological methods: The authentic and rapid screening of foods for Y. enterocolitica has become an important issue with changing scenario of global trade. In recent time several new approaches have been used for detection of different bacteria from foods. These methods include ELISA, IFT, nucleic acid probes, PCR etc.Among themELISAand PCR are most frequently used techniques [76]. The standard microbiological methods for detection of Y.enterocolitica are sensitive and confirmatory but also vary laborious and time consuming. The detection and identification of Y.enterocolitica with conventional method takes 1 to 3 weeks. On the other hand Enzyme Linked Immuno Sorbent Assay (ELISA) has been developed as one of the most rapid , inexpensive, sensitive and specific technique for detectingmany food borne pathogens. The sensitivity of the technique varies from 104 to 107 CFU per ml depending on quality of antibody and assay [76] and microorganism can usually be detected in very short time.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitation of human IgM, IgG, and IgA antibodies against Y. enterocolitica and total of 423 sera samples were tested. The results obtained with the ELISA were compared with the results of the conventional tube agglutination test. Antibodies of the IgM class persisted only for 1 to 3 months after onset of the disease. The persistence of the IgG and IgA antibodies was variable and not parallel with each other. Remarkably, all three patients in which the disease was complicated with arthritis had IgA Yersinia antibodies at the end of the follow-up period of 9 to 14 months, and in those without arthritis the IgA antibodies disappeared within 3 months after onset of the disease [77].

Enzyme immunoassay (EIA) was developed to detect antibodies in pigs against the lipopolysaccharidic antigen of the three serotypes of Yersinia enterocolitica mostly associated with human infections. This test appeared as a valuable tool in conjunction with culture, to identify pigs or herds infected by strains of Y. enterocolitica associated with human infections [78].

Swine are implicated as the principal animal reservoir for plasmid-bearing Yersinia enterocolitica (YEP+) strains that are pathogenic to humans. The use of PCR for detection of YEP+ strains in naturallycontaminated pig feces was conducted [79]. The samples were first enriched in Irgasan ticarcillin potassium chlorate broth for 48 h at 25C and then tested by multiplex PCR, PCR-ELISA, and fluorogenic 52 nuclease PCR assays. Three different primer sets for amplification of the ail gene sequences were used in these three assays. Three out of 50 (6%) samples were positive for YEP+ strains using the multiplex PCR targeting the chromosomalail (170 bp) and plasmid virF (591 bp) genes. Two of the 3 samples positive by the multiplex PCR were also positive by the PCR-ELISA method using primers targeting the ail gene (425 bp). The multiplex PCR was found the most reliable and sensitive assay for detecting YEP+ strains in feces among the three assays evaluated.

An antigen capture immuno assay was developed using LcrV, as the target antigen secreted by Yersinia spp. [80]. They generated anti-LcrV monoclonal antibodies (MAbs) and screened for the ability to bind bacterially secreted native Yersinia pestis LcrV. They used anti-LcrV MAb 19.31 as a capture antibody and biotinylated MAb 40.1 for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay was found to be 0.1 ng/ml. This assay detected LcrV fromhuman sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV

ACompetitive Indirect ELISAwas developed against whole cells of Y. enterocolitica O:3, O:9 and also a group of pathogenic Y. enterocolitica strains (serotypes O:3, O:5,27, O:8. and O:9). The immunoblotting analysis of Yersinia lipopolysacchides separated by SDS-PAGE showed that IgG against the single serotype O:3 interacted with high-molarmass LPS of O:3 whereas other antibodies were bound to low-molar-mass LPS of serotypes O:3,O:5,27, O:9 and strain Y. enterocolitica (CNCTC Y 2/68) [76].

A study was conducted in between October 2007 and March 2008, on153 wild boars aged between 6 months to 2 years slaughtered in Switzerland for detection of Y. enterocolitica infection [81]. Prevalence ofenteropathogenic Yersinia in tonsils and faeces was studied using culture and PCR methods and in tissue fluid of tonsils by an ELISA system. Prevalence of anti-yersinia antibodies in tissue fluid was 65%.Detection rate of enteropathogenicYersinia in tonsil of 153 wild boars by real-time PCR was 44%. Prevalence was shown to be significantly higher in tonsils than in faeces.

3. Molecular techniques: Numbers of molecular techniques like restriction endonuclease analysis (REA), amplified fragment length polymorphism (AFLP), DNA hybridization, Polymerase chain reaction (PCR) are in use and being most sensitive, specific and reliable test, PCR is usually accepted for detection of pathogens in food. In a study, PCR method was compared with the culture method for detecting pathogenic Y. enterocolitica [82] and PCR assay revealed a significantly (P< 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). This indicates higher sensitivity, with respect to number of samples and sites identified as positive.

A PCR method was developed for direct detection of Y. enterocolitica from natural samples without extraction of DNA. The method targetted the yad A-gene located on the virulence plasmid, which is essential for full pathogenecity of Yersinia enterocolitica [83].

The use of 16S RNA gene PCR assay to confirm the diagnosis of Y. enterocolitica was recommended [84, 85]. The prevalence of pathogenic Y. enterocolitica in pigs in United States using a fluorogenic 5' nuclease PCR assay and a culture method was reported. The mean prevalence was 13.10% when both PCR and culture positive results were combined. The PCR assay indicated a contamination rate of 12.35% compared to 4.08% by culture method. They demonstrated higher sensitivity for PCR method as compared to culture for detecting pathogenic Y. enterocolitica [82].

Real-time PCR was found to be 100% specific to virulent Y. enterocolitica with sensitivity in pure cultures and stool to 102 CFU/ml and 103 CFU/g, respectively.All the diarrheal positive stools samples were successfully detected by both real-time PCR and culture methods, where as no positive sample was found by these 2 methods from healthy people, which sufficiently showed the consistency as 100%. Therefore, because of its convenience in operation and time saving, it can be used as an efficient method to detect pathogenic Y. enterocolitica from diarrhea stools [86].

CONCLUSION

Being an emerging pathogen worldwide, Y. enterocolitica demands a special attention. The high prevalence rate has led to development of new techniques of detection which have higher sensitivity, accuracy, cost effectiveness and prompt results. The conventional culture methods are obsolete and more effective serological and molecular techniques are substituting it but still its relevance cannot be ruled out.

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Author affiliation:

ARORA, D., 1 MEHTA, N.2* AND SAINI, R.3

1Department ofVeterinary PublicHealth and Epidemiology, College ofVeterinarySciences, LLRUVAS, Hisar;

2Department ofLivestock Products Technology, College of Veterinary Sciences, LLRUVAS, Hisar 125 001;

3Department of Zoology, College of Basic Sciences and Humanities, CCSHAU,

Hisar 125 001. E. mail: nmvets220@gmail.com

Received: January 6, 2012;Accepted: February 4, 2012

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