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Comparison between standard in vitro virulence associated assays and human coproantibody siga production.. Fletcher, Kathleen Margaret 1987

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COMPARISON BETWEEN STANDARD IN VITRO VIRULENCE ASSOCIATED ASSAYS AND HUMAN COPROANTIBODY SIGA PRODUCTION AS PREDICTORS OF YERSINIA ENTEROCOLITICA AND YERSINIA ENTEROCOLITICA-LIKE ORGANISM ASSOCIATED MOUSE VIRULENCE AND HUMAN DISEASE PRESENTATION  By KATHLEEN MARGARET FLETCHER (B.Sc, The University of B r i t i s h Columbia, 1985) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Medical Microbiology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1987 c Kathleen Margaret Fletcher  In  presenting  degree  at  this  the  thesis  in  University of  partial  fulfilment  of  of  department publication  this or of  thesis for by  his  or  her  DE-6(3/81)  representatives.  PATHOLOGY  OCTOBER 9, 1987  for  an advanced  Library shall make  it  agree that permission for extensive  It  this thesis for financial gain shall not  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  that the  scholarly purposes may be  permission.  Department of  requirements  British Columbia, I agree  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head  of  copying  my or  be allowed without my written  -  I.  A semi-quantitative which d i s t i n g u i s h e s  two  r e c o v e r e d : those who secretory  IgA  indirect  immunofluorescence assay was from whom y e r s i n i a e  produce a s t r o n g y e r s i n i a e s p e c i f i c those who  do not.  developed are  coproantibody  This  SIgA response  appeared to be y e r s i n i a e s p e c i f i c as f a e c a l supernatant c o n t r o l s p a t i e n t s whose s t o o l s where shown to y i e l d n e g a t i v e or p o s i t i v e f o r S a l m o n e l l a , Campylobacter, or C l o s t r i d i a were SIgA Organisms incidence was  not  associated  virulence. and  i s o l a t e d from p a t i e n t s w i t h h i g h  of v i r u l e n c e associated  Clinical  SIgA t i t r e . yersiniae No  This  a higher  assays  of  shown to e x i s t between SIgA  s t a n d a r d of b a c t e r i a l  examination o f p a t i e n t s  documented a s t r o n g  cultures  c h a r a c t e r i s t i c s a l t h o u g h SIgA response  a s s o c i a t i o n was  mouse v i r u l e n c e , the g o l d  from  negative.  SIgA t i t e r s had  w i t h most o t h e r commonly r e c o g n i z e d  A strong  titre  virulence.  culture p o s i t i v e with y e r s i n i a e  a s s o c i a t i o n between acute e n t e r i c i l l n e s s and  a s s o c i a t i o n was  not dependant on  the  high  cultured  species.  single jji v i t r o virulence associated  r e l i a b l e p r e d i c t o r of animal v i r u l e n c e . Y . f r e d e r i k s e n i i and non-pathogenic  one  i n man,  Y.kristenseni i , was  -  ABSTRACT  types o f p a t i e n t s  (SIgA) response and  i i  The  assay was  v i r u l e n c e of  previously  a l s o documented.  found to be nine  thought to  be  a  - i i i II.  TABLE OF CONTENTS  page I.  Abstract  II.  Table of Contents  III.  L i s t of Tables  iv  IV.  L i s t of Figures  v  V.  Introduction A. Yersinia C h a r a c t e r i s t i c s B. Virulence Associated C h a r a c t e r i s t i c s C. Coproantibody Secretory Immunoglobulin A  VI.  ii iii  1 4 8  Methods A. Specimens and Organisms B. Organism Associated C h a r a c t e r i s t i c s 1. Autoagglutination 2. Calcium Dependance 3. Congo Red Absorption 4. Plasmid P r o f i l e 5. Mouse Virulence C. Human Coproantibody SIgA Response D. Murine Serology E. Diagnostic Test Evaluation  13 14 14 14 16 16 17 18  VII.  Results  20  VIII.  Discussion  24  IX.  Summary  28  X.  Bibliography  29  XI.  Appendix A  39  XII.  Appendix B  40  XIII.  Appendix C  41  XIV.  Acknowledgments  53  12  - iv III.  LIST OF TABLES  Table §  page  I.  Biochemical Characteristics of Y e r s i n i a Species  42  II.  Biochemical Features of Yersinia Biogroups  43  III.  Summary of Patient and Organism C h a r a c t e r i s t i c s  44  IV.  Relationship Between I n V i t r o Yersinia Virulence Associated Assays and Acute Enteric Disease (All Isolates Included)  46  V.  Relationship Between In V i t r o Y e r s i n i a Virulence Associated Assays and Acute Enteric Disease (Excluding Strains Isolated From Patients With Inflammatory Bowel Disease)  47  VI.  Relationship Between In V i t r o Y e r s i n i a Virulence Associated Assays and Mouse Virulence (All Isolates Included)  48  VII.  Relationship Between I n Vi tro Y e r s i n i a Virulence Associated Assays and Mouse Virulence (Excluding Strains Isolated From Patients With Inflammatory Bowel Disease)  49  VIII.  Comparison of P o s i t i v e Virulence Associated C h a r a c t e r i s t i c Expression Between IgA P o s i t i v e (2+) and Negative (1+ or -) Strains  50  IX.  Comparison of Mouse Serological Response to Human Intestinal Response  51  Comparison of Days Cold Enrichment to Recovery of IgA Stimulating Organisms  52  X.  - V -  IV.  LIST OF FIGURES  Figure § 1  page Typical Indirect Immunofluorescence SIgA Assay Results  21  - 1 -  V.  INTRODUCTION  The genus Yersinia consists of gram negative fermentative b a c i l l i belonging to the Enterobacteriacae family.  O r i g i n a l l y members of the  genus Pasteurella, these organisms were assigned to a new genus in 1944 based on their oxidase negativity (1), and later, in 1964,  they were  separated into three species: Y.pestis, Y.pseudotuberculosis. and Y . e n t e r o c o l i t i c a (2). Plague in man,  Y.pestis is the known causative agent of Bubonic  Y.pseudotuberculosis  is associated with granulomatous  lesions of the intestine, and Y.enterocolitica is associated with a v a r i e t y of enteric disorders including diarrhea, abdominal pain, g a s t r o e n t e r i t i s , and mesenteric lymphadenitis (3-6).  A.  Yersiniae Characteristics The f i r s t report of Y.enterocolitica-related enteric i l l n e s s came  from Carlsson et a! in 1964 who mesenteric  recovered the organism from the  lymph nodes of a patient with acute terminal i l e i t i s (7).  In  1966 Winblad e_t a_i documented patients culture and sero-positive with V . e n t e r o c o l i t i c a who had presented with acute diarrhea or a p p e n d i c i t i s - l i k e symptoms (8).  Although the majority of  Y . e n t e r o c o l i t i c a isolates reported were primarily r e s t r i c t e d to Northern Europe, Canadian isolates were f i r s t reported in 1967  (9).  Since then  Y . e n t e r o c o l i t i c a has been increasingly associated with acute,  - 2s e l f - l i m i t e d enteric i l l n e s s and several p o s t - i n f e c t i o n syndromes, such as p o l y a r t h r i t i s and erythema nodosum (5,10-13). also been associated with pseudoappendicitis surgery (14).  Y . e n t e r o c o l i t i c a has  i n children resulting in  In addition, severe yersiniae associated septicaemia in  children and immunosuppressed patients has been documented (15,16). In 1980,  a series of atypical Y.enterocolitica-1ike organisms were  subclass i f i e d on the basis of DNA-DNA h y b r i d i z a t i o n homology (17) and unique biochemical properties into three new species: Y.frederikseni i . V.kristenseni i . and Y.intermedia  (18-20).  Table I outlines the  biochemical properties of these y e r s i n i a e and those of two additional species, Y.ruckeri and Y.aldovae (21), solely found as aquatic isolates.  Y . e n t e r o c o l i t i c a does not refer to a single organism.  Instead, this species may be subdivided by biochemical r e a c t i v i t y into 8 biotypes  (Table II) and by 56 1ipopolysaccharide 0 antigens and 19  f l a g e l l a r H antigens  into serotypes  (22).  When l a b e l l i n g a s t r a i n  however, only the biotype and 0 antigen type are routinely included. Phage typing systems which further d i f f e r e n t i a t e the serotype/biotypes have also been developed,  but are usually reserved for epidemiologic  studies (23). Serotype  (24) and biotype (25) have both been associated with the  organism's pathogenic  potential.  In 1983,  the World Health Organization  outlined the syndromes associated with various serotypes 0:3  and 0:9 were termed pathogenic  (10).  Types  as they were associated only with the  " c l a s s i c " g a s t r o e n t e r i t i s syndrome in man.  "Other" serotypes, ie 4, 5,  6, 7, 8, 13, etc, were found only in healthy carriers or cases of  - 3 supra-infection and were therefore considered non-pathogenic.  This same  report noted that strains belonging to Biotype 1 were not usually associated with human disease.  Since then there have been several  reports of infection with these "non-pathogenic" strains (26-31), ie Biotype 1 strains have been implicated i n septicaemia cases (28), 0:5, 0:5,27, 0:6,30, 0:6,31, and 0:21 in human e n t e r i t i s cases (32-35), and 0:8 in food bourne outbreaks  (36).  The varied reports of pathogenic  potential may be the result of varied patient ages across the studies, i.e. C a p r i o l i et a i noted a r i s e in the incidence of "other " serotype with increasing patient age.  It is important to note that some  "pathogenic" strains have also been recovered from healthy human and animal c a r r i e r s (10,37,38).  Currently the most commonly pathogenic  strains are 0:3, 0:5,27, 0:8, and 0:9. Yersinia species are p s y c h o t r o p i c and w i l l therefore grow at temperatures  lower than competing  flora.  In 1974 Eiss (45) documented a  40% increase in yersiniae recovery after cold enrichment, however the usefulness of cold enrichment has been questioned.  in the recovery of pathogenic  organisms  Van Noyen (46) reported that cold enrichment did  not s i g n i f i c a n t l y increase recovery of serotypes 0:3 and 0:9 although increased recovery of other serotypes was found. Y . e n t e r o c o l i t i c a sensu s t r i c t o , and the newly defined strains have been isolated from a variety of environmental and c l i n i c a l sources including human and animal stools, f i s h , small mammals, water, milk, raw and cooked pork, tofu, and ice cream (39-44).  Y.kristenseni i .  Y.frederikseni i , and Y.intermedia have primarily been isolated from the  - 4 environment or skin and wound infections and were not o r i g i n a l l y associated  with enteric i l l n e s s  (18-20).  Recently, however, these  organisms have been recovered from patients presenting with acute enteric symptoms (6,32).  B.  Virulence  Associated Characteristics  Virulence been associated  of Y.enterocolitica and related organisms has repeatedly with a v a r i e t y of i n vivo and j_n v i t r o c h a r a c t e r i s t i c s .  In vivo assays are animal model based, and i n v i tro assays range from d i f f e r e n t i a l media to plasmid analysis. In vivo virulence has been defined keratoconjunctivitis  in guinea pigs  death after intraperitoneal  as the a b i l i t y to cause  (Sereny Test)(47) , to cause mouse  i n j e c t i o n (48), and to cause mouse diarrhea  or to invade the intestine and deep organs (spleen and l i v e r ) of rabbits and mice after oral challenge (49-51).  The guinea pig Sereny Test is  limited in i t s usefulness as a yersiniae virulence test as some commonly pathogenic strains ( i . e . serotypes 0:3, 0:9, and 0:5,27) f a i l c o n j u n c t i v i t i s response (50).  to evoke a,  However, when mice are u t i l i z e d in place  of guinea pigs the assay's s e n s i t i v i t y does increase s i g n i f i c a n t l y (52).  Overall  the mouse is currently the animal model of choice as  mouse symptoms following  inoculation v i a numerous routes c l o s e l y mimic  those of humans, i.e. intravenous or i n t r a g a s t r i c inoculation y i e l d s mouse i n t e s t i n a l lesions h i s t o l o g i c a l l y similar to those seen in humans (50,53).  Injection of d i l u t e b a c t e r i a l suspensions intraperitoneally  (i.p.) into mice w i l l detect  the virulence of numerous serotypes.  The  - 5 spectrum of the i.p. assay can be further improved by the addition of iron dextran to the inoculum  (54).  Oral inoculation mouse models with  subsequent mouse diarrhea better p a r a l l e l the events of human disease, but onset of mouse diarrheal symptoms may be d i f f i c u l t to assess with accuracy.  I n f i l t r a t i o n of mouse deep organs, l i v e r and spleen,  following oral challenge with Y e r s i n i a species provides the simplest method of assessing virulence.  This model correlates well with animal  models, yet has an easier, more definable end point, i . e . <10 orgs/spleen considered avirulent and 50-100 orgs/spleen considered virulent. In v i tro virulence associated c h a r a c t e r i s t i c s include a requirement of calcium for growth (55), a b i l i t y to autoagglutinate (56), absorption of a haemin type dye, Congo Red (57), hydrophobicity (58), expression of outer membrane proteins (74), and carriage of a 40-48 Megadalton (Mda) DNA plasmid  (59), a l l of which are expressed  in v i r u l e n t strains at  37°C but not at 25°C. The requirement  of calcium for growth by v i r u l e n t Y e r s i n i a species  was f i r s t reported in 1959 by Higuchi et aj_ (60).  They documented  v i r u l e n t Y.pestis strains requiring the cation for growth in l i q u i d media at 37°C.  When starved for calcium these v i r u l e n t strains  undergo r e s t r i c t i o n , a slow down of metabolism, involving a reduction in stable RNA and nucleotide triphosphate formation and decreased energy stores.  adenylate  Growth on calcium d e f i c i e n t media at 37°C is therefore  pin-point in nature.  Although no mechanistic role for calcium  dependance has been elucidated, this phenomenon may relate to the low i |  intraphagosomal  Ca  ||  and Mg  ion concentrations encountered during  - 6 ingestion by mammalian c e l l s  (55).  Since 1959 calcium dependance of  yersiniae has been frequently associated with the organism's a b i l i t y to cause disease in animals  (55) and man (61).  c h a r a c t e r i s t i c has been assigned  More recently this  to a s p e c i f i c , highly conserved 9kb  region on the virulence associated plasmid (62).  It is important to  note that this c h a r a c t e r i s t i c has been detected in environmental non-pathogenic isolates as well as c l i n i c a l  isolates  (63,64).  Autoagglutination of Yersinia species in tissue culture media at 3 7 ° C was reported by Laird and Cavanaugh in 1980 (56). that v i r u l e n t strains had a greater avirulent s t r a i n s .  They noted  tendency to clump at 3 7 ° C than  In 1985 Prpic et a l (58) described autoagglutination  by Laird and Cavanaugh's method as a r e l i a b l e predictor of mouse virulence in organisms carrying an additional virulence associated c h a r a c t e r i s t i c , Congo Red absorption (see below).  Autoagglutination has  been associated with the expression of two outer membrane proteins, V and W antigens,  o r i g i n a l l y defined in Y.pestis and is thought to result  from development of a protein fiber matrix linking the c e l l s (62,65).  together  As with calcium dependance, autoagglutination has been  associated with the virulence plasmid although in this case no specific l o c i has been elucidated  (65,66).  Y . e n t e r o c o l i t i c a species do not generally produce exogenous siderophores and therefore r e l y on the presence of free f e r r i c iron in the environment, or uptake of siderophores released by other organisms in their v i c i n i t y , (17).  i . e . desferrioxamine released by Streptomyces  Absorption of Congo Red is thought to p a r a l l e l  pilosus  iron absorption,  - 7 a c h a r a c t e r i s t i c with great advantages to organisms attempting survive in the mammalian host.  Absorption was  to  o r i g i n a l l y associated  with virulence in Y.pestis (67,68), but has more recently been documented in Y . e n t e r o c o l i t i c a species (57).  In 1987,  the expression o f  a series of high molecular weight Iron Regulated Proteins (IRPs) on the outer membrane of Y e r s i n i a species under iron starvation conditions documented (69).  was  Such expression could account for iron or congo red  uptake although absorption studies on these organisms were not included in their report.  In addition, aerobactin producing  Y.frederikseni i , Y.kristenseni i . and Y.intermedia reported  (70).  Although Y . e n t e r o c o l i t i c a and  strains of  have recently been  V.pseudotuberculosis  strains examined in the same study did not produce the hydroxymate siderophore,  i t s role in potential iron absorption should not be  ignored. Several studies have documented a family of 40-48 Mda present  plasmids  in Y . e n t e r o c o l i t i c a strains that can be associated with  virulence (48,52,60,71,72,73,74).  The f i r s t of this series was  documented during a milk related y e r s i n i o s i s outbreak in 1980  (47).  Its  association with virulence and tissue invasiveness was established through ethidium bromide curing experiments (47), and at this time i t was  found that repeated  the plasmid.  subculture at 37°C also resulted in curing o f  Since 1980,  50-100% DNA  homology has been shown to exist  within Y . e n t e r o c o l i t i c a plasmids and 50% DNA  homology between  Y . e n t e r o c o l i t i c a and Y.pestis plasmids, perhaps indicating a common ancestral o r i g i n (75).  In addition to calcium dependance and  - 8autoagglutination plasmid carriage has been associated with  expression  of a unique series of outer membrane proteins (74), c y t o t o x i c i t y to human e p i t h e l i a l c e l l s  (73,75), and i n h i b i t i o n of phagocytosis and  chemiluminescence of normal human neutrophils (76).  It is important to  note that non-pathogenic strains have also been shown to carry plasmids within this size range, and that plasmid-less pathogenic strains have been documented (77).  A recent study has suggested the existence of  movable genetic elements in Yersinia species that could account f o r the presence of plasmid  c h a r a c t e r i s t i c s in the absence of a plasmid (78).  Each of the _in v i tro c h a r a c t e r i s t i c s discussed above has some a b i l i t y to d i f f e r e n t i a t e organisms with potential virulence in man (79), but none considers the human response to the organism. The f i r s t objective of this study was to compare the i n v i tro virulence associated c h a r a c t e r i s t i c s of Y . e n t e r o c o l i t i c a and Y.enterocolitica-1ike organisms recovered  from symptomatic patients to  animal virulence, the gold standard of bacterial virulence, and to the disease presentation in humans.  C.  Coproantibody Secretory Immunoglobulin A In the c e l l s of the i n t e s t i n a l  lamina propria (Gut Associated  Lymphoid Tissue) there is a predominance of Immunoglobulin A (IgA) producing (79).  plasma c e l l s , accounting  for >80% of the Ig secreting c e l l s  Two IgA monomers are linked v i a d i s u l f i d e bonds to a 15,000mw  J-chain and a c t i v e l y transported out of the lamina propria (79).  - 9 E p i t h e l i a l c e l l s synthesize  a glycoprotein or secretory component  which in turn acts as a basal receptor f o r the IgA dimer. (SIgA) i s then transported lumen and released.  through the e p i t h e l i a l c e l l  This complex  layer to the gut  This type of active transport ensures i t s presence  in large quantities in i n t e s t i n a l  secretions.  Of the immunoglobulins, SIgA is the best suited to deal with antigenic material  in the gut lumen as i t r e s i s t s phagocytosis and  proteolysis by i n t e s t i n a l enzymes, is quadravalent, and has an a f f i n i t y for the mucosa (80).  SIgA i s thought to be the most important  resistance mechanism for b a c t e r i a l elimination as i t can both agglutinate  the organisms and act as an immune b a r r i e r in the intestine  by preventing  adherence and thereby invasion of the organisms (81).  This type of blocking a c t i v i t y is preferred in the gut as i t does not activate any further antimicrobial a c t i v i t y , i.e. complement a c t i v a t i o n , that could lead to degranulation Several  recent  and tissue damage.  studies have documented a r i s e in serum t i t r e of  yersiniae s p e c i f i c antibody early in i n f e c t i o n (82-84) primarily accounted for by IgA class antibodies  in p a r t i c u l a r the 11S or dimer  form normally found in secretions, however the r e l i a b i l i t y of serological diagnosis of yersiniae i n f e c t i o n remains controversial. Agglutination assays, either by tube or m i c r o t i t r e , have great potential for c r o s s - r e a c t i v i t y as whole organisms are u t i l i z e d although high antibody t i t e r s can be considered t i t e r s may r e s u l t from some underlying  (85).  In addition,  diagnostic, low antibody  i l l n e s s , i . e . co-infection or  immunodeficiency, and cannot exclude y e r s i n i o s i s .  -10In addition, agglutination assays detect primarily IgM and IgG as IgA does not agglutinate.  In fact, the presence of IgA may  even decrease  the apparent t i t r e of IgM and IgG by binding to antigenic sites and blocking IgM and IgG interactions (84).  Although ELISA assays provide a  more sensitive method for detecting class s p e c i f i c antibody, they s t i l l do not overcome the problems associated with IgA blocking (85,86).  In  addition to methodological problems, no c r i t e r i a exist for ascribing significance to an increased IgA t i t r e (87), i . e . healthy individuals often exhibit high background antibody levels to y e r s i n i a e .  Therefore  the diagnostic applications of serum IgA levels in a case of suspected y e r s i n i o s i s are limited, and any serology for this purpose requires at least one sera from both acute and convalescing phases  (13,88).  Overall, in spite of the d i f f i c u l t i e s discussed, an increased IgA t i t r e has been implicated in the prediction of post-infection aseptic a r t h r i t i s , thereby remaining important  (85,87).  In 1971 Reed and Williams (89) documented the presence of Secretory IgA (SIgA) class s h i g e l l a agglutinating antibodies in faeces from patients with recent onset diarrhea.  In the past 25 years several other  investigators have reported coproantibody responses to enteric in r e c t a l mucosa and faeces using indirect immunofluorescence (90-92).  organisms assays  Although no mechanistic role has been suggested, a good  association between coproantibody production and stage of enteric i l l n e s s was  clearly  demonstrated.  The second, o b j e c t i v e of this study was an immunological  to determine whether or not  marker of human immune response, coproantibody SIgA,  correlated f i r s t with human disease presentation and secondly with any of the established virulence associated markers including mouse virulence. It was suggested by Sarasombath et aj. in 1987 (93) that systemic antibody responses and i n t e s t i n a l SIgA responses were linked during the course of S.typhi infections. yersiniae i n f e c t i o n .  The  Such a linkage p o t e n t i a l l y exists during  third objective  of this study was to compare  organisms e l i c i t i n g varied human i n t e s t i n a l serological t i t e r s in a mouse model.  immune responses to  - 12 VI.  A.  METHODS  SPECIMENS AND ORGANISMS  Faecal specimens were received in the UBC-HSCH Microbiology Laboratory in Cary-Blair Transport  Media or s t e r i l e containers and  routinely processed for detection of enterics as previously described (6).  Briefly,  faecal material was plated on CIN Y e r s i n i a Selective Agar  (PML Microbiologicals) for 18-24 hours at 37°C.  Remaining specimen  was incubated  at 4°C in broth culture and subcultured  and  Colonies with the c h a r a c t e r i s t i c bulls-eye appearance were  14 days.  a f t e r 1, 3, 7,  screened on T r i p l e Sugar Iron, Urea, Lysine, Indole, and m o t i l i t y assays (PML M i c r o b i o l o g i c a l s ) . Enterobacteriacae  Strains were further characterized on API 20E  System (API Laboratory Products). A l l  Y . e n t e r o c o l i t i c a strains were then biotyped and serotyped  by an  independent reference  simultaneously  laboratory.  Faecal specimens were  examined f o r the presence of parasites, but v i r a l cultures were not included. Original faecal specimens were maintained at -4°C u n t i l bacteriology was completed.  When a Yersinia species was recovered, the  faeces was processed for coproantibody SIgA detection.  Organisms were  maintained at -70°C i n Glycerol and Dimethyl sulfoxide (DMSO) u n t i l assayed f o r virulence. and  C l i n i c a l h i s t o r y of a l l patients was reviewed  i s presented in Table 3.  - 13 In cases where the organisms were isolated and frozen prior to the onset of this study, faecal supernatant was not available.  These  organisms were included in the virulence testing for comparison purposes, but were not tested for SIgA.  B.  ORGANISM VIRULENCE ASSOCIATED ASSAYS  1.  Autoagglutination Interpretation of autoagglutination is commonly based on subjective observation of complete  tube clearing.  Several  authors have expressed d i f f i c u l t y in assessment of autoagglutination with Laird and Cavanaugh's method (56), i . e . strains f a i l i n g to grow in tissue culture media, high rate of f a l s e p o s i t i v e s , varying results dependant on inoculum size, and f a u l t y interpretation as a result of p a r t i a l l y cleared tubes (94,95).  For these reasons the method of Laird and  Cavanaugh (56) was modified to include a spectrophotometric analysis of clearing.  Organisms were grown overnight in  tryptone yeast extract broth (pH 7.0)  (Difco) at 25°C,  centrifuged, washed in phosphate buffered saline (PBS), and 9 resuspended  to McFarlane Standard 3 (10  orgs/ml).  The  culture was divided, incubated 18 hours at 25°C or 37°C, and the o p t i c a l density (O.D.) read at 460 nm.  Known  autoagglutination positive and negative strains (Strains 700L and 700S, Dr.C.Pai, Calgary) were included in ten experimental  - 14 runs and the difference between twice their Standard Error of the Mean (Appendix A) values was calculated to be 0.1 O.D. units.  Strains where the O.D. at 25°C exceeded that at  37 C by 0.1 were interpreted as autoagglutination p o s i t i v e . 2.  Calcium Dependance Direct plating on Magnesium Oxalate Agar (Appendix B) as described by Higuchi and Smith (96) was used to detect calcium dependant s t r a i n s .  Colonies from organisms requiring calcium  for growth (Calcium Dependant) were <1 mm diameter after 36 hours at 37°C and those not requiring calcium (Calcium Independent) were >1 mm 3.  diameter.  Congo Red Absorption The Congo Red Acidmorpholinepropanesulfonic Acid agar  (Appendix  B) described by Prpic et aj. (57) was used to assess absorption of Congo Red.  Organisms that a c t i v e l y take up the dye form red  colonies after 36 hours at 37°C and are scored as p o s i t i v e , those that do not form white or s l i g h t l y pink colonies and are scored as negative. 4.  Plasmid P r o f i l e Presence of a 40-48 Mda plasmid was determined with a modified Birnboim and Doly Alkaline Lysis Extraction method (97). Organisms were grown overnight in beef heart infusion broth (Difco) at 25°C and harvested by centrifugation, washed, and 9 resuspended  to 10  orgs/ml  in PBS.  C e l l s were resuspended in  lOOul fresh, c h i l l e d Lysis Buffer (50mM Glucose, lOmM EDTA,  - 15 25mM Tris-HCl  (pH 8.0), and 4mg/ml Lysozyme) and allowed to  stand 5 minutes at room temperature walls.  to weaken the c e l l u l a r  200ul of fresh, c h i l l e d Alkaline Buffer (0.2 N NaOH and  1% SDS) was added, mixed twice by inversion, and allowed to stand 5 minutes on ice to l y s i s c e l l s .  150ul fresh, c h i l l e d  Acid Buffer (60 ml Potassium Acetate, 11.5 ml G l a c i a l Acetic Acid and 28.5 ml d i s t i l l e d ^ 0 )  was added, inverted and  gently vortexed for 10 seconds, and allowed to stand 5 minutes on ice to p r e c i p i t a t e chromosomal DNA.  Samples were  centrifuged (12,000 xg for 5 min) and the supernatant added to an equal volume of Phenol/Chloroform.  Samples were then  vortexed and centrifuged (12,000 xg for 2 min) to extract contaminating proteins.  The supernatant was removed and  allowed to stand in 2 volumes of 70% ethanol at -20°C for 20 minutes to precipitate plasmid DNA.  Samples were  recentrifuged, additional ethanol poured o f f , and the p e l l e t a i r dried.  P e l l e t s were resuspended  in 40ul of RNase Buffer  (30mM T r i s (pH 8.0), 5mM EDTA, 50mM NaCl, and 20 ug/ml RNase) to eliminate contaminating RNA. of Stop Mix  15ul of DNA solution and 5ul  (50 ml Glycerol, 7ml EDTA (0.5 M), 5mg Bromphenol  Blue, in 100 ml D i s t i l l e d HgO)  were loaded on a T r i s Borate  Buffered Agarose Gel (8.9mM T r i s Base, 8.9mM Boric Acid, 0.2mM EDTA, and 0.6% agarose  in d i s t i l l e d HgO).  Gels were  electrophoresed for 2 hours at 50mV on a Mini-Gel  Apparatus  (Bethesda Research Laboratories) and post-stained in 5ug/ml  - 16 ethidium bromide f o r 20 minutes.  Standards included T4, T5,  T7, and lambda phage DNA (Sigma) and a s t r a i n known to carry the 40Mda plasmid (700S).  Those strains with plasmids banding  in the same region as the control s t r a i n were considered plasmid carrying. 5.  Mouse Virulence For animal virulence assessment was modified.  the method of Bakour et aj_ (50)  Adult Swiss mice (Charles River) were dehydrated  for 24 hours then allowed to drink ad 1ib from a 0.1% peptone  9 broth containing 10 orgs/ml, from an overnight tryptone soy agar plate (Difco), f o r 36 hours.  Mice were s a c r i f i c e d by  carbon dioxide and their spleens homogenized then plated on C1N y e r s i n i a selective agar (PML Microbiologicals) f o r 24 hours at 37°C.  Colonies with the c h a r a c t e r i s t i c bulls-eye  appearance  were further characterized on TSI and Urea slants (PML Microbiologicals).  A s t r a i n was defined as mouse v i r u l e n t i f  the homogenized spleen contained more than 100 organisms. C.  HUMAN C0PR0ANTIB0DY SIGA RESPONSE  Intestinal SIgA response was assessed with a newly established semi-quantitative indirect immunofluorescence  assay.  Faecal samples  were stored at -4°C u n t i l a yersiniae species was recovered. Faecal specimens were then diluted 1:1 with PBS, centrifuged (30 min at 15,000xg) and the supernatant stored at -70°C.  200 ul of s e r i a l  - 17 -  supernatant  d i l u t i o n s (1/10,1/50,1/100) were incubated with 40 ul of  9 o 10 orgs/ml i n PBS on covers l i p s (18 hours at 25 C). Covers l i p s were a i r dried, dip washed in d i s t i l l e d water, and incubated with 120 ul of FITC labelled goat anti-human IgA (Sigma) (30 min at 37°C).  Slips  were dip washed, a i r dried, inverted and mounted for fluorescence microscopy.  Results were defined as follows: no fluorescence - ; single  organisms 1+; homogeneous microagglutination 2+ (Figure 1).  D.  MURINE SEROLOGY  Adult Swiss mice were o r a l l y inoculated with IgA 2+, 1+, and strains as described above.  Animals were s a c r i f i c e d 7 days after  inoculation and their heart blood immediately withdrawn.  Blood was  centrifuged and sera stored at -70°C u n t i l time of assay. Formalinized antigen was prepared  as described by Winblad (98).  Organisms were grown 1-2 days on Blood Agar plates (PML Microbiologicals) at room temperature, harvested by centrifugation, and washed in saline before suspending in 10% Buffered Formalin.  A loopful  of antigenic material was streaked on Blood Agar plates and incubated overnight at 37°C to check s t e r i l i t y .  P r i o r to the microagglutination  assay the antigenic material was washed in saline and resuspended to McFarlane Standard 5.  0.5 ml of sera was s e r i a l l y d i l u t e d in duplicate  round bottom m i c r o t i t r e plates (Becton Dickinson) and 0.5 ml of formalinized antigen was added to each well.  Plates were spun at 700  - 18 rpm  for 10 minutes.  agglutination  E.  The inverse of the last d i l u t i o n showing halo-type  was recorded as the antibody t i t r e .  DIAGNOSTIC TEST EVALUATION  C l i n i c a l evaluation of any diagnostic accuracy, that (99).  i s , how close  test is based on the test's  is the test measurement to the true value  The true value in many cases is unreadable, therefore new tests  are compared against established  or "gold standard" tests.  In this  study, two gold standards of organism virulence were u t i l i z e d . virulence  Animal  is a widely accepted standard of organism pathogenicity, and  association  with acute human enteric symptoms further approximates the  true human pathogenic potential of the organism. When selecting a test for use i t becomes important to know the p r o b a b i l i t y that the test w i l l be positive when applied that  to an organism  is t r u l y v i r u l e n t , i.e. i t s s e n s i t i v i t y , and the probability  the test w i l l be negative i f applied avirulent,  i.e. i t s s p e c i f i c i t y .  selecting a test for use, test r e s u l t .  to an organism that  that  is t r u l y  Although these values are useful when  they are of limited use in evaluating a single  In that case i t is necessary to know the p r o b a b i l i t y  an organism w i l l be v i r u l e n t  that  i f the test result is p o s i t i v e , that i s ,  the predictive value of the test needs to be calculated. predictive value refers to the r a t i o of true positives  Positive  to test  positives  and negative predictive value refers to the r a t i o of true negatives to test negatives.  Predictive values are influenced  not only by the  - 19 s e n s i t i v i t y and s p e c i f i c i t y of the test, but also by the p r o b a b i l i t y o f virulence  in the population prior to testing (99). Testing of a  population with a high incidence values.  of positives increases  the predictive  Appendix C shows sample calculations of positive predictive  value, negative predictive value, s e n s i t i v i t y , and s p e c i f i c i t y for the Coproantibody SIgA assay.  However, since this study was concerned  primarily with the p r o b a b i l i t y that an organism i s pathogenic in man when the test value i s p o s i t i v e , only positive predictive values for remaining assays are reported in the text.  - 20 -  VII.  RESULTS  Figure 1 i l l u s t r a t e s typical fluorescence microscope f i e l d s for a 2+, a 1+, a negative, and a negative  in phase contrast.  A 2+ result is  defined as large clusters of agglutinating organisms fluorescing bright apple green and is indicative of a strong s p e c i f i c IgA class antibody response (Figure IA). A 1+ result is defined as single fluorescent organism and is indicative of a weak s p e c i f i c antibody response (Figure IB).  When the cultured yersiniae species stimulates no antibody  response, the f i e l d fluorescence  is apparently  can not be accounted for by an absence of bacteria however  as organisms are detected ID).  empty (Figure 1C). This absence of  in phase contrast of the same f i e l d  (Figure  Supernatant only and organism only control s l i d e s (photos not  included) showed some homogeneous background fluorescence, but no organism-like  structures.  Faecal supernatants from salmonella 65, 66, 67), or C l o s t r i d i a  (Pt# 64),  Campylobacter  (Pt#  (Pt# 68) associated diarrhea patients and  asymptomatic, organism-negative patients (Pt# 69, 70, 71) were examined for  cross-reactive agglutination with y e r s i n i a .  agglutinating c r o s s - r e a c t i v i t y was found.  No s p e c i f i c  However,  non-agglutinating  halo-type staining of single organisms was detected when supernatants were tested against control organisms including Campylobacter,  Clostridia,  and Escherichia c o l i .  salmonella,  - 21 -  • Figure 1 - Typical Indirect Immunofluorescence SIgA Assay R e s u l t s . A, ++, Homogenous M i c r o a g g l u t i n a t i o n B, +, Single Organism and Non-Specific Background; C, - , No S p e c i f i c Fluorescence; D, - i n Phase Contrast, Non-clumping Organisms Coating S l i d e .  Table III contains a complete l i s t i n g of a l l patients and organism c h a r a c t e r i s t i c s used i n t h i s research and referred to i n the t e x t . Table IV documents the a b i l i t y of the v i r u l e n c e associated assays to d i f f e r e n t i a t e organisms from patients with acute e n t e r i c symptoms. Although not absolute, the p o s i t i v e p r e d i c t i v e value of SIgA response for acute disease (P.P. - 85.7%) was greater than that of any single in v i tro (Autoagglutination - 56.0%, Calcium Dependance - 56.5%, Congo Red - 58.5% , Plasmid Carriage - 53.7%) and p a r a l l e l e d that of mouse v i r u l e n c e ( P . P . - 73.7%).  Of note, exceptions to t h i s a s s o c i a t i o n  included patients # 6, 8, and 47 who suffered inflammatory bowel disease  - 22 yet e l i c i t e d a strong SIgA response 10, 24, and 25 who SIgA response.  (see discussion) and patients #9,  presented with acute diarrhea yet had a low or absent  Patient #10 was  shown to be Clostridium d i f f i c i l e  toxin  p o s i t i v e , although the bacterium was not cultured, and patient #9 carried B. hominis. When organisms  recovered from patients with inflammatory bowel  disease are excluded from data analysis on the basis that these patients may be presenting with diarrheal symptoms as a result of underlying i l l n e s s , SIgA production remains a better predictor of acute enteric symptoms (P.P. - 75.0%) than any other i n v i t r o assay (Autoagglutination - 52.2%, Calcium Dependance - 52.3%, Congo Red - 55.3%  , Plasmid  Carriage - 51.4%) and s t i l l p a r a l l e l s the gold standard of mouse virulence (P.P. - 70.6%)(Table V). Overall positive predictive values (P.P.) of the i n v i t r o virulence associated assays for mouse virulence were: Autoagglutination - 44.0%; Calcium Dependance - 39.1; Congo Red Absorption - 34.1%; and Plasmid Carriage - 29.3%  (Table VI), although these values fluctuated s l i g h t l y  with the yersiniae species. al  These results agree with those of Prpic e_t  (58), which found autoagglutination to be the most r e l i a b l e predictor  of mouse.virulence  in congo red positive organisms.  The newly  established coproantibody SIgA assay predicted mouse virulence better than any of the established virulence associated assays (P.P. 85.7%)(Table VI) and i f organisms  recovered from patients with  inflammatory bowel disease are again excluded the SIgA assay appears to be an absolute predictor of mouse virulence (Table VII).  - 23 Organisms cultured from SIgA positive (2+) patients have a higher incidence of some virulence associated c h a r a c t e r i s t i c s .  Table VIII  documents no s i g n i f i c a n t difference between SIgA 2+ and SIgA 1+/2 strains in expression of congo red absorption (X =0.00), calcium 2 2 dependance (X =0.14), and plasmid carriage (X =0.15), however, a 2 s i g n i f i c a n t difference was apparent with mouse virulence (X =17.65; p<0.002) and autoagglutination (X =5.39; p<0.05). 2  Table IX relates the a b i l i t y of organisms to stimulate a human coproantibody response to their a b i l i t y to e l i c i t a serological in another animal, the mouse, s i m i l i a r l y o r a l l y challenged. d e f i n i t e conclusions could be drawn from only four organisms,  response  Although no i t was  noted with interest that organisms coming from SIgA producing patients (2+) e l i c i t e d high antibody responses from low SIgA producers response.  in the mouse whereas those coming  (1+ or -) e l i c i t e d l i t t l e or no antibody  These findings are consistent with those of Sarasombath et al  (93) that a c o r r e l a t i o n responses does e x i s t .  between i n t e s t i n a l and systemic antibody Simultaneous  human sera was not available from  these patients, therefore similar experiments  i n man were not included  in the study. Table X compares the days cold enrichment the type of SIgA stimulation.  required f o r recovery to  The number of SIgA stimulators d i d not  increase or decrease substantially from 0 to 14 days and although a s l i g h t increase in SIgA non-stimulators was seen in the f i r s t week, this increase d i d not persist into the second week.  Overall, 58.3% of the  mouse v i r u l e n t , antibody-indueing yersiniae were recovered only after cold  enrichment.  VIII.  DISCUSSION  The virulence test results in this study agree with those of Kay et al  (100) that no single virulence associated assay correlates well with  yersiniae virulence as assessed by the mouse model.  In addition, this  study demonstrates a r e l a t i o n s h i p between coproantibody SIgA stimulation, mouse virulence, and acute human enteric i l l n e s s . As discussed e a r l i e r , V.frederikseni i , Y.kristenseni i . and V.intermedia were not o r i g i n a l l y considered recently been recovered  enteric pathogens but have  from patients presenting with acute diarrhea.  Prpic e_t aj_ (58) found that some V. enterocol i t ica-1 ike organisms expressed some of the j_n v i tro virulence associated c h a r a c t e r i s t i c s , but were s t i l l mouse avirulent.  S i m i l a r l y , this study documents the  expression of one or more _in v i tro c h a r a c t e r i s t i c s in Y.frederikseni i . Y.intermedia, and Y.kristenseni i species.  It also documents the  existence of Y.frederikseni i . Y.kristenseni i , and atypical Y . e n t e r o c o l i t i c a strains ( i . e . 0:41,43, 0:36, etc) which are mouse v i r u l e n t and immunostimulatory i n humans. previously discussed observation  This i s consistent with the  that strains of Y . e n t e r o c o l i t i c a other  than 0:3, 0:5,27, etc, may be pathogenic in man. Although the usefulness question,  of cold enrichment techniques is s t i l l in  this study found i t to be important in recovery of some  e n t e r i c a l l y pathogenic y e r s i n i a e , i.e. 58.3% of the mouse v i r u l e n t ,  - 25 immunostiraulatory yersiniae were recovered  only after cold enrichment.  The existence of coproantibody SIgA in faecal specimens following enteric infections was Banvard (101)  documented as early as 1947 when Harrison &  reported the detection of agglutinating antibody in faecal  specimens from patients with both acute and chronic b a c i l l a r y dysentery.  Since then several studies have reported the presence of  high coproantibody t i t e r s in faeces patients with u l c e r a t i v e c o l i t i s and l i v e S. typhi vaccine  (91,92).  (102)  and  i n t e s t i n a l mucosa (90)  of  in volunteers o r a l l y immunized w i t h  In the current investigation the  presence of high SIgA t i t e r s in faecal specimens has been documented and strongly associated with acute enteric i l l n e s s and carriage of mouse v i r u l e n t organisms.  Of note, when patients with inflammatory bowel  disease were excluded from analysis because their underlying represented  disease  confounding data, the p o s i t i v e predictive value of SIgA for  mouse virulence became 100%.  In addition, this association was  not  dependent on the cultured yersiniae species. In 1951 Barksdale et aj. (102) noted that c e r t a i n strains of coliform b a c i l l i contained s h i g e l l a and salmonella  antigenic components common to c e r t a i n  species.  Such cross-reactions could  conceivably  limit the diagnostic value of a coproantibody assay, therefore appropriate  controls, including supernatants from  salmonella,  Campylobacter, or C l o s t r i d i a diarrhea patients and enteropathogen negative  asymptomatic patients, were included in this study.  Since  these specimens were negative, a strong agglutinating SIgA response appeared to be yersiniae s p e c i f i c . supernatants in this study was  However, as the number of control  low, no overall s p e c i f i c i t y could be  - 26 determined. Two exceptions to the association between SIgA response, acute disease, and mouse virulence in the current study were found in patients with Crohn's Disease and acute c o l i t i s , both of whom exhibited a strong SIgA response to an apparently avirulent yersiniae s t r a i n .  This  observation was consistent with Barksdale et ai's case of a chronic u l c e r a t i v e c o l i t i s patient from whom neither s h i g e l l a nor salmonella were isolated, yet whose coproantibodies agglutinated three salmonella species and four or more s h i g e l l a species (102).  Toivanen et aj_ (103)  suggested that persistence of yersiniae within the intestine, i . e .  in  e p i t h e l i a l or lymphoid tissues, could provide a stimulus f o r prolonged antibody production in cases of reactive a r t h r i t i s .  Such a mechanism  may also be involved in maintenance of antibody production in inflammatory bowel disease.  Monteira et a l (92) suggested that strains  of enteric bacteria which would produce only transient reactions i n n o n - c o l i t i c patients may be able to e l i c i t antibody production in mucosa of c o l i t i c patients.  Further explanations include the p o s s i b i l i t y that  mouse-virulent organisms  could lose their  temperature-dependent  virulence plasmid while in the gut lumen or that strains of yersiniae may exist which are immunostimulatory mouse model.  in humans yet inactive in the  The second explanation is supported by Hancock et al's  (104) finding that certain strains of mice are naturally resistant to some yersiniae s t r a i n s .  For the aforementioned  reasons, the  coproantibody SIgA assay as outlined here may produce f a l s e positives and should not be r e l i e d on to predict the animal virulence potential of  - 27 yersiniae strains recovered from patients with known inflammatory bowel disease . Carriage of yersiniae in the presence of another causal agent would account for cases of acute diarrhea where low or absent SIgA responses were detected.  The mouse avirulent nature of the cultured yersiniae  species in these cases and the observation that some of these serotypes are  normally found only in c a r r i e r s or in suprainfections (10) supports  this concept.  Two  such patients (Pt# 9, 10) carried b a c t e r i a l or  p a r a s i t i c pathogens which may have accounted for their symptoms.  No  additional organisms were cultured from the remaining patients (Pt# 24, 25), however, since electron microscopy of faecal samples was not done v i r a l l y induced e n t e r i t i s could not be excluded. A number of patients culture positive with a mouse v i r u l e n t yersiniae carried a concurrent parasite, i . e . B.hominis. D . f r a g i l i s , or I.butchli i .  These patients may represent concurrent yersiniae and  p a r a s i t i c infection, or infection with one organism predisposing the patient to the other.  The actual sequence of infection, however, could  not be determined from a single faecal specimen.  In these cases the  coproantibody SIgA assay may be of use in d i f f e r e n t i a t i n g transient non-immunostimulatory intestinal  yersiniae isolates from those e l i c i t i n g a strong  immune response.  -  IX.  In  summary t h i s  indirect  t h e s i s has o u t l i n e d  the use of a  assay without the need f o r  secretory  mouse v i r u l e n c e  than any o f  and appeared to be y e r s i n i a e  T h i s assay was  the e s t a b l i s h e d i n v i t r o specific.  In a d d i t i o n ,  i n man, was documented.  are  coproantibody  A positive  o f acute human e n t e r i c  n i n e Y . f r e d e r i k s e n i i and one Y . k r i s t e n s e n i i , non-pathogenic  specific  (SIgA) response and those who do n o t .  SIgA response was a b e t t e r p r e d i c t o r  the performance of a  from whom y e r s i n i a e  those who produce a s t r o n g y e r s i n i a e IgA  semi-quantitative  repeat specimens.  shown to d i s t i n g u i s h two types o f p a t i e n t s recovered:  -  SUMMARY  immunofluorescence assay which f a c i l i t a t e s  serological  28  (2+)  i l l n e s s and  assays o f  virulence  the v i r u l e n c e  p r e v i o u s l y thought  of  to be  - 29 -  X. BIBLIOGRAPHY  1  Van Loghem J J . The c l a s s i f i c a t i o n of the plague b a c i l l u s . of Microbiology and Serology, 10, 15, 1944.  2  Frederiksen U. A study of some Versinia pseudotuberculosis-1ike bacteria ("Bacterium enterocoliticum" and "Pasteurella X"). Proceedings of the XIV Scandinavian Congress of Pathology and Microbiology, Oslo, 103, 1964.  3  Mollaret HH, Thai E. Genus XI Yersinia. In RE Buchanan^ N Gibbons (eds) Bergey's Manual for Determinative Bacteriology 8 ed. 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Edwards^nd Ewing's I d e n t i f i c a t i o n of Enterobacteriacae. 4 ed E l s e v i e r , New York. 461, 1986.  39  APPENDIX A  EVALUATION OF AUTOAGGLUTINATION STANDARDS  Value  Autoagglutination  +  Number of Runs  Mean Value  10  10  0.0425  0.1268  0.0156  0.0224  Standard Error of the Mean Upper Boundary  0.0737  Lower Boundary  -  Twice the Standard Error of the Mean  0.0992  0.1729  =0.10  APPENDIX B  MAGNESIUM OXALATE AGAR  880 ml  D i s t i l l e d Water  40 gm  Blood Agar Base  Bo i1, Coo1, and Add: 40 ml  MgCl - 6H 0  80 ml  Sodium Oxalate  2  2  Autoclave 20 min at 121°C.  CRAMP AGAR  15 gm  Noble Agar  2 gm  Galactose  2 gm  Casamino Acids  1000 ml 10 ml  D i s t i l l e d Water Congo Red (5,000 ug/ pH 7.0  Autoclave 25 min at 121°C  - 41 -  APPENDIX C DIAGNOSTIC TEST EVALUATION SAMPLE CALCULATIONS  2x2  DATA TABLE  Gold Standard Value  + Diagnostic Test Value  +  a  b  -  c  d  a b c d  = = = =  # # # #  True Positives False Positives False Negatives True Negatives  Positive Predictive Value = a /a+b Negative Predictive Value = d /d+c S e n s i t i v i t y = a/a+c S p e c i f i c i t y = d/d+b EXAMPLE: 2x2  DATA TABLE FOR SIGA ASSAY Gold Standard Value  Diagnostic Test Value  + 1 2 -  2 0  14  P o s i t i v e Predictive Value = 12/12+2 = 85.7% Negative Predictive Value = 14/14+0 = 100% S e n s i t i v i t y = 12/12+0 = 100% S p e c i f i c i t y = 14/14+2 = 87.5%  TABLE I BIOCHEMIGAL CHAMCTERISTTCS OF YT^RSINIA SPECIES ^  Characteristic Motility Lysine Decarboxylase Ornithine Decarboxylase Urease Citrate Voges-Proskauer Indole Rhamnose Sucrose Cellobiose Melibiose Sorbose Sorbitol  2 1  '  1 0 5  ^  Y.pestis Y.pseudo- Y.entero- Y. i n t e r - Y.freder- Y.kris Y. Y. tuiberculosis c o l i t i c a media i k s e n i i t e n s e n i i ruckeri aldovae + + + + + + V + -  -  -  +  -  +  +  +  + + + + + + + + + +  -  --  V  + +  V  -  +  -  -  + +  -  -  -—  +  —  +  -  -  +  +  +  +  V  + + + + +  -  + +  -  V  +  +  -  + + +  + V  -  -  --  -  +  -  -  -  + + +  —  --  +  TABLE I I BICCHEMICAL FEATURES OF YERSINIA BIOGROUPS^ ^ 64  Criaracteristic  Biogroup 1  Lipase A c t i v i t y (Hydrolyzes Tween) E s c u l i n Hydrolysis Indole Production Acid Production From Xylose Voges-Proskauer Test Ornithine [Decarboxylase, Acid Production from Trehalose, and N i t r a t e Reduction Pymzinaraidase A c t i v i t y  +  "American" Strains  + -  1 2 S A ^  5  3  -  -  -  -  —  "/+  —  —  —  —  —  —  v  +  +  +  —  —  + + +  + + +  + +  +  + +  +  + +  +  +  +  + +  1 - Positive f o r a c i d production from sorbose and i n o s i t o l 2 - Negative f o r a c i d production from sorbose and i n o s i t o l  4  2  —  —  '-  -  44  TABLE I I I ~ SUMMARY OF PATIENT AND ORGANISM CHARACTERISTICS  Patient # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36  Species  Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.enterocolitica Y. e n t e r o c o l i t i c a Y. e n t e r o c o l i t i c a Y. e n t e r o c o l i t i c a Y. e n t e r o c o l i t i c a Y. errterocolitica Y.enterocolitica Y. f r e d e r i k s e n i i Y.f r e d e r i k s e n i i Y.f r e d e r i k s e n i i Y.f r e d e r i k s e n i i Y.frederiksenii Y.frederiksenii Y.f r e d e r i k s e n i i Y. f r e d e r i k s e n i i Y.frederiksenii Y.frederiksenii Y. f r e d e r i k s e n i i Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.f r e d e r i k s e n i i Y.frederiksenii Y.frederiksenii  Biotype Serotype 1,0:6,31 1,0:6,36 1,0:7,13 4,0:3 4,0:3 l,nt l,nt 1,0:5 1,0:4,32 1,0:6,30 1,0:7,8 1,0:7,13 1,0:36 1,0:41,43 l,nt 1,0:5 1,0:7,8 1,0:5,27 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND  Synptoms  Calcium Congo Red Autoagglu Plasmid Mouse IgA Response Virulence Dependance Absorption - t i n a t i o n P r o f i l e  Acute Diarrhea ++ Abdcminal Pain ++ Acute Diarrhea ++ Acute Diarrhea ++ Acute Diarrhea ++ Acute C o l i t i s ++ Acute Diarrhea ++ Crohn's Disease ++ Acute Diarrhea + Acute C o l i t i s + Fever NYD + Crohn's Disease + Weight Loss + Abdominal Pain + Rectal Bleeding + Abdominal Pain Abdominal Pain Septicaemia ND Acute Diarrhea ++ Acute Diarrhea ++ Hyporat^remia ++ Acute Diarrhea -HAcute Diarrhea ++ Acute Diarrhea + Acute Diarrhea Abdominal Pain Chronic Diarrhea Crohn's Disease Reactive A r t h r i t i s ND Acute Diarrhea ND Acute Diarrhea ND AIDS ND Acute C o l i t i s ND Acute Diarrhea ND Acute Diarrhea ND Abdominal Pain ND  +  + + + + + +  -+ + + + + +  + -  + + + +  -  +  —  +  D.fragilis  +  + +  +  +  +  +  —  —  —  —  —  •  —  Other Orqanism  + -  -  — — —  -  -  +  —  p• _ i  —  —  —  —  —  —  —  +  —  —  —  +  + +  +  —  —  —  —  —  +  +  —  +  -  —  —  —  —  —  + + + +  + +  +  —  —  + -  +  -  —  + + + + +  +  + + —  —  — •  -  —  —  —  +  + +  —  —  + + + + +  —  —  —  +  -  +  ;  + + + + —  -  + + + + —  —  B.harunis C.diff.Toxin  B.hcminis —  D. f r a g i l i s —  Salmonella — — — — — — — —  Trophozoites —  —  —  —  +  +  —  —  —  B.hcaninis  -  I.butchlii  +  +  + + +  —  —  +  +  —  —  B.haninis S.ireuchin —  -  -  45  TABLE I I I - SUMMARY OF PATIENT AND ORGANISM CHARACTERISTICS - CONT'D  Patient # 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72  Species  Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y.frederiksenii Y. k r i s t e n s e n i i Y.kristensenii Y. k r i s t e n s e n i i Y. k r i s t e n s e n i i Y.kristensenii Y.kristensenii Y.kristensneii Y.kristensenii Y.kristensenii Y.intermedia Y.intermedia Y.intermedia Y.irrtermedia Y.intermedia Y.intermedia Y.intermedia Y. intermedia S. enter i d i d i s C. j e j u n i C. j e j u n i C. j e j u n i C.difficile E.coli Negative Negative Negative  Biotype Serotype ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0157:H7 ND ND ND  Symptoms  Mouse Calcium Congo Red Autoagglu Plasmid IgA Response Virulence Dependance Absorption - t i n a t i o n P r o f i l e  Acute Diarrhea Abdorrtinal Pain Chronic Diarrhea Chronic Diarrhea Abdominal Pain Acute Diarrhea Abdominal Pain Acute Diarrhea Acute Diarrhea Acute Diarrhea Acute C o l i t i s Acute Diarrhea Abdominal Pain Acute Diarrhea Rectal Bleeding Acute Diarrhea AMcminal Pain Abdominal Pain Acute Appendicitis Nausea Acute Diarrhea Acute C o l i t i s Acute Diarrhea Abdominal Pain Acute Diarrhea Acute Diarrhea Abdominal Pain Acute Diarrhea Acute Diarrhea Acute Diarrhea Acute Diarrhea Acute Diarrhea Acute Diarrhea Asymptomatic Asymptomatic Acute C o l i t i s  ND ND ND ND ND • ND ND ND ND ND  ++  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND  -* -*  -** -4c*  -** -**  -* -* -*  -  -  -  +  -  -  -  --  -  ND ND ND ND ND ND ND ND ND  * - Tested against y e r s i n i a i s o l a t e s from patient # 1. ** - Tested against yersiniae i s o l a t e s from patients # 2, 4, 11, 18.  +  -  +  -  +  --  + + + + +  --  +  -  +  -  -ND ND ND ND ND ND ND ND ND  _  +  _  + + + + + + +  +  —  + + + +  —  + + + +  —  +  —  —  — ;  + + + + + + + +  +  + + +  —  —  + +  +  —  —  +  — :  —  +  + + + + + + + + + +  — —  —  + + + + + + —  + +  ND ND ND ND ND ND ND ND ND  Other Organism  i  — •  — — —  + —  — — —  ND ND ND ND ND ND ND ND ND  ND ND ND ND ND ND ND ND ND  — — —  B.hcminis Isospora —  Giardia — — —  B.hcminis — —  C. j e j u n i — — —  S.typimuriui — — — —  D. f r a g i l i s — — — — — — — — — — — —  -  - 46 -  TABLE IY RELATIONSHIP BETWEEN IN VITRO YERSTNTA VIRULENCE ASSOCIATED ASSAYS AND ACUTE ENTERIC DISEASE (All Isolates Included) (# True P o s i t i v e s / Test Positives)  Species  Autoagglu Congo Red Calcium Plasmid SIgA Mouse - t i n a t i o n Absorption Dependance Carriage Response Virulence  Y.enterocolitica Y.frederikseni i Y.kri stensenii Y.intermedia  3/6 7/14 3/4 1/1  4/8 13/21 3/5 4/7  4/5 5/12 3/5 1/1  4/6 9/19 5/8 5/8  7/8 4/5 1/1 ND  6/8 7/10 1/1 ND  Total  14/25  24/41  13/23  23/41  12/14  14/19  (56.0)  (58.5)  (56.5)  (53.7)  (85.7)  (73.7)  Value  {%)  - 47 -  TABLE V RELATIONSHIP BETWEEN IN VITRO YERSINIA VIRULENCE ASSOCIATED ASSAYS AND ACUTE ENTERIC DISEASE (Excluding Strains Isolated From Patients With Inflammatory Bowel Disease) (# True P o s i t i v e s / Test Positives)  Species  Autoagglu Congo Red Calcium Plasmid SIgA Mouse - t i n a t i o n Absorption Dependance Carriage Response Virulence  Y.enterocolitica Y.frederikseni i Y.kristenseni i Y.intermedia  3/6 7/14 2/3 ND  4/8 11/19 3/5 3/6  3/4 5/12 2/4 VI  3/5 8/19 4/7 4/7  5/6 4/5 0/1 ND  5/7 6/9 1/1 ND  Total  12/23  21/38  11/21  19/37  9/12  12/17  (55.3)  (52.3)  (51.4)  (75.0)  (70.6)  Positive Predictive Value (96) (52.2)  - 48 -  TABLE VI RELATIONSHIP BETWEEN IN VITRO YERSINIA VIRULENCE ASSOCIATED ASSAYS AND MOUSE VIRULENCE (All Isolates Included) (# True P o s i t i v e s / # Test Positives)  Species  Autoagglu -tination  Congo Red Absorption  Calcium Dependance  Plasmid Carriage  SIgA Response  Y.enterocolitica Y.frederiksenii Y.kristenseni i Y.intermedia  4/6 6/14 1/4 0/1  4/8 9/21 1/5 0/7  3/5 5/12 1/5 0/1  4/6 7/19 1/8 0/8  7/8 5/5 0/1 ND  Total  11/25  14/41  9/23  12/41  12/14  (34.1)  (39.1)  (29.3)  (85.7)  P o s i t i v e Predictive Value (%) (44.0)  - 49 -  TABLE VII RELATIONSHIP BETWEEN IN VITRO YERSINIA VIRULENCE ASSOCIATED ASSAYS AND MOUSE VIRULENCE (Excluding Strains Isolated From Patients With Inflammatory Bowel Disease) (# True P o s i t i v e s / # Test Positives)  Species  Autoagglu -tination  Congo Red Absorption  Calcium Dependance  Plasmid Carriage  SIgA Response  Y.enterocolitica Y.frederikseni i Y.kristenseni i Y.intermedia  4/6 6/14 1/3 ND  4/8 8/19 1/5 0/6  3/4 5/12 1/4 0/1  4/5 6/18 1/7 0/7  6/6 5/5 ND ND  Total  11/23  13/38  9/21  11/37  11/11  (34.2)  (42.9)  (29.7)  (100)  Positive Predictive Value (%) (47.8)  - 50 -  TABLE VIII  COMPARISON OF POSITIVE VIRULENCE ASSOCIATED CHARACTERISTIC EXPRESSION BETWEEN IGA POSITIVE (2+) AND NEGATIVE (1+ OR -1 STRATNS (# of Isolates With Positive Reaction)(%)  IgA Response  2 + 1+  /0  # Organisms  Autoagglu -tination  Congo Red Absorption  14 14  9(64.3) 2(14.3)  6(42.9) 5(35.7)  Calcium Dependance 7(50 0) 7(50.0)  Plasmid Carriage  Mouse Virulence  7(50.0) 5(35.7)  12(85.7) 0(0)  - 51 -  TABLE IX COMPARISON OF MOUSE SEROLOGICAL RESPONSE TO HUMAN INTESTINAL RESPONSE  SIgA Response  2+ 2+ 1+ 0  Species  Y.enterocolitica Y.enterocolitica Y.enterocolitica Y.frederiksenii  Serotype  0:6,36 0:7,13 0:5 ND  Murine Serum Antibody T i t r e  1/1024 1/2048 1/1 0  - 52  TABLE X COMPARISON OF DAYS COLD ENRICHMENT TO RECOVERY OF SIGA STIMULATING ORGANISMS  # Days  0 1-7 8-14  IgA Stimulators Recovered 5 5 4  IgA Non-Stimulators Recovered 2 7 5  - 53 -  ACKNOWLEDGEMENTS  This work was supported by a grant from the B r i t i s h Columbia Health Care Research Foundation (#5-52403).  For his valuable advice and assistance in many aspects of this thesis I would l i k e to thank Dr.M.A. Noble.  For their technical  assistance in yersiniae i s o l a t i o n , serotyping, and fluorescence photography I also thank R.L. Barteluk, S. Toma, and A.W. Vogl respectively.  

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