Molecular Biogrouping of Pathogenic Yersinia enterocolitica ...

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Abstract. Yersinia enterocolitica (YE) is associated with several inflammatory gastrointestinal disorders. Pathogenic YE organisms are classified as biogroup 1B.
Microbiology and Infectious Disease / MOLECULAR BIOGROUPING OF YERSINIA ENTEROCOLITICA

Molecular Biogrouping of Pathogenic Yersinia enterocolitica Development of a Diagnostic PCR Assay With Histologic Correlation Laura W. Lamps, MD,1 Jennifer M. Havens,1 Lori J. Gilbrech, MT(ASCP),2 Peter H. Dube, PhD,3 and Margie A. Scott, MD1,2 Key Words: Yersinia enterocolitica; Virulence; Polymerase chain reaction; PCR; Granuloma; Enterocolitis; Appendicitis; Biogroup DOI: 10.1309/A8JJPGGGWXYLF48A

Abstract Yersinia enterocolitica (YE) is associated with several inflammatory gastrointestinal disorders. Pathogenic YE organisms are classified as biogroup 1B (high-virulence [HV] serovars) or biogroups 2 through 5 (low virulence [LV]). We developed the first molecular assay designed to distinguish between these groups and correlated the molecular results with histologic patterns of inflammation. Eleven known pathogenic YE culture isolates (6 biogroup 1B and 5 biogroups 2-5) and 6 YE-positive archival cases were subjected to polymerase chain reaction analysis using primer pairs targeting a strain-dependent variable region, allowing discrimination between biogroups with a single assay. All 11 known culture isolates were confirmed. Of the 6 archival cases, 4 were LV and 2 were HV. Histologic correlation revealed granulomatous inflammation in the LV cases and suppurative inflammation in the HV cases. This novel assay is useful for diagnosis using culture samples and archival tissues. It also could yield important information correlating YE epidemiology, pathogenesis, and morphology because these preliminary data suggest that LV strains may be associated with chronic granulomatous processes and HV strains with suppurative inflammation.

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Yersinia enterocolitica is a common gram-negative, foodborne enteric pathogen found in water, dairy products, and meats.1-7 It is one of the most common causes of food-borne gastroenteritis in Western and Northern Europe; the incidence also is increasing in the United States and Canada, although this may be a result of improved surveillance and detection methods.4,8,9 Y enterocolitica comprises a heterogeneous collection of more than 50 strains with variable pathogenicity in humans and animals. The concept and measurement of virulence in Y enterocolitica originally was established in murine experiments, and Y enterocolitica organisms traditionally are classified as highly virulent (lethal to mice), poorly or low virulent (nonlethal to mice), and avirulent (environmental or commensal strains).10,11 Pathogenic Y enterocolitica generally are classified as belonging to 1 of 2 groups, the high-virulence (HV) biogroup 1B or the low-virulence (LV) group comprising biogroups 2 through 5, based on the biochemical properties of the strain. Common human pathogenic strains include serovars O:8 and O:13, classified in the HV group, and strains O:1, O:3, and O:9, which are classified in the LV group.10-12 Previous serologic, microbiologic, and molecular studies have implicated pathogenic strains of Y enterocolitica as causative agents in numerous gastrointestinal inflammatory diseases, including appendicitis (granulomatous and suppurative), gastroenteritis, ileitis, colitis, and mesenteric lymphadenitis.4,8,11,13-18 The spectrum of illness caused by Y enterocolitica is extremely variable, ranging from acute self-limited gastroenteritis to fatal dissemination and sepsis. Food-borne outbreaks have been associated with virtually all pathogenic serovars. Serovar O:8 has characteristically been associated with more catastrophic human infection, whereas O:3 and O:9 have been linked to milder cases.4,10,11 © American Society for Clinical Pathology

Microbiology and Infectious Disease / ORIGINAL ARTICLE

The study and clinical identification of Y enterocolitica have challenged investigators in the past because it is fastidious and often difficult to culture. The use of serologic studies is limited by numerous factors, including cross-reactivity with similar organisms.1,4,11,14 The recent development of sensitive, specific polymerase chain reaction (PCR) assays for the detection of Yersinia organisms has greatly improved investigators’ ability to study this organism in fresh and fixed samples.15,19-21 Because Y enterocolitica is a common foodborne pathogen and is implicated in such a wide range of gastrointestinal diseases, the development of a PCR assay that could be used to assign Y enterocolitica–positive specimens to a particular biogroup has significant implications for clinical diagnosis, microbiologic research, and epidemiologic studies. The ability to use such an assay on formalin-fixed, paraffin-embedded pathologic materials also would allow association of specific biogroups with particular disease phenotypes in pathologic specimens. Our goal, therefore, was to develop a molecular biogrouping assay for use with archival specimens and culture isolates.

Materials and Methods Specimen Preparation of Clinical Culture Isolates and Archival Patient Specimens Eight pathogenic strains (O:1, O:2, O:4, O:13, O:15, O:20, O:21, and O:34) of Y enterocolitica were obtained from the Centers for Disease Control and Prevention (CDC), Atlanta, GA. Organisms were received on agar slants and subsequently cultured using tryptic soy broth tubes that were inoculated with each strain and incubated at 35°C overnight. Inoculates were subjected to Vitek biochemical analysis for confirmation (Biomerieux, Durham, NC). Three additional strains (O:3, O:8, and O:9) were obtained from the American Type Culture Collection (ATCC Nos. 700822, 29913, and 55075, respectively). These specimens were received as freeze-dried organisms, which subsequently were rehydrated with phosphate-buffered saline (PBS) before culture. After rehydration, tryptic soy medium was inoculated with each strain and incubated at 35°C overnight. These isolates also were confirmed and characterized using the Vitek biochemical analysis system. All inocula were spun down, rinsed, resuspended in 1 mL of PBS and subjected to lysis digestion (see “DNA Extraction”). Six archival, formalin-fixed, paraffin-embedded patient specimens that had tested positive for pathogenic Y enterocolitica chromosomal DNA using PCR analysis (see “Initial PCR Assay for the Y enterocolitica ail Gene”)15,20,21 also were retrieved. Of the 6 cases, 3 were granulomatous appendicitis, 1 was malacoplakia of the appendix and right colon, 1 was a colon

resection from a patient with acute Y enterocolitica enterocolitis with perforation and sepsis, and 1 was a Y enterocolitica soft tissue abscess. DNA Extraction DNA was extracted from the CDC and ATCC reference strains as follows: Aliquots (25 µL) from each culture specimen were suspended in TRIS-EDTA buffer containing 10 mg/mL of Proteinase K (Invitrogen, Carlsbad, CA) in a total volume of 200 µL and incubated at 37°C for 1 hour. DNA was captured and purified using silica resin (Qbiogene, Carlsbad, CA). Archival patient specimen DNA was similarly extracted from two 25-µm sections of formalin-fixed, paraffin-embedded tissue cut from the paraffin block, following deparaffinization and Proteinase K digestion at 55°C overnight. All DNA isolation procedures were executed in a class II biological safety cabinet in a room physically separated from the nucleic acid amplification area and the post-PCR area. Initial PCR Assay for the Yersinia enterocolitica ail Gene PCR for the detection of pathogenic Y enterocolitica DNA was performed using previously published primers and methods15,19-21 to confirm the presence of Y enterocolitica DNA. The oligonucleotide primers were designed to amplify a 170-base-pair (bp) region of the Y enterocolitica ail gene present only in pathogenic strains.19 Briefly, primers were 5' end labeled with γ– phosphorus 32 (32P) adenosine triphosphate, and DNA amplification was performed by PCR. Pure genomic DNA derived from cultures of Y enterocolitica (ATCC No. 29913) served as the positive control sample, and reagent blanks served as negative control samples. These particular oligonucleotide primers previously were tested against numerous other bacteria for specificity.15,19 A 99-bp fragment of the human β-actin gene served as a “housekeeping” gene to ensure that intact DNA was recovered from the archival tissues. Size separation of amplicons was performed on 8% native polyacrylamide gel followed by gel drying and autoradiography. Molecular Biogrouping PCR Assay Targeting the 23S rRNA Gene Following confirmation of pathogenic Y enterocolitica chromosomal DNA using the aforementioned method, specimens were subjected to PCR targeting fragments of the 23S ribosomal RNA (rRNA) gene to determine the biogroup. Design of Oligonucleotide Primers DNA sequence data for the Y enterocolitica 23S rRNA gene were obtained from GenBank and the Y enterocolitica Sequencing Group of the Sanger Institute (ftp.sanger.ac.uk/pub/pathogen/ye; Cambridge, England), which maintains the most current and complete Yersinia DNA library. Conserved and variable Am J Clin Pathol 2006;125:658-664

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regions then were identified by aligning published sequences of all Y enterocolitica serovars from biogroups 1B and 2 through 5. Novel primer pairs were designed that result in the amplification of a 182-bp region of the 23S rRNA gene for Y enterocolitica biogroup 1B and a 204-bp region of the 23S rRNA gene for Y enterocolitica biogroups 2 through 5. The assay produces amplicons of different sizes (182 vs 204 bp) based on differences in the sequence of the targeted region in HV vs LV Y enterocolitica, thus allowing distinction between biogroups using a single PCR reaction. The primers were tested against numerous other bacteria, including Yersinia pseudotuberculosis, Staphylococcus aureus, Enterococcus faecalis, Bacteroides fragilis, Pseudomonas aeruginosa, Proteus vulgaris, Enterobacter cloacae, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Shigella flexneri, and Campylobacter jejuni. Results indicated that the primers were specific because there was no amplification product generated from testing any of the other bacteria. PCR Amplification The DNA extraction procedure is detailed in the preceding text. Reaction mixes were prepared in a hood in a room separate from that used for extraction to prevent contamination. The PCR amplification conditions were optimized by varying the magnesium chloride concentration, annealing temperature, primer concentrations, and DNA template concentration. Following optimization, 50-µL total reaction volumes contained the following: 20 mmol/L of Reaction Buffer (Invitrogen, Carlsbad, CA); 1.5 mmol/L of magnesium chloride (Invitrogen); 125 nmol/L each of dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxythymidine triphosphate) (Amersham Pharmacia, Piscataway, NJ); 5 U of Platinum Taq DNA polymerase (Invitrogen); 30 pmol/L of each primer; and 6 µL of template DNA. Primers were 5' end labeled with γ-32P adenosine triphosphate. Reaction mixtures then were subjected to the following thermal cycling parameters in a PerkinElmer 9600 thermocycler, Perkin Elmer, Wellesley, MA: predenaturation for 1 minute at 94°C, then 25 cycles of 94°C for 30 seconds, 58°C for 1 minute, and 70°C for 2 minutes, followed by a final extension at 70°C for 5 minutes and then 4°C for infinity. Representative Y enterocolitica serovars from the HV (ATCC 29913) and LV (ATCC 55075) biogroups served as positive control samples, and multiple reagent blanks and negative amplification controls were included in every set of PCR reactions. The aforementioned 99-bp fragment of the human β-actin gene served as a housekeeping gene control to ensure intact DNA recovery. Amplicon Detection Following amplification, 25-µL aliquots were removed from each reaction mixture and examined by electrophoresis 660 660

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(200 V, 40 minutes) on 8% native polyacrylamide in 0.05 TRIS-borate-EDTA and stained with ethidium bromide. After confirmation of sample amplification, gels were rinsed and dried (dryer, Biorad, Hercules, CA) for 10 minutes, exposed to Kodak x-omat radiography film (Kodak, Rochester, NY) overnight at –20°C, and then developed. Restriction Fragment Length Polymorphism Analysis of PCR Amplicons for Product Confirmation Restriction sites for expected products for the 182- and 204-bp fragments were determined using published sequences and NEBcutter (New England Biolabs, Beverly, MA). Restriction sites were selected based on size for optimal detection. Based on the restriction site analysis, all PCR amplicons were restricted with BsaH I using standard digestion conditions (10-µL aliquot of PCR product, 1 U/µL of enzyme, and 0.2 µg/mL of bovine serum albumin in a 20-µL total reaction volume at 37°C for 1 hour). Restriction fragments were separated by gel electrophoresis using both 12% polyacrylamide and 3% agarose gels to confirm the results of the restriction analysis. Gels were stained with ethidium bromide and visualized under UV light. Sequencing of PCR Amplicons for Product Confirmation All Y enterocolitica PCR amplicons were purified with the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) to remove excess dNTPs (deoxynucleoside triphosphates), polymerases, salts, and primers. Eluted DNA then was run on a 3% agarose gel and subsequently excised and purified using the QIAquick Gel Extraction kit (Qiagen). Purified DNA then was quantitated and submitted to cycle sequencing using the BigDye Terminator kit (Applied Biosystems, Foster City, CA), and amplicons were subjected to Centrisep spin column (Princeton Separations, Adelphia, NJ) purification to remove unbound dye. Purified amplicons then were dried and resuspended in 20 µL of HiDi formamide (Applied Biosystems) and sequenced in both directions on an ABI Genetic Analyzer (Applied Biosystems) using both forward and reverse primers for the 23S rRNA gene. Resulting sequences were aligned, and a consensus sequence was generated using CLUSTALPROF (SDSC, San Diego, CA). Sequences then were compared with published GenBank sequences.

Results Microbiologic Culture Specimens All 11 ATCC and CDC known culture isolates were confirmed appropriately as 6 HV serovars (O:4, O:8, O:13, O:20, O:21, and O:34) and 5 LV serovars (O:1, O:2, O:3, O:9, and O:15), yielding expected amplicons of 182 and 204 bp, © American Society for Clinical Pathology

Microbiology and Infectious Disease / ORIGINAL ARTICLE

respectively. Representative examples are given in ❚Image 1❚. Products of the restriction fragment length polymorphism (RFLP) analysis for the 182- and 204-bp products matched expected restriction patterns, further confirming the specificity of the PCR assay (data not shown). As final confirmation, resultant sequences from the generated PCR amplicons (both microbiologic cultures and tissue samples) had 100% homology with published sequences and confirmed that the difference in amplicon size (182 vs 204 bp) allows distinction between biogroups. Archival Pathology Specimens Of the 6 archival, formalin-fixed, paraffin-embedded Y enterocolitica–positive pathology cases, 4 yielded DNA corresponding to LV biogroups, and 2 yielded DNA corresponding to the HV biogroup. Representative examples are given in ❚Image 2❚. RFLP analysis and sequencing again yielded products matching expected restriction patterns and published sequences, respectively. Before this analysis, it was known

0:8

0:20

204 bp 0:3

182 bp

0:1 0:9

only that these specimens were positive for Y enterocolitica chromosomal DNA, but the biogroup of the strain causing infection was unknown. These data show the usefulness of the assay for assigning a biogroup to Y enterocolitica DNA contained in archival tissue specimens. Histologic Correlation Following molecular analysis, the pathologic features of the 6 archival cases were reviewed. Of the 4 specimens containing LV biogroups DNA, 3 were cases of granulomatous appendicitis; these cases were part of a previous study documenting the association between Yersinia infection and granulomatous appendicitis using molecular methods.15 Prominent histologic features included epithelioid granulomas with lymphoid cuffing ❚Image 3❚, mucosal ulceration, and a transmural inflammatory infiltrate featuring numerous lymphoid aggregates with associated fibrosis. Of the 4 cases, 1 was a case of the rare histiocytic disorder malacoplakia ❚Image 4❚, which occurs most frequently in the urinary tract, but also is well documented in the gastrointestinal tract. Malacoplakia has been associated with numerous bacteria, including Yersinia,21 and is thought to result from a macrophage defect in the processing of bacterial lipopolysaccharide. The 2 specimens harboring HV biogroup DNA consisted of 1 case of severe Y enterocolitica enterocolitis with perforation and sepsis and 1 case of a suppurative soft tissue abscess due to Y enterocolitica infection ❚Image 5❚. In these cases, the neutrophil was the predominant inflammatory cell, and there was associated tissue necrosis. No granulomas or histiocytic

❚Image 1❚ Polymerase chain reaction gel showing confirmation of 5 known culture isolates using biogrouping assay. Lanes 1 and 3 show the 182-base-pair (bp) amplicon present in high virulence (biogroup 1B) strains, and lanes 2, 4, and 5 show the 204-bp amplicon present in low virulence (biogroup 2-5) strains.

NC + Case HV

HVC

+ Case + Case LV LV

LVC 204 bp

182 bp

❚Image 2❚ Polymerase chain reaction gel showing analysis of archival specimens. Lane 1, negative control sample; lane 2, the 182-base-pair (bp) amplicon present in a case containing high-virulence group 1B DNA; lane 3, the high-virulence Yersinia enterocolitica control sample; lanes 4 and 5, cases containing the 204-bp amplicon indicating low-virulence DNA; and lane 6, the low-virulence Y enterocolitica control sample.

❚Image 3❚ Granulomatous appendicitis that was positive for low-virulence Yersinia enterocolitica DNA. Note epithelioid granulomas with giant cells and surrounding lymphoid tissue (H&E, original magnification ×100).

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A

B

❚Image 4❚ Gastrointestinal malacoplakia that was positive for low-virulence Yersinia enterocolitica DNA. A, Note diffuse histiocytic infiltrate filling submucosa and infiltrating mucosa (H&E, original magnification ×40). B, Characteristic calcified Michaelis-Gutmann bodies (H&E, original magnification ×400).

infiltration were seen in these 2 cases associated with HV Y enterocolitica. Although the cases numbers are small, these data suggest a striking difference between the inflammatory patterns in LV and HV Y enterocolitica infection: LV Y enterocolitica seems to correlate with chronic granulomatous processes, whereas specimens positive by PCR for HV Yersinia enterocolitica correlate with suppurative inflammation and tissue necrosis.

❚Image 5❚ A suppurative soft tissue abscess that was positive for high-virulence Yersinia enterocolitica DNA. Note the suppurative center with surrounding early fibrosis (H&E, original magnification ×100).

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Discussion Our knowledge and understanding of the wide spectrum of clinical and pathologic conditions caused by Y enterocolitica infection is rapidly evolving and has been aided greatly by molecular tools that have become available only relatively recently. By using a PCR assay that targets the ail gene present in invasive Y enterocolitica strains, our group previously detected pathogenic Y enterocolitica in several gastrointestinal and extraintestinal inflammatory conditions, including granulomatous appendicitis, malacoplakia, Yersinia abscesses, and Yersinia sepsis.15,20,21 We subsequently undertook development of a molecular biogrouping assay that would allow analysis of microbiologic culture specimens and archival, formalin-fixed, paraffin-embedded pathology specimens for diagnostic and research purposes. To our knowledge, this is the first assay of its type, and all of the culture isolates and archival pathology specimens analyzed in this study were successfully amplified and assigned to the appropriate biogroup. Although our number of cases (11 culture isolates and 6 archival cases) is relatively small, we believe the usefulness and specificity of this novel assay were demonstrated successfully on microbiologic and archival specimens. We subsequently have applied the assay to numerous cases of Crohn disease that are known to harbor Y enterocolitica, and more than 98% of these cases featuring chronic granulomatous inflammation contain LV biogroup DNA20 (L.W.L., unpublished data, March 2005). Cases harboring HV DNA are much more difficult to find because these often are selflimited processes that are not cultured or do not come to biopsy or resection. © American Society for Clinical Pathology

Microbiology and Infectious Disease / ORIGINAL ARTICLE

In the past, there have been several limitations to the assays available for diagnosis and study of Y enterocolitica. Yersinia often is difficult to culture from patient samples (particularly for laboratories with limited experience with Yersinia) without cold enrichment and selective media, and isolation from other fecal flora may be difficult.1,4,12,14 In addition, culture alone cannot distinguish between pathogenic and nonpathogenic strains of Y enterocolitica. A 4-fold elevation in serum antibody titers historically has been considered diagnostic of Y enterocolitica infection, and serologic studies have yielded much of the epidemiologic and demographic data used in studying Y enterocolitica and potential food-borne epidemics with which it has been associated.1,4,7,10,14 However, serum antibodies may be present in asymptomatic persons, and false-negative test results may be encountered in children and immunocompromised patients. In addition, numerous serologic cross-reactions with similar bacteria have been described, including Salmonella, Brucella, and Morganella species, as well as enterohemorrhagic E coli.1,8,14 Because epidemic Y enterocolitica infections often have been linked to particular biogroups and serotypes, the development of a molecular assay that can effectively assign pathogenic Y enterocolitica to the correct biogroup could yield important information regarding food-borne outbreaks and patterns of geographic distribution. Because this assay has been optimized for use in fixed, processed tissue samples, as well as in bacterial culture specimens, it is suitable for use with microbiologic specimens, fresh tissue samples, and archival patient specimens. Its suitability for use with archival pathology materials also will allow retrospective testing of tissue samples, which is particularly useful given the common clinical practice of initiating antibiotic use before obtaining cultures or not obtaining cultures at all. In addition to the successful molecular segregation of pathogenic Y enterocolitica biogroups, this also is the first study to demonstrate differences in histologic patterns associated with the different biogroups. We broadly observed 2 different types of inflammatory lesions in the archival human specimens: granulomatous inflammation associated with LV strains and suppurative inflammation associated with HV strains. These morphologic data support previous in vitro studies demonstrating that genomic differences between Y enterocolitica strains may be responsible for differences in clinical and pathologic manifestations of disease, which has profound implications for the diagnosis and epidemiology of Y enterocolitica infection.12,22 This is the first assay developed for molecular biogrouping of pathogenic Y enterocolitica. Furthermore, the molecular segregation of Y enterocolitica serovars into LV and HV biogroups seems to correlate with differences in morphologic patterns of inflammation. These differences illustrate the need for further studies in several areas, including animal studies

that will allow morphologic comparison using larger case numbers. It is hoped that these initial observations linking molecular and histologic differences between Yersinia biogroups might ultimately result in improved microbiologic diagnostic abilities, yield important information regarding the epidemiology, pathogenesis, and histologic patterns of Y enterocolitica infection, and further elucidate the range of inflammatory diseases with which Y enterocolitica is associated. From the Departments of Pathology, 1University of Arkansas for Medical Sciences and 2Central Arkansas Veterans Health Care Systems, Little Rock; and 3Microbiology, the University of Texas Health Science Center at San Antonio. Supported in part by grant IBD-0095 from the Broad Medical Research Program, Los Angeles, CA. Presented in part at the United States and Canadian Academy of Pathology, San Antonio, TX, March 2005. Address reprint requests to Dr Lamps: Dept of Pathology, University of Arkansas for Medical Sciences, 4301 W Markham St, Slot 517, Little Rock, AR 72205.

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13. Attwood SEA, Cafferkey MT, Keane FBV. Yersinia infections in surgical practice. Br J Surg. 1989;76:499-504. 14. Baert F, Peetermans W, Knockaert D. Yersiniosis: the clinical spectrum. Acta Clin Belg. 1994;49.2:76-84. 15. Lamps LW, Madhusudhan KT, Greenson JK, et al. The role of Yersinia enterocolitica and Yersinia pseudotuberculosis in granulomatous appendicitis: a histologic and molecular study. Am J Surg Pathol. 2001;25:508-515. 16. Saebo A, Lassen J. Acute and chronic gastrointestinal manifestations associated with Yersinia enterocolitica infection: a Norwegian 10 year follow-up study on 458 hospitalized patients. Ann Surg. 1992;215:250-255. 17. Schapers RFM, Reif R, Lennert K, et al. Mesenteric lymphadenitis due to Yersinia enterocolitica. Virchows Arch A Pathol Anat Histol. 1981;390:127-138. 18. Paff JR, Triplett DA, Saari TN. Clinical and laboratory aspects of Yersinia pseudotuberculosis infections, with a report of two cases. Am J Clin Pathol. 1976;66:101-110.

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19. Nakajima H, Inoue M, Mori T, et al. Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method. J Clin Microbiol. 1992;30:2484-2486. 20. Lamps LW, Madhusudhan KT, Havens JM, et al. Pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis DNA is detected in bowel and mesenteric nodes from Crohn’s disease patients. Am J Surg Pathol. 2003;27:220-227. 21. Havens JM, Montgomery E, Greenson JK, et al. Pathogenic Y. enterocolitica DNA is detected in gastrointestinal malakoplakia [abstract]. Mod Pathol. 2003;16:1200A. 22. Denecker G, Totemeyer S, Mota LJ, et al. Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells. Infect Immun. 2002;70:3510-3520.

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