YERSINIA ENTEROCOLITICA

Hin-chung Wong Department of Microbiology

Soochow University _____________________________________________________________ 1. INTRODUCTION 2. TAXONOMY AND GENERAL CHARACTERISTICS 2.1. Biofilm

2.2. Antibiotic Resistance 3. BIOTYPING AND SEROTYPING

4. PHAGE TYPING 5. GROWTH AND SURVIVAL 5.1. Nutritional Requirements 5.2. Acidity 5.3. Temperature 5.4. Sodium Chloride 5.5. Organic Acids and Salts 5.6. Radiation 5.7. Competitive Microorganisms 5.8. Controlled atmosphere 5.9. Essential oils 5.10. Physiology 6. OCCURRENCE IN FOODS AND ENVIRONMENT 6.1. Animal 6.2. Water and Soil 6.3. Isolation from Foods 7. ISOLATION AND IDENTIFICATION 7.1. Selective Enrichments 7.2. Selective Differential Platings 7.3. Identification 7.4. Membrane Filter Method 7.5. DNA Hybridization Method 7.6. Commercial Rapid Detection Kit

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7.7. DNA microarray 7.8. Real-time PCR 8. PATHOGENICITY AND VIRULENCE FACTORS 8.1. Clinical Manifestations 8.2. Effects of Sublethal Stresses on Virulence 8.3. Enterotoxin 8.4. Autoagglutination 8.5. Invasiveness 8.6. Congo Red and Crystal Violet Binding 8.7. Calcium Requirement 8.8. Surface Hydrophobicity 8.9. Mouse Lethality 8.10. Requirement of Iron

8.11. Phospholipase 8.12. Type III secretion system 8.13. A three-dimensional collagen gel model

9. ROLE OF PLASMIDS IN VIRULENCE 9.1. Proteins Encoded by Virulence-associated Plasmids 9.1.1. Excreted Proteins versus Surface Proteins 9.1.2. Proteins Associated with Fibrillae, Adhesion and Autoagglutination 9.1.3. Proteins Associated with Serum Resistance 9.2. Molecular Manipulation of Plasmids 10. MOLECULAR STUDY OF INVASIVENESS

10.1. Flagellar master regulator 11. CONCLUSIONS 12. REFERENCES ________________________________________________________________ 1. INTRODUCTION Yersinia enterocolitica was discovered by Schleifstein and Coleman in 1939 in USA. Most of the reports about this bacterium were published since the early 1960s. In the last four decades, it is popularly known as an important foodborne pathogen. In fact, Y. enterocolitica and Campylobacter jejuni can be regarded as the "pathogenic bacteria of the 1980s" (Swaminathan et al., 1982).

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Since Y. enterocolitica is one of the few pathogenic bacteria which can grow at refrigerating temperature, foods contaminated initially with even low levels of this pathogen may serve as medium for proliferation and vehicle of disease. 2. TAXONOMY AND GENERAL CHARACTERISTICS The Genus Yersinia currently placed in the family Enterobacteriaceae comprises three major pathogenic bacteria, namely, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. The species Y. enterocolitica is characterized by Gram negative rods (0.99-3.54 μm x 0.52-1.27 μm), arranged singly or in short chains or heaps. Coccoid forms predominate in old cultures grown at 22-25C on selective differential media used for the isolation or enteric organisms (Swaminathan et al., 1982). Strains of Y. enterocolitica are biochemically heterogeneous. Typical strains of Y. enterocolitica ferment sucrose but cannot utilize rhamnose, citrate, α-methyl glucose or melibiose. Although most strains of Y. enterocolitica are o-nitrophenyl β-D-galactopyranoside (ONPG)-positive they do not possess β-galactosidase, except for those strains which contain the plasmid lac+. Atypical strains of Y. enterocolitica may be divided into four major groups, two of which are sucrose negative and two of which are rhamnose positive (Table 1) (Swaminathan et al., 1982).

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On the basis of DNA hybridization studies, it is concluded that the biochemically viariant strains (rhamnose positive, sucrose negative, etc.) are more closely related to Yersinia than any other Genus in the Enterobacteriaceae. Different names like Y. enteritidis, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. rhamnophilica etc. are suggested for the atypical strains (Table 2)(Bissett et al., 1990; Swaminathan et al., 1982). Y. enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were differentiated by the electrophoretic polymorpism of acid phosphatase, esterases, and glutamate and malate dehydrogenases (Goullet and Picard, 1988).

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By cross-streaking method, three clinical isolates of Y. enterocolitica produced inhibitory substances (bacteriocin-like material) at room temperature, and these substances were active against a variety of clinical isolates and their plasmid-cured derivatives at both room temperature and 37C (Cafferkey et al., 1989). Methodology Cross-streaking Method The strain to be tested is inoculated as a 1.5-cm-wide steak across tryptone soya blood agar plates and incubated overnight at room temperature. The inoculum is removed with a glass slide and the remaining viable cells are killed by exposure to UV for 30 min. The indicator strains are streaked singly at right angles to the original inoculum and incubated at 37C overnight. Inhibition zones are observed (Cafferkey et al., 1989). The first restriction endonuclease YenI was indentified in Y. enterocolitica (only in O8 strains) with or without the virulence-associated plasmid. YenI was very stable during the purification and storage at 4C or lower temperatures. YenI required 10 mM Mg2+ for cleavage of lambda DNA with the highest activity at pH 7.5 to 8.1 in the presence of 50 mM NaCl. Cleavage patterns with YenI and PstI were identical, showing that YenI is an isoschizomer of PstI, the cutting site is CTGCA/G. YenI has high activity at low temperature (Miyahara et al., 1988). Some Y. enterocolitica strains produce hemagglutinins. Production of the mannose-resistant hemagglutinin (MRHA) was affected by the culture media, e.g. none of the autoagglutination-positive strains produced MRHA in either nutrient broth or on colonization factor agar. In contrast a distinct autoagglutination-associated MRHA was detected after growth in Eagle minimal essential medium (Kapperud and Lassen, 1983). Enterobacterial common antigen (ECA) was found in the outer membrane and also in the cytoplasm of Y. enterocolitica by electron microscopy with the use of ECA specific antibodies and secondary antibody labelled with gold

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(Acker et al., 1986), or by the immunoferritin technique (Acker et al., 1981). ECA is a surface antigen common to almost all Enterobacteriaceae and consists of an amino sugar heteropolymer. The ECA on the cell surface of Y. enterocolitica Ye75S (smooth cell) is covered by O-specific chains of the lipopolysaccharide if grown at 22C and is therefore not accessible to ECA antibodies. It becomes accessible, however, when O-chains are lacking (R mutants) or when they are reduced in size or amount (growth at 40C) (Acker et al., 1981). 2.1. Biofilm Y. enterocolitica biovar 1B is one of a number of strains pathogenic to humans in the genus Yersinia. It has three different type III secretion systems, Ysc, Ysa, and the flagella.The effect of flagella on biofilm formation was evaluated. In a panel of 31 mutant Y. enterocolitica strains, the mutations that abolish the structure or rotation of the flagella greatly reduce biofilm formation when the bacteria are grown under static conditions (Table 3). These results were further evaluated by assessing biofilm formation under continuous culture using a flow cell chamber. The results confirmed the important contribution of flagella to the initiation of biofilm production but indicated that there are differences in the progression of biofilm development between static growth and flow conditions (Kim et al., 2008).

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2.2. Antibiotic Resistance

To obtain basic data for future resistance monitoring programs, 386 Y. enterocolitica strains from human patients, raw retail pork and pig feces were tested for their susceptibilities to 16 antimicrobial agents and two antimicrobial growth promoters (carbadox and olaquindox). No strains were resistant to ceftriaxone, cefuroxime, ciprofloxacine, gentamicin, kanamycin, neomycin or polymyxin. Although in Switzerland carbadox and olaquindox were used as growth promoters for pigs for over 25 years, all strains were susceptible to them. In contrast, there were high levels of resistance to ampicillin, cefalothin and amoxicillin/clavulanic acid. Less than 10% of clinical isolates and strains from pig feces were resistant to streptomycin, sulfonamide, trimethoprim/ sulfamethoxazole, tetracyclin, trimethoprim and chloramphenicol, but strains from retail pork were all susceptible to these antimicrobial agents. This finding suggested that pork is probably not a major source of Y. enterocolitica that cause human infections in Switzerland (Baumgartner et al., 2007).

3. BIOTYPING AND SEROTYPING

Different biotyping schemes are proposed for the Y. enterocolitica strains (Table 4) (Stern and Pierson, 1979; Swaminathan et al., 1982).

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The commonly used biotyping scheme is adopted from Wauters (Table 5) (FDA).

Table 5. Biotype scheme(a) for Y. enterocolitica Reaction for biotypes(b)

Biochemical test 1A 1B 2 3 4 5 6 Lipase + + - - - - - Esculin/salicin (24 h) +/- - - - - - - Indole + + (+) - - - - Xylose + + + + - V + Trehalose + + + + + - + Pyrazinamidase + - - - - - + β -D-Glucosidase + - - - - - - Voges-Proskauer + + + +/-(c) + (+) -

a Based on Wauters. b ( ) = Delayed reaction; V = variable reactions. c Biotype of serotype O:3 found in Japan.

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Y. enterocolitica is represented by six biovars viz. 1A, 1B and 2-5. Some biovar 1A strains, despite lacking virulence plasmid (pYV) and chromosomal virulence genes, have been reported to cause symptoms similar to that produced by isolates belonging to known pathogenic biovars.

Y. enterocolitica possess lipopolysaccharide O-antigens similar to other Gram negative bacteria. Specific antigens are found in strains of this species. A total of 34 O factors and 19 H factors were identified. The strains of Y. enterocolitica are grouped into 11 serotypes, mainly based on somatic antigens, and H antigens do not appear to be important in typing of strains. Serotype 5 is divided into two subgroups (Table 6) (Swaminathan et al., 1982). A revision of the original Wauters et al. serotyping scheme for Y. enterocolitica was proposed by Aleksic and Bockemuhl, with the exlusion of those O and H antigens which are not associated with the typical Y. enterocolitica, and contained 18 serogroups containing 20 O factors (Aleksi:c and Bockem:uhl, 1984).

Y. enterocolitica serogroups O:1,2a,3, O:3, O:5,27, O:8, and O:9 have been

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commonly associated with disease. Throughout most of the world (Europe, Japan, some part of Canada), the disease-causing O:3 strains predominate, along with the O:9 serotype strains. In the U.S., the O:8 serotype is most frequently incriminated, with the O:5,27 and O:6,30 strains less frequently seen. The O:3 and O:9 strains are rarely observed within the U.S. The O:8 strains are seldom found outside the U.S. and in western Canada (Schiemann et al., 1981; Stern, 1982). Bissett et al. studied over 300 strains of Yersinia spp. excluding Y. pestis and Y. pseudotuberculosis in U.S. and found that O:3 predominated increasing dramatically during 1984 to 1989 (Fig. 1). Of the remaining Y. enterocolitica isolates, over 40% were identified as belonging to serogroups generally considered to be nonpathogenic (Bissett et al., 1990).

4. PHAGE TYPING Twenty four bacteriophages were isolated from raw sewage and chosen as being the most useful for differentiating strains within the four Yersinia species (Y. enterocolitica, Y. kristensenii, Y. intermedia, and Y. frederiksenii). This set of phages typed 92% of Y. enterocolitica, 100% of Y. kristensenii strains, 97% of Y. frederiksenii strains, and 90% of Y. intermedia strains (Table 7) (Baker and Farmer, III, 1982).

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Methodology Phage Typing Bacterial lawns are prepared, and the phages are dropped (104 PFU/drop) onto the lawns with the applicator, 60 drops per plate (Fig. 2, 3). After the drops have dried for about 15 min at room temperature, the plates are incubated overnight and examined for lysis.

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5. GROWTH AND SURVIVAL 5.1. Nutritional Requirements

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Y. enterocolitica is not a nutritionally fastidious organism when grown at 28C but some strains may require additional nutritional factors for growth, for example thiamine, alanine, methionine, cysteine, glutamic acid, pantothenic acid, and niacin. Y. enterocolitica usually grows in minimum glucose medium at 28C, but not at 37C (Swaminathan et al., 1982). 5.2. Acidity The pH range for the survival and growth of Y. enterocolitica appears to be 4.6-9.0 (Stern et al., 1980b) with the optimum range being pH 7.0-8.0. When incubated at 4C, strains of Y. enterocolitica grow slowly at pH values of 5.2 and 5.4 and show heavy growth at pH 5.6-7.6 (Swaminathan et al., 1982). 5.3. Temperature The growth range of Y. enterocolitica in nutrient broth is about 1-44 C. Most strains grow at 25-39 C, but not at 43C. Significant growth was obtained in meat extract broth or in sterile milk at 4C, and raw or cooked meat at 7C for several days (Olsvik and Kapperud, 1982; Swaminathan et al., 1982). Toxin production by this pathogen is affected by growth temperature and the composition of culture medium. Toxigenic Y. enterocolitica produced heat-stable enterotoxin in milk at 25C, but not at 4C (Francis et al., 1980). Strains which grew well at 4C in milk did not produce significant amount of toxin to be detected by infant mouse assay (Olsvik and Kapperud, 1982). Most Y. enterocolitica cells will be killed or injured when being stored during frozen storage at -20C. When ground beef inoculated with Y. enterocolitica was stored at -20C for 30 days, approximately 83% of the inoculated cells were destroyed and 24% of the survivors were sublethally injured (Swaminathan et al., 1982). In phosphate buffer, freezing at -18 and -75C for 1 h and followed by ambient thawing resulted in 7 and 42% cell inactivation (killing) and 55 and 83% cell injury, respectively (Table 8) (El-Zawahry and Grecz, 1981).

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Y. enterocolitica could be injured by sublethal heat treatment. When the O:3, O:8, and O:17 cultures were thermally stressed in 0.1 M phosphate buffer, pH 7.0, at 47C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. The injured cell could form colony on brain heart infusion agar, but not on trypticase soy agar plus bile salt (Table 9) (Restaino et al., 1980).

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Y. enterocolitica is not heat resistant bacteria, with D value at 62.8C for 15 enterotoxigenic and 6 non-enterotoxigenic cultures ranged from 0.7 to 17.8 sec in sterile whole milk, the heat-treated cells were counted on tryticase soy agar with yeast extract (Francis et al., 1980), it indicates that it does not survive pasteurization. In another report (Lovett et al., 1982), three raw milk isolates of Y. enterocolitica had D values at 62.8C from 0.24 to 0.96 min in sterile whole milk (Table 10). However, if the initial level of Y. enterocolitica is very high, complete destruction may not occur during pasteurilization (Swaminathan et al., 1982). Sublethal injury of Y. enterocolitica may occur when the cells are treated at 47C for 12-70 min (Swaminathan et al., 1982).

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5.4. Sodium Chloride Y. enterocolitica strains can grow in Brain Heart Infusion broth containing 5% sodium chloride at 3 or 25C (Stern et al., 1980b). It suggests that this pathogen may be resistant to some common methods of food preservation. 5.5. Organic Acids and Salts Trypticase soy broth with 0.1 or 0.2% sorbate adjusted to pH 5.5 with any of the acids was bacteriostatic to Y. enterocolitica, and the organic acids, specifically citric and lactic, potentiate the antimicrobial action of the potassium sorbate (Restaino et al., 1982). 5.6. Radiation Y. enterocolitica were found to be among the most radiation sensitive of foodborne microorganisms with D values in trypticase soy broth in the range 0.7-11.8 krad, and the D value doubled in ground beef (Swaminathan et al., 1982). In phosphate buffer, D value of Y. enterocolitica was 10, 14.3, and 24

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krad when irradiation was carried out at 0-2, -18, and -75C, respectively, and 32, 42 and 54%, respectively, of cells were injured (inability to form colonies in agar containing 2.5% NaCl) (Fig. 4, Table 11)(El-Zawahry and Grecz, 1981). Under low dose (60 krad), Y. enterocolitica were sublethally injured (Swaminathan et al., 1982).

Y. enterocolitica is more sensitive to UV than many of the pathogens associated with waterborne disease outbreaks. The UV dose required for a 3-log

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reduction (99.9% inactivation) of Y. enterocolitica O:3 was 2.7 mWs/cm2. No association was found between the sensitivity of Y. enterocolitica to UV and the presence of a 40- to 50-Mdal virulence plasmid (Butler et al., 1987). 5.7. Competitive Microorganisms When competitive microorganisms (Micrococcus varians, Alcaligenes faecalis, Achromobacter pestifer, and Bacillus cereus) and Y. enterocolitica were added to pasteurized milk and held at 3C, these common spoilage competitive organisms were isolated, while Y. enterocolitica was not present among the colonies randomly picked (Stern et al., 1980c). It shows that Y. enterocolitica is a poor competitor. 5.8. Controlled atmosphere The influence on Y. enterocolitica counts of a gradual increase of carbon dioxide concentrations (percentage by volume in air) during packaging and storage of ground pork meat artificially contaminated with this pathogen was evaluated. Ground meat was packaged under customary conditions using modified atmospheres with various carbon dioxide percentages (0, 30, 50, 70, and 100% CO2 by volume; for atmospheres of less than 100% CO2, the rest of the gas was O2). The packs were stored at 2C for 12 days. Y. enterocolitica counts were not significantly different (P > or = 0.05) in the ground pork packaged under the various CO2-enriched atmospheres. The growth of Y. enterocolitica was nearly entirely inhibited in all tested modified atmospheres containing the protective CO2. However, in ground pork packaged with 100% oxygen, there was a significant decrease (P < or = 0.05) for Y. enterocolitica from 4.30 log CFU/g (day 0) to 3.09 log CFU/g at the end of the storage time (day 12). The decrease was presumably due to the marked increase in aerobic plate count seen only in those packages stored under 100% O2. Packaging with high CO2 concentrations had significant inhibitory effect (P < or = 0.05) on the growth of mesophilic aerobic bacteria (Strotmann et al., 2008). 5.9. Essential oils

Experiments were conducted to determine the effectiveness of oregano and nutmeg essential oils (Eos) on the growth and survival of Y. enterocolitica and Listeria monocytogenes in broth culture and in Iranian barbecued chicken.

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Ready-to-cook Iranian barbecued chicken was prepared according to the common practice with 1, 2, and 3 microl/g of oregano and nutmeg EOs. The test and control (without EOs) samples were inoculated with Y. enterocolitica and L. monocytogenes to a final concentration of 6 to 7 log CFU/g and stored at 3, 8, and 20C. Microorganisms were counted just before and at 24, 48, and 72 h after storage. In the broth culture system, the nutmeg EO had a greater effect on L. monocytogenes (MIC = 0.20 nicrol/ml) than did the oregano EO (MIC = 0.26 microl/ml). However, the oregano EO had a greater effect on Y. enterocolitica (MIC = 0.16 microl/ml) than did the nutmeg EO (MIC = 0.25 microl/ml). In ready-to-cook Iranian barbecued chicken, the log CFU per gram of both bacteria after up to 72 h of incubation was not decreased significantly by various combinations of oregano and nutmeg EOs (1, 2, and 3 microl/g) and storage temperatures (3, 8, and 20C) when compared with control samples (without EOs). Although examination of spices in culture media can yield accurate microbiological data, without complementary tests in foods these data are of limited value for assessing food safety (Firouzi et al., 2007). 5.10. Physiology Y. enterocolitica tolerates osmotic stress by intracellular accumulation of osmolytes, such as betaine. The proP gene and proU operon of Y. enterocolitica were sequenced, and single (ProP- ProU+ and ProP+ ProU-) and double (ProP- ProU-) mutants were generated. Upon exposure to osmotic or chill stress, the single and double mutants demonstrated a reduction in betaine uptake compared to that in the wild type (Fig. 5), suggesting that proP and proU play a role in betaine uptake during osmotic and chill stress responses of Y. enterocolitica (Annamalai and Venkitanarayanan, 2009).

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6. OCCURRENCE IN FOODS AND ENVIRONMENT 6.1. Animals The evidence is not yet complete as to whether humans serve as reservoirs of Y. enterocolitica. It is isolated from low percentage of asymptomatic humans. However, it appears that the animal kingdom is a significant reservoir. Some members of the animal kingdom harbor unique serotypes of Y. enterocolitica which have not been implicated in human infections. The swine has been implicated as a major reservoir of Y. enterocolitica seotypes involved in human infections although a definite connection between the isolation of Y. enterocolitica from the swine and human illness remains to be

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established. The incidence of Y. enterocolitica in swine varies not only from country to country but also within a country. The rate of isolation of Y. enterocolitica from tonsils and tongues of pigs is generally greater than the rate of isolation from cecal or fecal materials. Y. enterocolitica serotype O:3 has been almost exclusively isolated from swine in some European countries, like Denmark, Belgium and Sweden. Some investigators concluded that O:3 strain is a normal inhabitant of the oral cavity of swine, and also involved in human infection. Examination of the throat flora from swine in Ontario for Y. enterocolitica found the incidence of serotype O:3 to vary from 20% for tonsils to 50% for throat swabs and 55% for tongues. In contrast, there were no isolations of serotype O:3 from throat swabs taken from swine in the western provinces of Canada. This incidence of serotype O:3 in swine correlates well with the human incidence of the same serotype which is 81% for all human isolations of Y. enterocolitica in the eastern provinces and 4% in the western provinces of Canada. The opposite relationship is true for serotype O:5,27. Majority of O:3, O:5,27 were positive for autoagglutination, a test which has been associated with virulence. The results suggest that swine are an important source of human infections with both O:3 and O5,27 (Table 12) (Schiemann and Fleming, 1981).

In Guangxi, mainland China, Y. enterocolitica were isolated from 48.4% of the swine with diarrhea, most of the isolates were O:3 with two isolates

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belonged to serotype O:9 (Zheng, 1987). These two serotypes are considered to be pathogenic for humans.

In another study in China, Y. enterocolitica (1,295 strains) was isolated from diarrhea patients, livestock, poultry, wild animals, insect vectors, food, and the environment. They were studied for epidemiology distribution using bacterial biochemical metabolism tests, their virulence genes, and pulsed-field gel electrophoresis (PFGE) sub-typing. The data showed that 416 of the 1,295 strains were pathogenic, where the pathogenic Chinese isolates were of serotypes O:3 and O:9. These two serotypes were found in livestock and poultry, with swine serving as the major reservoir. The geographic distribution of pathogenic isolates was significantly different, where most of the strains were isolated from the cold northern areas, whereas some serotype O:3 strains were recovered from the warm southern areas. By the analysis of the data of the Ningxia Hui Autonomous Region, the phenomenon of 'concentric circle distribution' was found around animal reservoirs and human habitation. The clustering of PFGE showed that the patterns of the pathogenic strains isolated from diarrhea patients were identical compared to those from the animals in the same area, thus, suggesting that the human infection originated from the animals (Wang et al., 2009).

In many years of surveillance in China for Y. enterocolitica, no pathogenic

O:8 strains have been found where the isolated O:8 serotypes lacked the major virulence genes and in contrast to O:3 and O:9 strains, none of the O:8 isolates were from humans. These O:8 isolates lack ail, ystA, yadA and virF genes but possess the ystB gene and all belong to Biotype 1A. These O:8 strains did not kill mice and could protect immunized mice against challenge with a pathogenic O:8 strain. Compared to the Chinese pathogenic O:3 and O:9 strains which have similar pulsed-field gel electrophoresis patterns, the 39 Chinese O:8 animal and food isolates were different from the pathogenic O:8 reference strains. This suggests the O:8 strains lacking virulence determinants may not disseminate rapidly in humans and are maintained in animal reservoirs; and therefore exhibit higher variance and divergence from the virulent type (Wang et al., 2008). Sixteen different isolates of Y. enterocolitica were recovered from porcine tongues, including six O:8, four O:6,30, two O:3, and one each of O:13,7, O:18, and O:46 (Doyle et al., 1981). All the serotype O:8 isolates were virulent to mice, causing the death of adults after oral challenge (Doyle et al., 1981).

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In a cross-sectional study, individual pigs on eight swine operations were

sampled for the presence of Y. enterocolitica. On each farm, both feces and oral-pharyngeal swabs were collected from pigs in five different production phases: gestating, farrowing, suckling, nursery, and finishing. A pig was considered positive if either sample tested positive. Of the 2,349 pigs sampled, 120 (5.1%) tested positive, and of those, 51 were ail positive (42.5% of Y. enterocolitica isolates). On all farms, there was a trend of increasing prevalence as pigs mature. Less than 1% of suckling piglets tested positive for Y. enterocolitica. Only 1.4% (44.4% of which were ail positive) of nursery pigs tested positive, but 10.7% (48.1% of which were ail positive) of finishing pigs harbored Y. enterocolitica. Interestingly, gestating sows had the second highest prevalence of Y. enterocolitica at 9.1% (26.7% of which were ail positive), yet Y. enterocolitica was never detected from the farrowing sows (Bowman et al., 2007). Dogs have also been incriminated as potential reservoirs. Isolation of Y. enterocolitica from small rodents, cows, horses, sheep, monkeys, deer and snails have also been reported. Y. enterocolitica or related species were isolated from 50% of cows in Scotland, and the isolates varied in serotypes (Davey et al., 1983). Y. enterocolitica were isolated from wild animals (Kaneko and Hashimoto, 1981; Kato et al., 1985), e.g. from 16 of 495 small wild animals (mainly mice) and from 1 of 38 foxes (Kaneko and Hashimoto, 1981). The Y. enterocolitica isolates were O:6, O:5A, O:4, and O:9. 6.2. Water and Soil Y. enterocolitica has been isolated from water by a number of investigators and note that water may be a reservoir for this pathogen. In general, isolates of Y. enterocolitica from water differ from those implicated in human disease. One investigator reported that a strain isolated from well water was capable of survival and proliferation in sterile water at 4, 25 or 37C. However, other investigators have reported that Y. enterocolitica does not survive or multiply in water at low temperatures in the absence of organic matter. Chao et al. reported that population of Y. enterocolitica decreased rapidly in river water and it is chiefly regulated by predators and toxin producers (Chao et al., 1988).

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When it was introduced into soils and air dried slowly, only 0.1% of the original population added still remained viable by day 10 (Chao et al., 1988). 6.3. Isolation from Foods Y. enterocolitica has been isolated from milk and milk products, egg products, raw meats (beef, pork, lamb) and poultry, vegetables and miscellaneous prepared food products. However, most of these isolates are atypical strains and usually non-serotypable. Most of these isolates lack any pathological significance in humans (Table 13) (Prpic et al., 1985). Therefore, isolation of Y. enterocolitica from a food is insufficient to ascribe any pathogenic significance to such isolation (Swaminathan et al., 1982).

From Milk and Milk Products Y. enterocolitica has been isolated from raw milk in many countries, like Australia, Canada, Czedhoslovakia, and USA. There were also a few reports on the isolation of this pathogen from pasteurized milk. It may be due to the malfunction in the pasteurization process leading to inadequate treatment or post-process contamination, or it may be due to the contamination of heat-resistant strains of Y. enterocolitica. However, heat-resistant strains have not been reported.

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Stern reported that Y. enterocolitica could grow in whole milk at 3C (Stern, 1982). Also the reduction of psychrotrophic bacteria in milk after pasteurization would enable a poor competitor and opportunistic pathogen such as Y. enterocolitica to grow better in pasteurized than in raw milk. So, the presence of this pathogen in pasteurized milk should be a cause for concern. Y. enterocolitica was isolated from 9.2% of cheese curd samples in Canada (Swaminathan et al., 1982). From Meat and Poultry Products Y. enterocolitica are commonly detected in meat and poultry products (Table 14). The level of this pathogen was found consistently in high numbers on vacuum-packed meats with a pH above 6 held at low temperature (Swaminathan et al., 1982). Growth of this pathogen is enhanced in cooked meats or at low temperature whereas competitive microorganisms are inactivated. Table 14. Incidence of Y. enterocolitica in meats

Food item Incidence rate % Investigators Pork 34.5 Leistner et al. 1975 49 Schiemann 1980 Swine carcass

tongue & trim 18.6 (Harmon et al., 1984) Swine throat 9 (Stern, 1981) Swine throat 53 Wauters & Janssens 1976

Pig tonsils 29.6 Hanna et al 1980 Pork tongues 65 Schiemann 1980 Pork,ground 60 Schiemann 1980

Pork, processed 7 Schiemann 1980 Chicken 28.9 Leistner et al. 1975 Beef 10.8 Leistner et al. 1975 14.6 Inoue & Kurose 1975

From (Swaminathan et al., 1982) and (Stern, 1981).

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Prevalence of pathogenic Y. enterocolitica in different sources in Bavaria is presented. The highest isolation rate of pathogenic Y. enterocolitica (67%) was found in tonsils of slaughter pigs. No pathogenic strains were isolated from cattle, sheep, turkey, and horses. ail-Positive Y. enterocolitica was detected in dogs (5%), cats (3%), and rodents (3%) by real-time PCR. Pathogenic Y. enterocolitica was isolated only from raw pork, especially from edible offal (51%). All pathogenic Y. enterocolitica isolates from nonhuman sources were belonging to bioserotype 4/O:3. All Y. enterocolitica 4/O:3 strains were susceptible to most of the tested antibacterial agents (Bucher et al., 2008). From Other Foods Strains of Y. enterocolitica have been isolated from oysters, mussels, shrimp, blue crab, fish, chicken salad and stewed mushrooms, and cabbage, celery and carrots (Swaminathan et al., 1982). 7. ISOLATION AND IDENTIFICATION At present, there are no completely reliable methods for recovering pathogenic Y. enterocolitica from foods and environmental samples. Procedures used may be different from country to country. 7.1. Selective Enrichments The food sample is usually blended for 2 min, or a surface swab is shaken, in a phosphate buffered saline (PBS) solution. Aliquot is usually cold enriched at 4C for up to 4 weeks. Enrichment in phosphate buffered saline at 4C was an ineffective method for the recovery of Y. enterocolitica from food sample and it takes a long time. Cold enrichment in PBS is useful for clinical samples for the isolation of O:3 strains. A number of selective enrichment media have been formulated, but each medium may not suitable for all the isolates of Y. enterocolitica. Addition of 1% sorbitol and 0.15% bile salts No.3 to PBS resulted in the enhanced recovery of Y.

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enterocolitica from meats, and addition of hemoglobin (0.1%) and potassium oxalate (0.2%) to the sorbitol-bile salts-PBS medium has been recommended for vegetable products (Swaminathan et al., 1982). A modified Rappaport's medium, termed magnesium chloride-malachite green-carbenicillin (MMC) medium was reported to increase the number of isolations of Y. enterocolitica O:3 and O:9 from clinical specimens (incubated at 22C). However, it was shown to be inhibitory to many isolates. Two modified selenite media, prepared by supplementing PBS (pH 7.5) with malachite green (20 μg/ml), carbenicillin (10 μg/ml) and sodium selenite (150 mg or 250 mg/100 ml) were reported to be effective in recoverying Y. enterocolitica from meat samples when incubated at 22C for 2-3 days (Swaminathan et al., 1982). A two-step enrichment procedure for recovery of Y. enterocolitica was developed (Schiemann, 1982). The first step is a preenrichment at 4C or 10C in yeast extract-rose Bengal broth for 3 days. The second step is a selective enrichment in bile-oxalate-sorbose broth with/without NaCl incubated at 22C for 3 days. This procedure showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods as compared to modified Rappaport broth and cold enrichment (Table 15) (Schiemann, 1982). The bile-oxalate-sorbose broth was modified by adding peptone (5 g/L) for water (fresh and marine) and other nonfood samples. Sample enriched in this modified medium at 22C for 48 h resulted in higher recovery (Weagant and Kaysner, 1983).

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Walker and Gilmour reported that pre-enrichment in trypticase soy broth for 24 h at 22C followed by selective enrichment in bile-oxalate-sorbose medium for 5 days at 22C allowed highest yield of Y. enterocolitica (Walker and Gilmour, 1986). Y. enterocolitica tolerates short exposures to weak alkali better than other members of the Enterobacteriaceae (Schiemann, 1983). So, the cold enriched cultures could be mixed with a solution of 0.5% potassium hydroxide in 0.5% sodium chloride before streaking for isolation on selective agars (Swaminathan et al., 1982). The alkalotolerance of Y. enterocolitica is affected by the phase of growth, temperature and medium of treatment. The addition of peptones to potassium hydroxide provided a protective effect. Log-phase cells were less alkalotolerant than cells in the stationary phase of growth. The rate of cell destruction was five times greater at 30C than at 20C(Schiemann, 1983). A new enrichment broth was derived from the modified Rappaport base by supplementing with Irgasan, ticarcillin, and potassium chlorate (ITC). Sample was inoculated into this medium and incubated at 24C for 2-3 days, and this medium was shown to be good for Y. enterocolitica O:3 (Wauters et al., 1988).

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7.2. Selective Differential Plating Y. enterocolitica can grow on many selective media commonly used for the isolation of enteric pathogens. Serotypes O:3, O:8 and O:9 grow well and form colonies on bismuth sulphite, endo, eosin methylene blue, MacConkey, desoxycholate-citrate, Salmonella-Shigella media, etc. However, some serotypes of Y. enterocolitica may not grow on all of these media, or they grow slowly and form tiny colonies and can be easily overlooked. A number of selective media have been formulated and compared: MT agar: a modified MacConkey agar containing Tween 80 and calcium chloride. Colony is about 2 mm, flat, wrinkled, surrounded by a sheen. DST agar: a modified DNAase agar and contains Tween 80, sorbitol, sodium lauryl sulphate and calcium chloride. Colony is translucent, colorless or pink with little or no lipolytic reaction or nuclease reaction. Modified Salmonella-Shigella agar with 2% deoxycholate. Y medium: contains sodium oxalate, sodium desoxycholate and bile salts and with peptone and casein hydrolysate. CIN agar: contains the selective agents celsulodin (4 or 15 mg/L), irgasan (4 mg/L), and novobiocin (2.5 mg/L). It is a widely accepted medium. CAL medium: contains cellobiose, arginine and lysine. Y. enterocolitica ferments cellobiose to produce acid and the resultant change in pH and the presence of neutral red in the medium impart a burgundy or red color to the colony (Swaminathan et al., 1982). DYS medium: like Y medium. It contains bile salts, sodium deoxycholate, sodium chloride, arabinose, arginine, lysine, neutral red in addition to peptone and casein hydrolysate. It was shown to be better than Y medium, MacConkey, Salmonella-Shigella medium (Agbonlahor et al., 1982). Nine selective media were compared (Walker and Gilmour, 1986). CIN agar, incubated for 48 h at 25C, allowed a high recovery of all the Yersinia spp. and

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was the most selective medium. Two different kinds of CIN agar, containing 4 mg (Difco) or 15 mg (Oxoid) of cefsulodin per liter, are commercially available. Seven selective differential plating media were also evaluated by Harmon et al. (Harmon et al., 1983). CIN agar was the most effective medium for the recovery of Y. enterocolitica. However, Y. enterocolitica O:12,25 was slightly inhibited on CIN agar. As evaluated by Head et al., CIN is the most effective medium for the recovery of Y. enterocolitica (Table 16) (Head et al., 1982).

However, CIN media inhibited the growth of Y. pseudotuberculosis and Y. enterocolitica biotype 3B serotype O:3 but not the growth of the other Yersinia organisms. Since the Y. enterocolitica 3B/O3 strains were resistant to cefulodin, Irgasan, and novobiocin at the concentrations used in these CIN media, it suggests that growth inhibition of Y. enterocolitica 3B/O3 is related to a component of the CIN base (Fukushima and Gomyoda, 1986). Direct KOH treatment of meat samples could be a valuable rapid method for direct isolation of Yersinia from meat contaminated with more than 100 cells per g. In another study, ground pork with artificially contaminated Y. enterocolitica was homogenized in 0.85% NaCl, treated/not treated with 0.72% KOH in 0.5% NaCl, and plated on CIN or MacConkey agar. The sample was also cold enriched in phosphate buffer for 1-14 days. All Yersinia strains were recovered from the pork samples contaminated with more than 100 cells per g after direct KOH treatment, without enrichment. However, virulent Yersinia isolates in pork

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samples contaminated with less than 104 cells per g were never recovered by using KOH postenrichment treatment (Fukushima, 1985).

Another selective agar medium for direct isolation of virulent Y. enterocolitica was formulated (Fukushima, 1985). This VYE agar consists of sodium deoxycholate, mannitol, esculin, ferric citrate, sodium chloride, neutral red, crystal violet, irgasan, cefsulodin, oleandomycin, and josamycin in addition to peptone and agar. This VYE agar proveded a quantitative recovery of 51 different strains of virulent Y. enterocolitica at 32C after incubation for 24 h. Virulent Y. enterocolitica formed red colonies is easily differentiated from most environmental Yersinia spp. and other gran-negative bacteria, which form dark colonies with dark peripheral zone as a result of esculin hydrolysis (Fukushima, 1987).

The selective agars most commonly used to isolate Y. enterocolitica in clinical, food and environmental samples, cefsulodin–irgasan–novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial species. A new agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica was developed. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes (Weagant, 2008).

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7.3. Identification For selective plates, colonies with characteristic appearances are screened through the triple sugar iron (TSI) or Kligler iron agar (KIA) slant. Presumptive Y. enterocolitica should present an acid-slant, acid-butt, no-gas, and no-H2S TSI test. KIA should yield an alkaline slant and acid butt with no gas or H2S production. Then the presumptive siolate should yield a positive urease test and a negative phenylalanine-deaminase test. Confirmation of a Y. enterocolitica isolate can be made through the use of a rapid bacterial identification kit such as the API 20E. A medium, lysine-arginine-iron agar (LAIA), was developed for the presumptive identification of Y. enterocolitica (Weagant, 1983). In this medium, lysine-iron agar is modified by the addition of 1% L-arginine, and allows for the testing of five biochemical characteristics in a single tube medium. Typical reactions of Yersinia spp. on LAIA are alkaline slant (purple), acid butt (yellow), no H2S (darkening of butt), or gas formation (no gas). PathoTec Test strips for ornithine decarboxylase (OD), Voges-Proskauer (VP), and urease test (UR) (General Diagnostics, Morris Plains, NJ) could be used for rapid confirming the identification Y. enterocolitica isolated from food enrichments on CIN agar (Devenish and Schiemann, 1981). Test strips are put into bacterial suspensions in phosphate-buffered saline and incubated at 22 or 35C for 6-24 h. The Y. enterocolitica are VP (22C), OD (22C), and UR (35C) positive. The most convenient method for accurate testing was the growth of the organism on blood agar at 35C for 22 h and incubation of the three PathoTec test strips at 22C for 24 h (Devenish and Schiemann, 1981). Also, virulence potential should be tested. There are a number of tests for virulence, e.g. enterotoxin, invasiveness, plasmid analysis, calcium dependency, hydrophobicity, autoagglutination, production of V and W antigens and other outer membrane proteins etc (Prpic et al., 1985). The most simple assay is autoagglutination. Two tubes of tissue culture medium are inoculated, and one is incubated at 23C and the second at 35C. If after overnight incubation the content of the tube at 35C has autoagglutinated while the tube held at 23C is turbid, the strain shows potential for virulence to humans (Stern, 1982). Discussion on the virulence factors is given in the following section.

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An Enzyme Immunoassay (EIA) for the detection of pathogenic Y. enterocolitica and Y. pseudotuberculosis was developed(Kaneko and Maruyama, 1989). Antiserum against plasmid-encoded proteins of serotype O:3 was prepared by immuning rabbit with formalinized-fixied cells which were grown at 37C for 24 h and the antiserum was absorbed with cells of plasmid-cured Y. enterocolitica cells grown at 37C. The antisera developed reacted with proteins released from O:3, O:5,27, O;8, and O:9 and strains of Y. pseudotuberculosis. Plasmid-cured Yersinia strains did not react in this EIA system (Kaneko and Maruyama, 1989). An objective Pyrolysis gas-liquid chromatography (PGLC) was used to differentiate between HeLa cell-invasive and noninvasive strains of Y. enterocolitica and between Sereny-positive and -negative strains, and the groups were separated by stepwise linear discriminate analysis (SLDA) and the results showed good correlation in prediction of the HeLa cell invasivity test (Stern et al., 1980a). However, it is not simple in a microbiology laboratory. 7.4. Membrane Filter Method For determining the low level of Y. enterocolitica in water sample, a membrane filter method is developed by Bartley et al. (Fig. 6) (Bartley et al., 1982). Bacteria collected on membrane are placed on Mye recovery broth containing deoxycholate as the selective agents and incubated at 25C for 48 h. The membrane is then placed on mYE lysine-arginine agar and incubated anaerobically at 35C for one h. The vivid yellow to yellow orange colonies (sorb+ lys- arg-) are marked. Then the membrane is placed on mYE urease broth, incubated at 25C for 5-10 min, and the deep blue marked colonies (urease+) are picked as presumptive Y. enterocolitica. The presumptive identification of Y. enterocolitica was accomplished in 50 h, and the rate of identity confirmation of typical colonies was 88%. The mean recovery rate of 15 strains from phosphate buffer suspensions was 91%, and quantitative recovery was demonstrated for low populations of organism in both laboratory-prepared and naturally occurring mixed cultures.

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7.5. DNA Hybridization Method The DNA colony hybridization method has two useful features: neither pure cultures nor physiologically stressful enrichments are necessary. The virulence determinants of Y. enterocolitica show a correlation with the presence of plasmids of 42 to 48 Mdal. So, virulent Y. enterocolitica can be detected by colony hybridization using a probe derived from such virulent plasmid. Hill et al. developed such DNA hybridization method for Y. enterocolitica (Hill et al., 1983; Jagow and Hill, 1986). The virulent plasmid pYV8081, 44 Mdal, was purified from Y. enterocolitica 8081 and digested with the BamHI and three fragments with molecular size 3.8, 4.3, and 5.0 kb were eluted, pooled and labeled with 32P and used as probe. The use of whole plasmid as a probe is not entirely satisfactory, possibly because of some genetic homology shown by pYV8081 and the host chromosomal DNA. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests (Hill et al., 1983). Later, Jagow and Hill tested the efficiency of enumeration of this colony hybridization method. Testing 11 artificially contaminated foods, the colonies observed ranged from 66 to 100% (average, 86%) and was influenced by the

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number of indigenous bacteria. The use of nitrocellulose filters and agar medium had little effect on efficiency of enumeration. For high indigenous bacteria (over 107 cfu/g), exposure of the sample for a few seconds to a 1:25 dilution of 0.5% KOH-0.5% NaCl before plating on nitrocellulose filters enhanced the selection of Y. enterocolitica (Jagow and Hill, 1986). 7.6. Commercial Rapid Detection Kit In 1982, the API Z system (France) was introduced as a screen for Salmonella and Shigella spp. The API Z system was subsequently renamed as Rapid SYS and extended the screening claim to include Y. enterocolitica. However, a evaluation test showed that this Rapid SYS system cannot be used in US for the detection of Y. enterocolitica. All the biotype 1 isolates produce lipase and could be potentially eliminated by the Rapid SYS system as nonpathogens. Pathogenic strains of Y. enterocolitica biotype 1 are rarely encountered in Europe, but they have been encountered frequently in the US (Mele et al., 1987). 7.7. DNA microarray

Four different food matrices (alfalfa, cilantro, mamey sapote, and mung bean) were contaminated with three different dilutions 106, 104, and 103 CFU/g of Y. enterocolitica. DNA was isolated from each food mix and used in chromosomal amplifications. The amplified DNA was used as templates in single PCR reactions of the four genes (virF, ail, yst, and blaA) followed by mixing the four reactions for one PCR primer extension reaction. The presence and the limit of detection of four genes in four food matrices were established by microarray hybridization. Data revealed the diversity of signal intensities. Neither the microarray chip hybridization nor the single PCR amplification could detect virF's presence located on a plasmid. Ail was detected in 103 CFU/g, whereas blaA and yst were detected from 105 to 106 CFU/g in all food matrices (Fig. 7, Table 17). Therefore, the ail gene could be the gene of choice in identifying Y. enterocolitica in alfalfa, cilantro, mamey, and mung bean. Other genes--blaA, yst, virF--exhibited wide variability in hybridization signals, highlighting the need of a better DNA purification step prior to DNA microarray hybridization (Siddique et al., 2009).

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7.8. Real-time PCR A TaqMan probe-based real-time PCR method for the detection of Y. enterocolitica was developed. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained (Table 18). The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene (Lambertz et al., 2008).

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8. PATHOGENICITY AND VIRULENCE FACTORS 8.1. Clinical Manifestations Gastroenteritis in humans is the major expression of the pathogenic Y. enterocolitica. Frequently reported sympotoms include: diarrhea, fever, vomiting, abdominal pain, nausea and headaches. Although relatively rare, fatality due to Y. enterocolitica does occur, but the patient's recovery is generally complete within 1 to 2 days (Stern, 1982; Stern and Pierson, 1979). In a human volunteer study, one individual took an oral dose of 3.5x109 cells of Y. enterocolitica, and the results were similar to those reported in other food and water-borne bacterial infections; the organism caused enterocolitis, darrhea and a fever. The acute symptoms ceased after 2 days of discomfort. Tenderness in the stomach and liver region lasted 4 weeks (Stern and Pierson, 1979). The organism must proliferate under favorable growth conditions and must be present in sufficient numbers to cause infectivity. Y. enterocolitica has been implicated in certain acute human diseases, such as enteritis, pseudoappendecitis, mesenteric lymphadenitis, terminal ileitis, and arthritis (Stern and Pierson, 1979). Y. enterocolitica accounted for 3.8% of the appendectomies performed becaused of the similarities in Y. enterocolitica enteritis and appendicitis. Indeed appendectomies were performed on 16 children after they contracted Y. enterocolitica enteritis in the New York chocolate milk outbreak (Stern and Pierson, 1979; Swaminathan et al., 1982). Y. enterocolitica has been isolated from the cerebrospinal fluid, blood, urine, and eyes of infected patients (Stern and Pierson, 1979). Y. enterocolitica has been occasionally occurring in donor blood from healthy donors or donors with a diarrhea history, such contaminated blood sometimes caused Yersinia bacteremia and death of the recipients (Table 19) (Jacobs et al., 1989).

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Tests on the pathogenicity of the species for mice and other laboratory animals have been studied. Intraperitoneal administration of Y. enterocolitica O:3 at a level of 3x109 cells caused the death of guinea pigs in 24-48 h while subcutaneous administration of the same organism at the same level had no observable effect on guinea pigs. Intravenous administration of Y. enterocolitica O:3 to rabbits caused death in 5-11 days. Marked inflammation was noted in the liver of the animals and a hemorrhagic-necrotic inflammation with ulceration was observed in the appendix. After intravenous administration of Y. enterocolitica O:8, a systemic, pyogenic infection involving the spleen, the liver and the lungs was observed in mice. The initial site of infection in mice after intragastric challenge with Y. enterocolitica O:8 was reported to be the Peyer's patches of the distal ileum. The primary lesions were found to appear as abscesses in the Peyer's patches and as caseous necrosis of the mesenteric lymph node (Swaminathan et al., 1982). Oral administration of Vwa plasmid (70-kDa) positive strains of Y. enterocolitica to mice resulted in systemic infection while the Vwa-negative strains did not (Bakour et al., 1985). After feeding Y. enterocolitica suspension for one day, these pathogenic bacteria were found in the spleen of the test animal next day.

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The course of bacterial penetration and spreading was determined by immunohistochemical staining and electron microscopy of mice after oral administration of Y. enterocolitica (Hanski et al., 1989). The bacteria entered Peyer's patches, which were about 1,000 times more heavily colonized than the surrounding epithelium of a comparable surface area. The number of bacteria in a single Peyer's patch was comparable to that in the rest of the ileal mucosa, nevertheless, the epithelial surface of the ileum is estimated to be 750 to 12,000 times greater than the surface of a single Peyer's patch (Fig. 8). The bacteria proliferated in the follicles, from which they spread into the lamina propria of the villi. At either site most of the bacteria multiplied extracellularly (Fig. 9), with only a small percentage observed to be present within the pahgocytes. The bacteria did not appear to be able to pass the intact basement membrane; hence, the integrity of the basement membrane is likely to play a role in derermining the route of entry and limit of spread of Y. enterocolitica infection (Hanski et al., 1989).

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Sereny test is commonly conducted to determine pathogenicity of Y. enterocolitica. The bacteria were inoculated into the conjunctival sac of guinea pig and observe keratoconjunctivitis for 7 days. Not all the pathogenic strains showed positive Sereny test (Swaminathan et al., 1982). Y. enterocolitica O:3, O:5B, O:8 and O:9 were found to be highly virulent in mice, rabbits, and monkeys, regardless of the origin of the strains. These strains caused lesions in the spleen, the liver and the intestine of these animals (Swaminathan et al., 1982). Generally no single current assay correlates with virulence in Y. enterocolitica. Among the isolates from human, two strains caused conjunctivitis in guinea pigs, 7 were lethal for mice, 54 invaded HEp-2 cells, 18 produced a heat-stable enterotoxin, 9 were calcium dependent, and 20 autoagglutinatied (Kay et al., 1983).

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Animals can be protected against Y. enterocolitica by pre-administration of Y. enterocolitica or Y. pseudotuberculosis. Mice given orally either the O:3, O:9 or O:5B of viable Y. enterocolitica showed protection upon subsequent oral challenge with another of these strains. Excretion of serovar O:3 in the feces was inhibited in mice surviving oral challenge with Y. pseudotuberculosis (Uchida et al., 1982). Mice vaccinated orally with heat-killed cells of O:3 of Y. enterocolitica were protected only against fecal excretion of the homologous serovar, whereas Formalin-killed cells provided cross-protection against O:9 and vice versa. Formalin-killed Y. pseudotuberculosis also provided cross-protection against Y. enterocolitica (Kaneko and Hashimoto, 1983). Virulence genes

Clinical isolates of pathogenic Y. enterocolitica in China cultured from the culture method were examined for virulence genes (inv, ail, ystA, ystB, ystC, yadA, virF) by PCR and for the presence of plasmid by four phenotypic tests. The positive rate of virulence genes tested in 160 isolates was inv (100%), ail (94%), ystA (93%), ystB (7.5%), ystC (5%), yadA (89%) and virF (82%) while the phenotypic test included autoagglutination (87%), binding of crystal violet (89%), calcium-dependent growth (74%) and Congo red absorption (78%), respectively. Not all pathogenic Y. enterocolitica necessarily carry all traditional virulence genes in both chromosomes and plasmids to cause illness. Perhaps, some of them, lacking some traditional virulence genes, contain other unknown virulence markers that interact with each other and play an important role in the diverse pathogenesis of pathogenic Y. enterocolitica (Zheng et al., 2008).

Virulence-associated genes viz. ail, virF, inv, myfA, ystA, ystB, ystC, tccC,

hreP, fepA, fepD, fes, ymoA and sat were studied in 81 clinical and nonclinical strains of Y. enterocolitica biovar 1A by PCR amplification (Table 20). All strains lacked ail, virF, ystA and ystC genes. The distribution of other genes with respect to clonal groups revealed that four genes viz. ystB, hreP, myfA and sat were associated exclusively with strains belonging to clonal group. The distribution of virulence-associated genes, however, did not differ significantly between clinical and nonclinical strains (Table 21). In strains of Y. enterocolitica biovar 1A, clonal groups seem to reflect virulence potential better than the source (clinical vs. nonclinical) of isolation (Bhagat and Virdi, 2007).

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8.2. Effects of Sublethal Stresses on Virulence In aquatic environments copper and other elements, even in trace amounts, present potentially important causes of bacterial injury. Extent of injury is usually determined by the numbers of differential CFU on nonselective tryptic lactose yeast extract agar (TLY) and a selective agar (TLY supplemented with 0.1% sodium deoxycholate). A sublethal concentration of copper (0.75 mg/L) caused substantial injury (87 to 95%) of Y. enterocolitica O:8 cells in 72 h at 4C without producing extensive cell death (Fig. 10, 11). Copper-injured cells had a higher LD50 dose in mice than uninjured cells (Table 22). This reduced virulence correlated with more rapid clearance of the injured cells from the blood of mice after intravenous inoculation (Fig. 12). A possibe role of the liver in this process was shown by significant cell accumulation in mouse livers when copper-injured Y. enterocolitica cells were administered, compared with uninjured bacteria. In vitro studies with isolated mouse liver membranes showed higher titers of aggregation with copper injured cells than control cells. The in vitro aggregation reaction and blood clearance activity in vivo were abolished by sugars that are known to interact with a hepatic lectin (Fig. 13). It suggested that copper-induced injury reduces the virulence of Y. enterocolitica and that the liver may be involved in nonimmune rapid clearance of the injured cells, probably by interaction with a hepatic lectin(s) (Singh et al., 1985).

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Both copper and chlorine caused injury of Y. enterocolitica. Injury of the exposed cells was further enhanced in the gastric environment of mice. The low gastric pH caused extensive loss of viability in copper-injured cells, and the lethality in the chlorine-injured cells was less extensive. The virulence of chlorine-injured Y. enterocolitica in orally inoculated mice was similar to that of the control culture (Singh and McFeters, 1987). 8.3. Enterotoxin Production of a heat-stable enterotoxin (ST) by Y. enterocolitica has been demonstrated (Stable at 100C for 20 min and at 120C for 15 min) (Okamoto et al., 1982; Swaminathan et al., 1982; Verma and Misra, 1984), and it is controlled by a chromosomal gene (Robins-Browne et al., 1985). Serotypes O:3, O:8 and O:9 are almost always enterotoxigenic. Production of heat-labile enterotoxin by Y. enterocolitica has not been demonstrated (Swaminathan et al., 1982). ST of Y. enterocolitica is excreted into the culture supernatant of the late-log phase of growth and increased lineally during the stationary phase of

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growth. The ST level becomes maximum at the decline phase of growth, and the ST is not detected in the lysate of bacteria obtained from the decline phase of growth (Okamoto et al., 1982). The heat-stable enterotoxin of Y. enterocolitica was purified by ultrafiltration with an Amicon HIP-10 hollow fiber, ethanol fractionation, protamine sulfate treatment, DEAE-Sephacel and hydroxylapatite column chromatographies, Sephacryl S-200 superfine gel filtration, and Bio-Gel P-10 filtration. The heat stability was also demonstrated (Okamoto et al., 1981). The molecular weight of purified ST was 9,000 by Sephadex G-100 superfine gel filtration. The purified ST was separated by isoelectric focusing into two active fractions, with pI's of 3.29 (ST-1) and 3.00 (ST-2), respectively (Okamoto et al., 1981). The ST produced by Y. enterocolitica was further purified, with molecular weight 97,000 by Sephadex G-75 gel filtration (Okamoto et al., 1982). The purified ST was stable to heating (100C for 20 min, 121C for 20 min) and did not lose its toxicity after treatment with protease, trypsin, lipase, phopholipase C, ribonuclease, deoxyribonuclease, β-glucosidase, and neuraminidase (Okamoto et al., 1981). The further purified ST was heat stable at 100C for 10 min between pH 2.2 and 8.0, but not at pH values greater than 9.0 or in 2N HCl (Okamoto et al., 1982). The biological activity of the purified ST was lost by treatment with 2-mercaptoethanol, suggesting that the ST contained disulfide bridges in the molecule which were required for the development of toxic activity (Okamoto et al., 1982). The crude enterotoxin of Y. enterocolitica is reported to be resistant to treatment at 121C for 30 min or storage at 4C for 7 months, and is stable in the pH range 1-11. Thus, enterotoxin of Y. enterocolitica may survive normal food processing and storage operations and the acid pH of the stomach and it is conceivable that illness may occur as a result of consumption of the preformed enterotoxin (Swaminathan et al., 1982). The minimal effective dose of purified ST was about 110 ng in the suckling mouse assay. Antiserum from guinea pigs immunized with the purified ST

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neutralized the activity of both Y. enterocolitica ST and E. coli ST (Okamoto et al., 1981). The minimal effective dose of the further purified ST was approximately 25 ng in the suckling mouse assay (Okamoto et al., 1982). Enterotoxin of Y. enterocolitica may not be related to the pathogenicity of this pathogen (Schiemann, 1981; Schiemann and Devenish, 1982). A strain of Y. enterocolitica O:3 that consistently produced ST at 22 but not at 37C and another strain of the same serotype which did not produce enterotoxin at 22 were both positive for autoagglutination at 35C. Both strains were infective for HeLa cells and pathogenic to guinea pig and mice. A control strain of O:3 positive for enterotoxin and HeLa cell infectivity but negative for autoagglutination was avirulent (Schiemann, 1981). As shown by Robins-Browne et al., enterotoxin has no role in the pathogenesis of yersiniosis, but there was eveidence that enterotoxin may promote intra-intestinal proliferation of Y. enterocolitica, thus favouring increased shedding of bacteria and encouraging their spread between hosts (Robins-Browne et al., 1985). 8.4. Autoagglutination The autoagglutination is mostly associated with clinical isolates while the hemagglutinin production is not (Kapperud and Lassen, 1983). The autoagglutination of Y. enterocolitica was dependent on the presence of the virulence plasmid and on the active growth of bacteria in tissue culture media at 37C. Synthesis of new virulence plasmid-associated surface factors was essential for autoagglutination (Skurnik et al., 1984). The autoagglutination associated protein is a 240,000 polypeptide, designated P1, and it could be dissociated under strongly reducing conditions into subunits of 52,500 Dal. Immunological related factor also occurred in Y. pseudotuberculosis (Skurnik et al., 1984). The motility of Y. enterocolitica at 22-25C and 35-37C can also be assayed on motility agar media, such as GI medium, motility medium S (Difco), motility test medium, motility indole ornithine medium and SIM medium (BBL) and confirmed by flagella stain or direct wet mount observation (D'Amato and Tomfohrde, 1981).

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8.5. Invasiveness Virulent Y. enterocolitica was reported to be invasive to HeLa and human epithelial (HEp-2) cell systems. Some investigators speculated that the infection of HeLa cells by Y. enterocolitica may be just an indication of the capacity of the bacteria to attach physically to the cultured mannalian cells: ingestion is probably an endocytic process initiated by the Hela cells (Swaminathan et al., 1982). However, LeChevallier et al. showed that invasion was more than simple association of the bacterium with the epithelial cell and involved a specific trigger to stimulate engulfment (LeChevallier et al., 1987). Inhibition of RNA synthesis by rifampin and protein synthesis by antibiotics inhibited the invasiveness but not the attachment of Y. enterocolitica to epithelial cells. Cell membrane from untreated as well as antibiotic (tetracycline and rifampin) treated cells added to the invasion assay blocked the invasiveness of virulent Y. enterocolitica, whereas membranes from chlorinated cells were unable to block invasiveness (Fig. 14, 15). The injury of Y. enterocolitica by chlorine inhibited invasiveness of this pathogen. Chlorine did not change the hydrophobicity or surface charge of injured Y. enterocolitica (LeChevallier et al., 1987).

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Also demonstrated by Schiemann and Nelson that surface protein structure may be important in mediating cell invasion by Y. enterocolitica. Coating of the bacteria with antibody (against formalized bacteria) did not greatly alter adhesion to HeLa cells; however, antibody against formalized bacteria inhibited HeLa cell invasion. Antibody against heat-killed bacteria had no inhibitory activity. Adsorption of the antiserum with lipopolysaccharide removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity (Schiemann and Nelson, 1988). Yersinia species (Y. pseudotuberculosis, Y. enterocolitica) are highly infective for HEp-2 cells but were unable to replicate extensively intracellularly as compare to enteroinvasive E. coli and Salmonella typhimurium (Fig. 16). However, Yersinia cells maintained intracellularly for prolonged periods without damage to the monolayer of cell culture (Small et al., 1987).

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Devenish and Schiemann reported on the development of a roller tube system for quantitative comparisons of in vitro infectivity of HeLa cells by Y. enterocolitica. Non-infective strains of Y. enterocolitica were reported to show a relative infectivity index of 3.0 or less while infective strains yielded a relative infectivity index of 3.7-5.0 by the roller tube technique (Schiemann and Devenish, 1982). Methodology Roller Tube System for Bacterial Invasion Cells of adjusted density are added to plastic tissue culture tubes. Bacterial suspension is added. The prepared tubes are placed on a roller apparatus and incubated at 35C for 30 min. After this infection period, 0.1 ml of gentamicin solution (2.5 mg/ml) is added to each tube, and then the tubes were

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incubated for 1 h. The supernatant is removed by centrifugation. Cells are sonicated within the tubes and then the invaded bacteria are enumerated (Schiemann and Devenish, 1982). The ability to associate with cultured cells is exhibited by all human pathogenic strains, regardless of carriage of the virulence plasmid, whereas most nonclinical isolates are unable to interact (Lassen and Kapperud, 1986). They found that (i) in serotype O:3, resistance to internalization was dependent upon prior growth at 37C and carriage of the virulence plasmid; (ii) in serotype O:9, this property was plasmid dependent but not temperature dependent; (iii) in serotype O:8, it was constitutive (not affected by growth temperature and plasmid). The ability of serotype O:3 to resist internalization was correlated with the expression of plasmid-associated fibrillae on the bacterial surface. No relationship between fibrillation and HEp-2 cell interaction was apparent for serotype O:8 or O:9. Serotype O:8 and O:9, unlike the O:3 strains studied, associated with HEp-2 cells in greater number after cultivation at 22C than after cultivation at 37C. Two chromosomal genes are involved in the invasiveness of Y. enterocolitica. The inv locus allows a uniformly high level of invasion in several tissue culture lines and is homologous to the inv gene of Y. pseudotuberculosis. The second locus, ail, shows more host specificity than inv. In Y. pseudotuberculosis, the inv encodes a surface protein of about 103,000 dal that promotes adherence to and invasion of tissue culture by these bacteria (Isberg et al., 1987; Miller and Falkow, 1988). 8.6. Congo Red and Crystal Violet Binding Binding of Congo Red is associated with virulent Y. enterocolitica containing 40-50 Mdal plasmid (Prpic et al., 1983; Prpic et al., 1985). Bacteria were suspended in buffer containing Congo red and incubated for 12 h, and the decrease of Congo red in the buffer was determined by spectophotometry. It was found that strains of Y. enterocolitica containing virulence plasmid and positive for several virulence assays could bind Congo red, while the plasmidless derivatives were avirulent and could not bind to Congo red (Table 23) (Prpic et al., 1983).

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Congo red acid-morpholinepropanesulfonic acid pigmentation agar (CRAMP) can be used to assay Congo red binding activity (Prpic et al., 1985). The Congo red could also be incoorporated into selective agar to form Congo red-magnesium oxalate agar (CRMOX) and virulent strains will form small red colonies (CRMOX+), while avirulent strains will form large colorless colonies (CRMOX-). As reported by Riley and Toma, 75.8% of the pathogenic serotypes of Y. enterocolitica were positive in this agar medium showing the presence of virulent plasmid in these strains, while 98% of nonpathogenic serotypes and strains of three other Yersinia species were negative (Riley and Toma, 1989). Virulence-plasmid-bearing strains of Y. enterocolitica can also be differentiated from their plasmidless derivatives by the binding of crystal violet (Table 24). The bacteria are plated on Brain Heart Infusion Agar (Difco) and incubated at 25 or 37C for 30 h, and flooded with crystal violet solution (85 g/ml) for two minutes. Dark violet virulent and white avirulent colonies were

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detected (Fig. 17). As with other plasmid-mediated properties of this organism, the binding of crystal violet occurs at 37C but not at 25C (Bhaduri et al., 1987).

Virulent (plasmid-associated) strains of Y. enterocolitica grown on RPMI 1640 agar (Flow Lab.) with 40 mM HEPES and 1.5% purified agar dissociated into small and large colonies. The autoagglutination test is regularly positive with small colonies and negative with large colonies. Avirulent Y. enterocolitica strains gave only large colonies on RPMI agar (Mazigh et al., 1983). 8.7. Calcium Requirement

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Higly virulent strains of Y. enterocolitica have an in vitro requirement for calcium at 37C but not at 26C. Avirulent wild strains of Y. enterocolitica do not have this calcium dependence. When grown on calcium-depleted media at 37C, the highly virulent strains yielded 0.5-6% large calcium non-requiring avirulent colonies; the remaining colonies were slow growing, calcium dependent and highly virulent. These slow growing calcium dependent colonies were highly virulent on intravenous inoculation, growing rapidly in the liver, spleen and lungs to produce multiple abscesses (Berche and Carter, 1982). The calcium dependency assay could be simplified by using a low-calcium, agarose-based medium of brain heart infusion with added magnesium. This medium effectively differentiated plasmid-bearing and plasmidless isolates (Bhaduri et al., 1990). Ca-independent mutants can be isolated by streaking the cultures onto magnesium oxalate agar (20 mM MgCl2, and 20 mM sodium oxalate) which consisted of blood agar base (BBL). Ca-dependent clones cannot form colony on this agar (Portnoy and Falkow, 1981) or formed small colonies on this MOX agar (Chang et al., 1984). A calcium-deficient brain heart infusion agarose was also used to differentiate Calcium-dependent strains which appeared to have small colonies (Fig. 18, Table 25) (Bhaduri et al., 1990).

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8.8. Surface Hydrophobicity Hydrophobicity was first indicated by the adherence of bacteria to polystyrene surface (polystyrene plate adherence, PSA). The plasmid-bearing cells of Y. enterocolitica from colonies grown at 37C on brain heart infusion agar plates adhered tenaciously to the plastic surface. On the other hand, the plasmidless cells were easily displaced from the surface by washing. The hydrophobicity can be determined by the partitioning method, nitrocellulose filter method, hydroxyapatite method, etc. (Lachica and Zink, 1984a). The adherence is affected by growth media (Lachica and Zink, 1984b). Plasmid-bearing cells grown on tryptic soy agar or at 22C were easily dislodged from the polystyrene surface (Lachica and Zink, 1984a). Xylene or some other water-immiscible solvent is mixed with an aqueous suspension of cells, the cells exhibited an affinity for one of the two phases upon partitioning, and can be quantitated by spectophotometry (Lachica and Zink, 1984a). Hydrophobicity of Y. enterocolitica can also be assayed by a Latex particle agglutination test (LPA) (Lachica and Zink, 1984b). Equal volume of latex

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suspension (about 108 particles/ml in 0.9% saline, 5.7 μm in diameter) and cell suspension were mixed and agglutination observed. A positive test was indicated by an immediate strong agglutination reaction which was easy to discern (Lachica and Zink, 1984b). A partial smooth-rough transition occurs in Y. enterocolitica grown at 37C. A rabbit antiserum prepared against 25C-grown bacteria contained antibodies directed mainly against the O-antigenic polysaccharide portion and to a smaller extent against the lipopolysaccharide (LPS). By contrast, a rabbit antiserum against 37C-grown bacteria contained antibodies directed mainly against the LPS. As demonstrated by immunodiffusion and hemagglutination inhibition tests, the immunogenicity of the LPS of Y. enterocolitica in vivo was similar to that of the bacteria grown in vitro at 25C (Kawaoka et al., 1983). 8.9. Mouse Lethality Virulence of Y. enterocolitica could be assayed by inoculating 107 cells intraperitoneally into pairs of BALB/c adult female mice pretreated with 5 mg of iron-dextran and 5 mg of desferrioxamine, and the mice are examined daily for up to 21 days (Prpic et al., 1985). Most mouse strains (C3H/HeN, BALB/c, BALB.B, DBA/2, A, Swiss, and SWR) were highly susceptible to infection (LD50, 2x102 to 6x102 administered intravenously) (Hancock et al., 1986). 8.10. Requirement of Iron Iron is an essential growth factor for nearly all bacteria, and the concentration of iron required is generally 0.4 to 4 μM. High-affinity iron chelators, known as siderophores, are produced by most pathogenic bacteria to survive in low-iron environment. Desferrioxamine B (Desferal) is a trihydroxamate siderophore, and it markedly increased the susceptibility of animals to yersiniosis (Fig. 19) (Robins-Browne and Prpic, 1985). In mice, iron-dextran reduced the median lethal dose of intraperitoneally administered Y. enterocolitica O:3 and O:9 (all with virulence plasmid) about 10-fold, whereas Desferal reduced this value more than 105-fold (Table 26) (Robins-Browne and Prpic, 1985). In vitro experiments indicated that Desferal promoted growth of Y. enterocolitica under iron-limiting conditions (Robins-Browne and Prpic, 1985). Clinically, it has

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been reported that overdose of iron and treatment with Desferal will result in Y. enterocolitica septicemia, since Y. enterocolitica does not produce siderophores but has receptors for the iron siderophore complex (Mofenson et al., 1987). Desferrioxamine- treated mouse was used as sensitive animal assay for the virulence of Y. enterocolitica (Mulder et al., 1989).

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Activity of Desferal as a siderophore can be demonstrated by a transferrin agar method (Fig. 20) (Robins-Browne and Prpic, 1985).

However, hydroxamate was detected in some Yersinia spp. other than Y. enterocolitica, and this siderophore is identical to aerobactin (from Enterobacter aerogenes). None of 50 Y. enterocolitica nor any of 5 Y. pseudotuberculosis isolates produced hydroxamates (Table 27) (Stuart et al., 1986).

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Under iron-starvation conditions, the different Yersinia species expressed various iron-regulated proteins (total cell proteins samples). Two high-molecular-weight outer membrane proteins (HMWPs) were synthesized in high-virulence-phenotype Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica but were absent in either low-virulence phenotypes or avirulent environmental strains (Carniel et al., 1987). These two HMWPs (about 190,000 and 240,000) were purified. They were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface (Carniel et al., 1989a). Gene coding for the 190,000-dal iron-regulated HMWP was cloned and screened by HMWP-specific antibodies. It is conserved among all highly pathogenic Yersinia species studied, but is missing in the low-virulence and nonvirulenct strains. The transcription of the HMWP gene is induced by iron starvation (Carniel et al., 1989b). 8.11. Phospholipase Analysis of the Y. pseudotuberculosis and Y. pestis genomes indicates that both species carry an identical copy of a gene that is predicted to encode a protein which shares 80% similarity to the Y. enterocolitica YplA, a secreted phospholipase that has been shown to contribute to virulence. In contrast to well tolerated production of the Y. enterocolitica YplA in E. coli, Y. pseudotuberculosis YplA expression was found to be toxic; however, cell viability could be restored if the Y. pseudotuberculosis YplA was expressed in the presence of its accessory protein YplB. In vitro, Y. pseudotuberculosis YplB was shown to reduce the activity of its cognate phospholipase in a dose-dependent manner. To determine whether the Y. pseudotuberculosis and Y. enterocolitica YplAs were secreted and regulated in a similar manner, secretion and promoter activity assays were performed. Unlike the situation apparent in Y. enterocolitica, expression of the Y. pseudotuberculosis yplA gene did not appear to be controlled by the flagellar regulon, nor did the phospholipase appear to be efficiently exported through the flagellar apparatus. These results indicate that the Yersinia YplAs vary in many of their attributes despite their high degree of amino acid homology (Meysick et al., 2009). 8.12. Type III secretion system

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Y. enterocolitica has three type three secretion systems, the flagellar, the

plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. A newly identified Ysa type III secreted protein, YspM, was identified.

Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspM(Y295)) was expressed. Site-directed mutagenesis of the histidine of YspM(Y295) ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s) (Witowski et al., 2008).

The Y. enterocolitica Ysa T3SS is such a system, where the apparatus genes, some regulatory genes, and four genes encoding secreted proteins (ysp genes) are contained in a single locus (Fig. 21). The remaining ysp genes and at least one additional regulator are found elsewhere on the chromosome. Expression of ysa genes requires conditions of high ionic strength, neutral/basic pH, and low temperatures (26C) and is stimulated by exposure to solid surfaces. The AraC-like regulator YsaE and the dual-function chaperone/regulator SycB are required to stimulate the sycB promoter, which transcribes sycB and probably yspBCDA as well. The putative phosphorelay proteins YsrRS (located at the distal end of the ysa locus) and RcsB, the response regulator of the RcsBCD phosphorelay system, are required to initiate transcription at the ysaE promoter, which drives transcription of many apparatus genes. Six unlinked ysp genes responded to NaCl and required YsaE/SycB, YsrRS, and RcsB for expression. Three ysp genes had unique patterns, one of which was unaffected by all elements tested except NaCl. Thus, while the ysp genes were likely to have been acquired independently, most have acquired a synchronous regulatory pattern (Walker and Miller, 2009).

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The Y. enterocolitica YtxR protein is a LysR-type transcriptional regulator that induces expression of the ytxAB locus, which encodes a putative ADP-ribosylating toxin. The ytxR and ytxAB genes are not closely linked in the Y. enterocolitica chromosome, and whereas ytxR is present in all sequenced Yersinia spp., the ytxAB locus is not. These observations suggested that there might be other YtxR-regulon members besides ytxAB. Microarray and reverse transcription-PCR analysis showed that YtxR strongly activates expression of the yts2 locus, which encodes a putative type 2 secretion system, as well as several uncharacterized genes predicted to encode extracytoplasmic proteins. Strikingly, under Ysc-Yop type 3 secretion system-inducing conditions, YtxR prevented the appearance of Yop proteins in the culture supernatant. Microarray and lacZ operon fusion analysis showed that this was due to specific repression of ysc-yop gene expression. YtxR was also able to repress VirF-dependent Φ(yopE-lacZ) and Φ(yopH-lacZ) expression in a strain lacking the virulence plasmid, which suggested a direct repression mechanism. This was supported by DNase I footprinting, which showed that YtxR interacted with the yopE and yopH control regions. Therefore, YtxR is a newly identified regulator of the ysc-yop genes that can act as an overriding off switch for this critical virulence system (xler-DiPerte et al., 2009). The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. A role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins) was demonstrated.

Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share the virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together

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with SycO, the chaperone of YopO. Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. In vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. SycO is integrated into the regulatory network of the Yersinia T3SS (Dittmann et al., 2007). 8.13. A three-dimensional collagen gel model A three-dimensional culture infection model (3D-CoG) was developed as the first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions. In this model, 105 Yersinia cells were suspended in liquid collagen solution containing bovine type I collagen at a final concentration of 1.7 mg/ml in RPMI 1640 medium adjusted to pH 7.4 (total volume, 66 μl). This solution was placed into a small self-constructed chamber (a tracking chamber) built by a hollowed coverslip on a glass slide and allowed to polymerize for 45 min (37°C; 5% CO2). The depth of the resulting collagen gel was about 400 μm. The remaining space in the tracking chamber was filled with RPMI 1640 medium, and thereafter, the chamber was sealed with wax (1:2, paraffin to vaseline) and incubated at 37°C in a cell culture incubator (Freund et al., 2008). Growth was checked microscopically (phase contrast) after different time points (with a conventional microscope) or followed by time-lapse video microscopy. To determine bacterial growth rates, yersiniae were released from the 3DCoG by using collagenase (Clostridium histolyticum collagenase) at a concentration of 1,000 U/ml in phosphate-buffered saline at 37°C. After digestion of the collagen gel, bacteria were washed with phosphate-buffered saline, and serial dilutions of the cell suspension (bacterial clusters and aggregates were dissociated, as checked by microscopy) were plated onto LB agar plates. CFU were counted after 48 h of incubation at 27°C. Collagenase treatment of yersiniae did not impair viability.

Surprisingly, plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37C. Motility at 37 C was abrogated

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in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37C in 3D-CoG (Fig. 22). Furthermore, the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, Ca2+-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG (Freund et al., 2008).

9. ROLE OF PLASMIDS IN VIRULENCE In 1980, Zink et al. discovered a virulence plasmid with a molecular weight of 41x106 in Y. enterocolitica O:8. This plasmid associated with tissue invasiveness as determined by Sereny keratoconjuntivitis test (Table 28) (Zink et al., 1980).

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Portnoy and Falkow demonstrated that 44-Mdal and 47-Mdal plasmid (designated as pYV plasmids) is present in virulent Y. enterocolitica and Y. pestis, respectively and associated with the Ca dependence phenotype (Portnoy and Falkow, 1981). The plasmids were found to share 55% DNA sequence homology. Mutants of Y. pestis that could grow on calcium-free medium were mostly cured of their 47-Mdal plasmid or with major deletion or insertion (Portnoy and Falkow, 1981). This virulenc associated plasmid was further characterized. It is associated with HEp-2 cell invasiveness, lethality for gerbils, production of three major outer membrane polypeptides (during growth at 37 but not at 25C) (Portnoy et al., 1981). The plasmid species associated with these properties ranged in molecular mass from 40 Mdal to 48 Mdal and comprised a family of related plasmids (Portnoy et al., 1981). In a study of 103 strains of Y. enterocolitica, Kay et al. found 10 strains to be lethal for mice and to possess 42- and 82-Mdal plasmid. A spontaneous derivative of one strain contained only the 82-Mdal plasmid and was lethal for mice. This 82-Mdal plasmid may be a new virulence-associated plasmid (Kay et al., 1982).

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V and W are antigens associated with virulence and were first known in Y. pestis and Y. pseudotuberculosis. Identical V and W antigens were speculated to occur in Y. enterocolitica accounting for both virulence and growth dependency. The virulence plasmids are associated with a number of temperature-inducible features of the bacteria: Ca2+-dependent growth at 37C (low calcium response), production of V and W antigens, autoagglutination, and the expression of outer membrane proteins (YOPs). The YOPs of Y. pseudotuberculosis and Y. enterocolitica can be separated into seven polypeptides (YOP1, YOP2a, YOP2b, YOP3, YOP4a, YOP4b, YOP5) with molecular weight 26,000 to 47,000 (Bo:lin et al., 1988). 9.1. Proteins Encoded by Virulence-associated Plasmids 9.1.1. Excreted Proteins versus Surface Proteins Y. enterocolitica with virulence-associated plasmid produce low to high molecular weight surface proteins associaed with adhesion, autoagglutination and invasion. Skurnik demonstrated that at least 16 polypeptides were apparently specified by the virulence plasmid when plasmid-bearing bacteria were grown at 37C or intraperitoneally in semipeable capsules. The different growth media used were also with added calcium. Also two chromosomally encoded polypeptides were expressed only at 37C, whereas the expression of eight polypeptides expressed at 22C was repressed at 37C (Skurnik, 1985). Chang and Doyle also demonstrated that the production of these specific outer membrane polypeptides is highly temperature dependent and only slightly affected by the inclusion of 10 mM Ca2+ (Chang and Doyle, 1984). Expression of the antigenic determinants(s) was temperature dependent, agglutination titers were lowest for cultures grown at 20C and highest for cultures grown at 35 to 40C (Doyle et al., 1982). Y. enterocolitica containing such plasmid also excreted proteins in calcium deficient medium. Heesemann et al. showed that strains harboring the virulence plasmids or the cointegrates apparently release about 10 proteins of discrete molecular weights. Protein release strongly increased after changing the medium from BHI to BHI-Magnesium Oxalate (MOX) medium. Five major

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proteins could be identified, with molecular masses of about 24, 32, 35, 46, and 49 kdal (Heesemann et al., 1984). Monoclonal antibodies directed against these plasmid-encoded released proteins were prepared and their activity among pathogenic Yersinia spp. were assayed (Heesemann et al., 1986). The monoclonal antibodies directed to a 36-kDa or a 46-kDa released protein of O:9 recognized also the corresponding proteins of other serotypes on Western blot analysis. However, a monoclonal antibody elicited against the 25-kDa protein of O:9 was species-specific, only reacted with the corresponding protein of Y. enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 (Heesemann et al., 1986). An exocellular antigen (protein) is associated with enterocolitis but absent from other serotypes or from other Yersinia spp. Both virulent Ca2+-dependent and avirulent Ca2+- independent isogenic pairs derived from the enterocolitis- associated serotypes synthesized the common antigen. Synthesis of this 24,000-dalton protein depended on the presence of metabolizable sugars and growth on solid medium at 37C (D:iaz et al., 1985). 9.1.2. Proteins Associated with Fibrillae, Adhesion and Autoagglutination Virulence-associated plasmid of Y. enterocolitica is correlated to autoagglutination and adherence to HEp-2 cell cultures, and these properties were lost by culturing at 37C in the absence of calcium (Vesikari et al., 1981). By insertional inactivation of genes located on the virulence plasmid, Kapperud et al. identified four plasmid-dependent, temperature-inducible properties related to the bacterial surface: (i) a fibrillar matrix covering the outer membrane, (ii) outer membrane protein YOP1 which is a structural component of the fibrillae, (iii) spontaneous autoagglutination, which is related to the fibrillae, and (iv) mannose-ressitant hemagglutination of guinea pig erythrocytes (Kapperud et al., 1987). Adhesion When the plasmid-containing strain was grown at 26C, the bacteria adhered to HeLa cells to a high degree. In contrast, when this strain was incubated at 37C in the same calcium-containing medium, it attached to the HeLa cells at a

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reduced level. When the pathogenic strain grown at 26C was given orally to Swiss albino mice, an infection was rapidly established and the mice died in short time. The culture grown at 37C was less virulent. These results suggest that the adherence properties of the bacteria may be of importance in the process of infection (B:olin et al., 1982). Also demonstrated by Martinez that the expression of the plasmid-mediated proteins on the outer membrane do not favor adherence of the bacteria to intestinal epithelial cells in vitro. Cultures grown at 25C adhered to Henle cell monolayers, whereas those grown at 37C did so much less effectively (Martinez, 1983). On the other hand, Schiemann et al. studied the surface properties of Yersinia species and epithelial cell interactions. They divided the process into three phenomena, association, attachment and invasion (intracellular) (Schiemann et al., 1987). Y. enterocolitica was more hydrophobic when grown at 35C than at 25C according to partitioning in a biphasic system, and attached strongly to both polystyrene and epithelial-cell monolayers. Attachment of Y. enterocolitica to epithelial cells probably involves non-specific surface properties that are not entirely explicable by hydrophobic and electrostatic interactions, whereas invasion of epithelial cells appears to resemble "receptor- mediated endocytosis". These are possibly low molecular-weight outer membrane proteins (YOPs). Fibrillae Mattrix Also from Y. enterocolitica O:3 that had host the virulence-associated 46-Mdal plasmid, one major outer membrane protein (47 kdal) was expressed when grown at 37C and not present in the plasmidless strain or in either strain grown at room temperature. A 200-kd protein also seen in the same preparations was shown to be an oligomer of the 47-kd protein by immunoblotting. As determined by electron microscopy and immulogical techniques, this major protein is probably the tack-like projections appeared on cells grown at 37C (Zaleska et al., 1985). Similar protein (Sarkosyl-insoluble) of 180 kdal was also demonstrated in pathogenic Y. pseudotuberculosis and Y. enterocolitica (Kapperud et al., 1985). This protein formed nonflagellar surface appendages,

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which appeared as a lawn of fine fibrillae (Fig.23), each having a diameter of 1.5 to 2.0 nm and a length of 50 to 70 nm (Kapperud et al., 1985; Lachica et al., 1984). This surface structure appeared to mediate autoagglutination of pathogenic Y. enterocolitica. Antibody of this protein inhibited hemagglutination (Kapperud et al., 1987; Lachica et al., 1984). This surface fibrillae structure is different from the rigid appendages (fimbriae) of Yersinia (Lachica et al., 1984). The production of fimbriae by Y. enterocolitica and Y. pseudotuberculosis was not correlated with the presence or absence of plasmids (Skurnik, 1984).

Insertional inactivation of the gene coding for YOP1, with resultant loss of the ability to express fibrillae, led to a signification reduction in the capacity of Y. enterocolitica, but not Y. pseudotuberculosis, to colonize the ileum of infected mice. In both Y. enterocolitica and Y. pseudotuberculosis, inactivation of the genes coding for Calcium dependency reduced the ability to maintain intestinal colonization, regardless of the ability to express fibrillae. Both surface fibrillae and Calcium dependency seem to reflect pathogenic determinants which are required for the establishment of Y. enterocolitica infection (Kapperud et al., 1987). 9.1.3. Proteins Associated with Serum Resistance The presence of the proteins on the bacterial surface appears to be involved in

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rendering the cells resistant to the bactericidal effects of serum, i.e., 37C-grown cells were resistant to serum killing and such resistance is associated with the virulence plasmid, and removal of the outer membrane proteins with pronase rendered them sensitive (Martinez, 1983). The surface protein P1 was demonstrated to be associated with the serum resistance of pathogenic Yersinia (B:olin et al., 1982; Balligand et al., 1985). A 30kb segment encoding the P1 protein of the virulence plasmid was cloned to E. coli expressing a 160-kdal YOP1 protein and proved to be related to the high degree of hydrophobicity, autoagglutinability, and resistance to serum killing (Martinez, 1989). However, by itself is not sufficient to specify the serum resistance property (Balligand et al., 1985). As shown by Bolin et al., YOP1 of molecular weight 140-kdal associated with the virulence plasmid of Y. pseudotuberculosis. This protein was induced within 2 min after a temperature shift from 26 to 37C. Similar protein occurred in Y. Enterocolitica (B:olin et al., 1982). This protein was also related to serum resistance. By transposon mutagenesis, Balligand et al. demonstrated that the largest outer membrane protein P1 is associated with the autoagglutination and resistance to human serum. However, the P1 was not sufficient by itself to specify the serum resistance property and a rapid autoagglutination of the host (Balligand et al., 1985). The binding ability to type I, II, and IV collagens is associated with the YOP1 protein. Curing of the virulence plasmid or Tn5 insertion in the gene encoding the YOP1 abolished the binding of all three collagen types to Y. enterocolitica and type I and II collagens to Y. pseudotuberculosis (Em:ody et al., 1989). As demonstrated by Lian et al. that Y. enterocolitica cells with expressed plasmid-mediated surface structure were much less sensitive to ingestion by human neutrophils (polymorphonuclear leukocytes, PMN) than those without it, and the resistance to phagocytosis was readily eliminated in a dose-dependent fashion by pronase treatment of whole cells, which was shown to remove plasmid-encoded outer membrane proteins (Lian et al., 1987). Ingestion and intracellular killing of E. coli were inhibited significantly in the presence of isolated outer membrane fragments derived from plasmid-bearing Y. enterocolitica cells. By injecting the bacteria into the backs of rabbits, the plasmidless strain was found almost entirely in PMNs or mononuclear cells. In

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contrast, the plasmid-bearing strain was found to be surrounded by, or interspersed with, PMNs and mononuclear cells; but most bacteria were extracellular, with little evidence of phagocytosis (Lian et al., 1987). 9.2. Molecular Manipulation of Plasmids The virulence plasmids of Y. enterocolitica cointegrated with a mobilizable vector were mobilized into other Y. enterocolitica strains, and found that the animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains, and other plasmid-mediated traits (calcium dependency, surface agglutinogens, cell adherence, etc.) were transferable to all Y. enterocolitica strains (Heesemann et al., 1984; Heesemann and Laufs, 1983). It shows that certain virulence factor is associated with chromosomal DNA. The structural genes of YOPs of Yersinia spp. and the V antigen of Y. pseudotuberculosis were cloned and mapped. Fragments of the virulence plasmids were cloned and the proteins expressed were determined by minicells and identified by immunoassay with specific antibody. The corresponding genes (for different YOPs and V antigen) were localized on pYV019 and pYV8081 of Y. pestis and Y. enterocolitica, respectively (Bo:lin et al., 1988). The virulence plasmids inY. pestis (pYV019), Y. pseudotuberculosis (pIB1) and Y. enterocolitica (pYV8081) are homologous to some extent. Genes are sometimes conserved in all these virulence plasmids (Bo:lin et al., 1988). No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, it is suggested that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. The gene encoding YOP5 protein encoded by pIB1 (of Y. pseudotuberculosis) was cloned on a mobilizable vector and introduced in Y. enterocolitica containing virulence plasmid with mutation in the homologous gene, the recombinant Y. enterocolitica secreted YOP5 and it showed that these systems

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are interchangeable (Bo:lin et al., 1988). By using transposon Tn2507 (with cat gene, chloramphenicol acetyltrans- ferase) mutagenesis of Y. enterocolitica W22703 (O:9), Mulder et al. (Mulder et al., 1989) identified an additional YOP protein called YOP20 and the mapping of genes encoding for YOP20, YOP44, YOP48, and V antigen. The V gene appeared to be part of an operon that also may contain yop37 and yop44. The transcription activity of the mutants was determined by assaying the activity of the cat gene. Transcription of this operon was activated by a temperature shift from 25 to 37C. At 37C, Ca2+ had a small decreasing effect but transcription still occurs. However, Yops were not released in those conditions. At 37C, mutants affected in this operon grew poorly, irrespective of the presence of Ca2+, or they even died in the presence of Ca2+. This operon was thus involved in the regulation by Ca2+, and it is then called car, for Ca2+ regulation. YOP20 or YOP48 were involved in virulence, since mutants affecting these two gene were markedly less virulent in desferrioxamine-treated mouse. Also by using transposon mutagenesis, Balligand et al. also determined the location of Ca2+ regulation (Balligand et al., 1985). By transposon mutagenesis, two mutants were affected in the properties of autoaggultination and resistance to human serum. Analysis of the OMP pattern of these two mutants revealed the absence of the YOP1. Complementation of one of these mutations with the cloned structural gene of YOP1 restored the wild-type phenotype (Balligand et al., 1985). Restriction fragment analysis of 18 virulence plasmids by using BamH1 digestion showed two types of plasmids, with a deletion of 4.4-kb BamH1 fragment in type II. However, no function differences between the strains bearing type I or type II plasmid were observed (Pulkkinen et al., 1986). The replication genes (rep) and the plasmid-stabilization system (par) of the virulence plasmid of Y. enterocolitica were determined by restriction endonuclease analysis and the replication function is thermosensitive. At 28C for 25-30 generations, the E. coli C600 losses the mini-derivatives of the virulence plasmid at frequenceies ranging from 10-18%, while at 40C, it was greater than 99% (Biot and Cornelis, 1988). Strains of Salmonella typhimurium harbouring stable aroA (encoding

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5-enolpyruvylshikimate 3-phosphate synthase) mutations are attenuated and excellent oral vaccines in mice and other animals. The aroA mutant of Y. enterocolitica failed to kill mice (Bowe et al., 1989). Attenuation is probably due to a requirement for one or more aromatic compounds, including para-aminobenzoic acid and dihydroxybenzoic acid, which are not readily available in mammalian tissues. The aroA genes from E. coli and S. typhimurium have been identified as the distal genes in an operon with serC (which encodes 3-phosphoserine aminotransferase). The aroA and serC genes of Y. enterocolitica have been sequenced with high (78% and 81%) homology with E. coli genes. Comparisons of serC and aroA of Y. enterocolitica, S. typhimurium, E. coli and Bordetella pertussis were done (O'Gaora et al., 1989). A new mercury-resistance transposon (Tn3926) from Y. enterocolitica was studied (Lett et al., 1985). This transposon has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. By comparing the restriction fragments with other transposon, the Tn3926 has high homology to Tn501 and Tn21, especially the fragment encoding the mercury- resistance, with low homology in regions encoding transposition functions (Lett et al., 1985). Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized. The conjugative plasmids pYE854 (95.5 kb)(Fig. 24) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Y. enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV (Table 29). The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation (Hammerl et al., 2008).

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10. MOLECULAR STUDY OF INVASIVENESS Recently, two chromosomal loci, inv and ail, which confers an invasive phenotype of Y. enterocolitica have been cloned and studied. A tissue culture

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invasion (TCI) model was used in assaying the invasiveness. In this assay, HEp-2 tissue culture cells are cultured in 24-well plates. After application of bacteria, the wells are washed with phosphate-buffered saline. The extracellular bacteria are killed by the use of gentamicin (100 μg/ml). The cells are lysed by Triton X-100 and the intracellular bacteria (invaded) are counted (Miller et al., 1989). These two genes were cloned into E. coli HB101 and these clones showed tissue invasiveness (Miller and Falkow, 1988). By hybridization with probes derived from these clones, Pierson and Falkow demonstrated that 35 nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. The inv-homologous sequences in these nonpathogenic strains are probably nonfunctional (Pierson and Falkow, 1989). The cloning of inv gene into these nonpathogenic strains yielded invasive strains. The ail gene is associated with the invasiveness of all the pathogenic strains(TCI+), with certain restriction fragment-linked polymorphisms (Miller et al., 1989). Nucleotide sequence of ail gene was determined in a region of 650 bp. The ail gene product determined by maxicells was a 17-kdal membrane-associated protein. The nucleotide sequence of the ail gene revealed a single unique open reading frame of 178 amino acids. A 23 amino acids signal sequence was identified by comparing the amino acid sequences deduced from the gene and the analysis of the purified protein (Miller et al., 1990). 10.1. Flagellar master regulator The Y. enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. Phenotypes of flhD and fliA mutants that result from the complex gene regulation were investigated with Phenotype MicroArrays (Biology). Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of

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the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants (Fig. 25). Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system. Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation (Fig. 26, 27) (Townsend et al., 2008).

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11. CONCLUSIONS Y. enterocolitica may be an important food-pathogen in Taiwan: (A) It is prinipally a zoonotic organism that has been isolated from a variety of animals, especially with swine. Swine raising is an important agricultural business in Taiwan and pollution of swine waste widely occurs. However, outbreak of yersinosis has not been directly associated with pork. (B) Symptoms of infection caused by Y. enterocolitica are often quite severe. However, fatality from gastroenteritis is rare. (C) High growth rate at low temperature. Y. enterocolitica is sensitive to heat, sodium chloride, and acidity and will generally be inactivated by environmental conditions that will kill salmonellae. It is important to eliminate the organism from foods (especially pork, milk, and foods that may have direct or indirect contact with porcine waste) by pasteurization or cooking. Care should be taken to avoid cross-contamination of processed, ready-to-eat foods with pork and porcine wastes, as well as animal and human fecal contaminations.

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