Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. NOVEL SCREENING METHODS FOR THE DETECTION OF YERSINIA ENTEROCOLITICA IN INFECTED BLOOD USED FOR TRANSFUSION A thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North New Zealand Christopher John Kendrick 2000 1 2 Abstract Between 1991-1996, 8 patients experienced rare life-threatening reactions that followed the transfusion of blood infected with Yersinia enterocolitica. The first reported case occurred in 1991 and was followed by seven others that direct! y caused or contributed to the death of 5 of 8 patients. Y. enterocolitica is a food and water borne infection of the gastrointestinal tract which in adults is often asymptomatic. An unknown number of those infected experience a period of self-limiting bacteraemia. The large volume of blood collected during donation phlebotomy may contain small numbers of bacteria that can increase in number during blood bank storage, producing potentially lethal levels of bacteria and toxin. Currently there are no reliable methods available to distinguish blood donations that present the greatest risk from those that present little risk. This thesis, reports on the evaluation of two techniques to prevent the transfusion of blood infected with Y. enterocolitica. The first, a molecular method, was used to amplify bacterial DNA in blood by Polymerase Chain Reaction (PCR). A 425 bp product was amplified from DNA extractions of infected blood. Results showed that the technical complexities of the methodology, together with poor sensitivity and the need for large­ scale donation sampling make PCR as applied for this purpose unattractive. An Enzyme Linked lmmunosorbent Assay was developed to detect current/recent infection with Y. enterocolitica in healthy blood donors. Polystyrene beads were coated with bacterial proteins to detect IgA antibody to Y. enterocolitica in human serum. The sera from donors of confirmed unit infections, paired sera from culture-proven Y. enterocolitica gastrointestinal tract infection and sera from volunteer blood donors were tested. Results showed that the sera of six bacteraemic blood donors tested contained elevated levels of IgA antibody. High rates of positivity (26/27), were detected in sera from culture­ confirmed GIT infection and a rate of 4.04% seropositivity was found among 495 blood donors enrolled in a clinical trial. Results showed a strong correlation between IgA seropositivity, and recipient risk associated with the transfusion of blood heavily infected with Y. enterocolitica. The work demonstrated how the use of a simple screening test for recent infection, could be used to exclude high risk donations and improve the safety of blood transfusion in New Zealand. 3 Acknowledgements Thank you to Dr. Paul O'Toole my supervisor for his expert scientific guidance during the research described in this thesis and his helpful suggestions in the preparation and proofing of this thesis. Thank-you also to the former Dept. of Microbiology and Genetics for providing seeding money to commence this research, and for the use of the laboratory in which the work was performed. Thanks also to Dr. Bart Baker, the staff of the Department of Transfusion Medicine, and Blood Donor Services, at Mid-Central Health Ltd., who were involved in the clinical trial of the assay undertaken at the Blood Centre. I also acknowledge the assistance of Mr. Brian Hanrahan, David Ackroyd and others from Abbott Diagnostics NZ. Ltd. for the generous support of the project and the provision of equipment and ELISA consumables and their assistance in the coordination of my visits to Sydney in October 1997 and to Abbott Diagnostics Division, Abbott Park, Tilinois, USA in February 1998. Thanks also to Dr. Andrea Ramirez, Auckland Regional Blood Centre; Andrew Mills, Waikato Regional Blood Centre; the staff of the Blood Bank at Taranaki Base Hospital; and the staff of the Masterton and Wellington Blood Centres , who provided serum samples from blood donors from time to time. I also acknowledge the cooperation of Dr. Arthur Morris and Diagnostic Laboratories, Auckland for making available paired sera from stool culture-positive Y. enterocolitica GIT patients. Thanks also to Michiko Kakubayashi , from the Dept. of Microbiology and Genetics for assistance with work on protein isolation and ELISA assay development and Thomas Edelmann for his assistance with the bacterial growth experiments conducted during his stay in the Dept. in 1997-98. Finally but not least a special thanks to my wife Bronwyn who has been a sounding board 4 for my thoughts and queries and who ably assisted with the performance of the IgA RP Bead ELISA assay trial at Mid Central Health Ltd. The research work presented in this thesis has received the financial assistance from the following grants : 1997 Massey University Research Fund (MURF) 1998 Palmerston North Medical Research Fund The research presented in this thesis has been granted ethical approval under the following applications: Massey University Ethics Committee ethics reg. no. HEC 95/48 Manawatu-Whanganui Ethics Committee. ethics reg. no. 17 /95 5 TABLE OF CONTENTS PAGE TITLE PAGE ... .. .. ........ ......... .............. .. ............ ... ....... ............. ........ .... .. ... .. .. ....... ... .......... ...... .... .. .. .... ... .......... 1 ABSTRACT .... ....... . .. ... . ............................................................... .......... ..... ... .......................... 2 ACKNOWLEDGEMENTS ...... ... .... . .. ..... .. .. .. ..... .. . ...... .. ... . ....... . .. ................. .... ... .... .......... .... ... . . 3 TABLE OF CONTENTS ... ............................. .. ............. ... ..... ........................ ........ .. ....... ............ . 5 LIST OF ILLUSTRATIONS .. .. ........ ... ...... ... .. ................. .. .. . ... .. .... .. ...... ............ ... .. ... .............. ...... . 9 LIST OFT ABLES ............. ... ............................ ............ ... .. ... .......... .............. ... ..................... . ... 10 LIST OF ABBREVIATIONS ........ ... .. .. ..... . .......... . ...................................................... ... .............. 11 LIST OF APPE DICES .............................. .. .. .... ... ......... .. .... ... ..... . ....... . .. .. . ......... ...... .. ........... ... 13 CHAPTER 1 TRANSFUSION TRANSMITTED ENDOTOXAEMIA CAUSED BY YERSINIA ENTEROCOLITICA INFECTED BLOOD IN NEW Z EALAND - A HISTORICAL REVIEW ..... ................... .. ...... .......................... .. ...... . ... .. ... 14 1 . 1 INTRODUCTION .................................................... .. . ..... ... ............ . ......... ... ... ...... . 14 1.2 B ACTERIAL CONTAMINATION OF DONATED BLOOD ...... .. ... ...... .. ....... .... .............. 16 1.2.1 Environmental bacteria as sources of infection .. ..... ....... ...... .... ............... 17 1.2.2 Commensal bacteria as sources of infection ........ ... ... ..... ...... .. ..... .... ..... . 17 1.2.3 Endogenous sources of infection ...... .... ....... ... ... .... ..... ... .... .... ........ .. ... ... 17 1.2.4 Gram-negative sepsis ........... ... ....................... ..... ... .. .............. ............. .... 18 1.2.5 Growth of Y. enterocolitica in blood bags .. .. .. .. .... ........................ ......... 19 1.3 FREQUENCY OF TRANSFUSION RELATED E 1DOTOXIC SHOCK ........ . .. .... .. .... ....... . 20 1.3. l United States of America .. .. .. .. ........ ...................... .. ... .. .. ... ... ............ ....... 20 1.3.2 Germany ......... ..... .. .. ..... ..... .. ..... ............ .............. ..... ........ ......... ... ... ... ... ... ... ............. .. 21 1.3.3 New Zealand .. ......... ...................... ............. .. ......... .. .. .. ....... ... .. ........ .. ..... . 21 1.4 TAXONOMY OF THE YERSINIAE .............. . .................... ..... .. . .... ...... ..... ... .... ..... ..... 26 1.5 BACTERIOLOGY OF Y. ENTEROCOLITICA .... ..... ..... . ..... ........ .. .... ..... ...................... 26 1.5.1 Biotypes .......... ....... ............... ... .. .......... ........ ........ ..... ....... ... ... ..... .......... .. 27 1.5.2 Serotypes ..... .......... ....... ...... ...... ............. ........ .... ............... .... ..... ........... ... 27 1.6 EPIDEMIOLOGY OF Y. ENTEROCOLITICA INFECTION .. ...................... . ................. ... 27 1.7 VIRULENCE FACTORS .... .. ........ ... .... ..... .. ............... .. ..... . ........ .. ........ . .. ... . ............. 28 1.7.1 PYVplasmid ..... ........................ ......... ..... .. ... ... ...... ......... ... .......... ..... ....... 28 l. 7 .2 Y op function .... ....... ..... .... .............. ... ...... ..... .. ......................................... 29 6 PAGE 1.8 HUMAN INFECTION WITH Y. ENTEROCOL/TICA . .. .... ........ . .................. . . . .. . ... ....... . . 32 1 .8.1 Mucosa] immune system ....................................... ............. ............. ........ 32 1.9 IMMUNE RESPONSE TO INFECTION .. ... .... ..... .. ... ............... .. ... ...... .. ... ................... 34 1.10 PA TH OGE ICITY OF Y. ENTEROCOLITICA .................... ............. ... ....... .. ... . ... ..... .. . 35 1.11 PERSISTENCE OF INFECTION .... .. .. ..... ........... .... ...... .... .... .. .... .. .. .... ......... .... ... ...... 36 1.12 Y. ENTEROCOLITICA BACTERAEMIA & SEPTICAEMIA .......................................... 37 1.13 LABORATORY DIAGNOSIS OF Y. ENTEROCOLITICA INFECTION .... ................ ........ 37 CHAPTER 2 GROWTH OF Y. ENTEROCOLITICA IN HUMAN AS-1 RED BLOOD CELLS DURING STORAGE AT 4°C . ...... ......... ..... ... .. .. ........ ...... .... .... 39 2.1 INTRODUCTION .................................................................................................. 39 2.2 MATERIALS ....................................................................................................... 39 2.3 METHODS ...... ...... ........ ... ......... .. .... ...... .................... .. ......... ..... ...... ... ... .... .... .... 42 2.3.1 Donor Blood Collection ........... .... .... .... .. ............ ...... ...... .......... .. .... .. ..... .42 2.3.2 Unit inoculation ...... ............... ............... .. .................. ............ .................. 42 2.3.3 Viable cell counts ................... ... ... .. ...... .. .... .................................... ........ 43 2.3 .4 PCR samples ........................................................................................... 43 2.4 RESULTS ........ ... .. ... .... ......................... .. .. .................... .................. .... ............. 43 CHAPTER 3 DETECTION OF Y. ENTEROCOLITICA IN HUMAN AS-] RED BLOOD CELLS BY PCR ... .................................... .. ............. ............. .... 46 3.1 L'1TRODUCTION ... ......... ... ............... ... ............................................. ... ................. 46 3.2 MATERIALS ........................... ... .............. .. ..... ... ..... .. .... ................ ...... ........ .... .... . 46 3.2.1 Agarose gel electrophoresis ... ..... ......... .. .. ... .............. .... ........ .. .... .... ........ .46 3.2.2 PCR reagents ........................................................................................... 47 3.2.3 DNA extractions ...................................................................................... 47 3.2.4 Culture media ......... ... .. ................... ............... .... ..... .. ......... ...... ........ .. ...... 42 3.2.5 Primer preparation ...... ... ............ ..... ..... .... .............. .... .. ........ ................. ... 48 3.3 METHODS ... ........... .. ...... ...... ..... ...................... ............... ..... .... ........ .. ..... ............. 49 3.3.1 DNA extraction ....................................................................................... 49 3.3.2 PCR reaction mix ....... ... .... ... .. .. ................ .. .... ..... .. ... ......... ..... ... ... ........... 50 3.3.3 PCR conditions .. ... ................ ...... .. ....................................... ............ ... .. ... 50 3.3.4 Agarose gel electrophoresis ................. .. ..... .. .. .... ..... .. ... ..... .................... .. 50 7 PAGE 3.4 RESULTS ...... .... ...................... .................... .... .................................................... 51 CHAPTER4 SEROLOGIC DETECTION OF Y. ENTEROCOL!T!CA INFECTION IN BLOOD DONORS BY IGA RP ELISA ASSAY ........................................... 53 4.1 lNTRODUCTIO .... .. .............................. .. ........................... .... ............................. 53 4.2 MATERIALS ....................................................................................................... 54 4.2.1 Released protein production .............................. ... ......................... ... ........ 54 4.2.2 SDS-PAGE .................................. .... ... .... ....... ............ ............. ....... .. ........ 55 4.2.3 Protein electro-blotting ............................................................................ 57 4.2.4 DAKO 0:3 LPS ELISA assay .................. .. ................................... ........... 57 4.2.5 IgA RP bead ELISA assay .......................................... .............................. 57 4.2.6 Clinical trial - blood collection ............................................................... 60 4.2. 7 Serum samples ............ ... ....... ...... .............. ..... ............ ... ........................... 60 4.2.8 Stool culture ................ ......... ... ........................ ................ ..... ... ... .............. 62 4.2.9 Blood culture ........................................................................................... 62 4.3 METHOD ............................................ .... .......... ..................................... ........ ... .. 63 4.3.1 Released protein preparation ................................................................... 63 4.3.2 Biorad protein assay ................................................................................ 63 4.3.3 SDS-PAGE ....... ...... .. ........ ... ... ... ................. ....................... ...................... 64 4.3.4 Protein electro-blotting ............................................................................ 66 4.3.5 DAKO 0:3 LPS ELISA assay .................................................................. 66 4.3.6 lgA RP bead ELISA assay ... .. ... ..... ... .......................... .... .... .... .... .............. 67 4.3.7 Stool culture ............................................................................................. 69 4.3.8 Blood culture .......................................................................................... 69 4.3.9 Blood collection ....... .. ........... ...... ...... ..... .................................. ...... ... ....... 69 4.4 RESULTS .. .... ......... ......... .................................................................................... 70 4.4.1 SDS-PAGE .................. ... ...................... ................. ........ .......................... 70 4.4.2 BIORAD protein assay ............................................................................... 71 4.4.3 ELISA blocking agents .............................................................................. 71 4.4.4 ELISA wash solutions ............................................................................... 72 4.4.5 ELISA sample testing ................................................................................ 72 CHAPTER 5 DISCUSSION ................................................................................................. 79 8 PAGE APPENDICES ............... . ... ... . ................ .. ......... . .. .... ......... . ...... . .. ..... .. .. ... .......... ... .. .... . ............. 88 REFERENCES .. . ..... .... .......................... .. ......... ..... . ...... .... ... .... . ... ........... ... ......... .. ..... . ........... 113 +++++++ Figure 1.1 Figure 1.2 Figure 1.3 Figure 2.1 Figure 3.1 Figure 4.1 Figure 4.2 Figure 4.3 LIST OF ILLUSTRATIONS Geographical distribution of blood unit infection with Y. enterocolitica in New Zealand. Proposed model of interaction between Yersinae and macrophages. EcoR1 restriction map of the virulence plasmid pYVe227 of Y. enterocolitica showing the location and direction of transcription of genes encoding secreted and structural proteins Yaps B, D, E, H, M, 0, P and Q, the V antigen (lcrV), the lipoprotein YadA, regulatory components (VirA, B, C, F) and replicative functions (repA, B, oriR). Graphical representation of Y. enterocolitica growth at 4°C following the inoculation of 30 cfu into 200ml of AS-1 human red blood cells. Representative gel following PCR amplification of ail gene from DNA extracts of citrate anticoagulated blood infected with Y. enlerocolitica. Geographical distribution and identification of group 1 serum samples. Diagrammatic representation of the Indirect ELISA bead assay developed for the Abbott Commander ELISA system. Predominant bands of the released proteins of p yy+ Y. enterocolitica (strain PN2) separated by SDS-PAGE. 9 PAGE 25 29 31 45 52 61 67 70 Table 1.1 Table 1.2 Table 2.1 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table 4.7 LIST OF TABLES Summary of NZ transfusion related endotoxaemias and unit infections. Yops and their function from "Foodborne Microorganisms of Public Health Significance". (Barton et al., 1997) Growth of p yy+ Y. enterocolitica in AS-1 red cells stored at 4°C over a period of 30 days. Reagents for SDS-PAGE gels. Protein quantitations of batches of released protein. Results of ELISA testing for serum IgA (cutoff 0.18) and IgG (cutoff 0.16) antibody to Y. enterocolitica 0:3 LPS (DAKO) in bacteraemic blood donors. Results of ELISA testing for serum lgA antibody (cutoff OD492 0.261) to Y. enterocolitica (released protein) in bacteraemic blood donors. Serum lgA antibody (cutoff OD492 0.248) to Y. enterocolitica RP' s from stool culture positive GIT infection. Summary of results of IgA RP ELISA assay, stool and blood culture testing on volunteer blood donors enrolled in stage 1 of the clinical trial. Summary of results from IgA RP assay, stool, blood cultures. 10 PAGE 25 34 44 65 71 72 73 74 76 77 AIDS AS-1 BHI BHI-B bp CIN CMI CPD CR-MOX DIC DNA EDTA EGTA ELISA GIT HBsAg HBV HCV HLA-B:27 IgA IgG IgM IL-1 INF-y kb kD lcr LPS MIS MMWR MWM LIST OF ABBREVIATIONS Acquired Immune Deficiency Syndrome Additive solution - 1 Brain heart infusion Brain heart infusion broth Base pair Cefsulodin-irgasan-novobiocin Cell mediated immunity Citrate phosphate dextrose Congo red, magnesium oxalate Disseminated intravascular coagulation Deoxyribonucleic acid Ethylenediaminetetraacetic acid Ethylenebis( ox yeth ylenenitri lo )-tetraacetic acid Enzyme linked immunosorbent assay Gastrointestinal tract hepatitis B surface antigen hepatitis B virus hepatitis C virus Human leucocyte antigen B:27 Immunoglobulin A Immunogfobulin G Immunoglobulin M Interleukin- ] Gamma interferon Kilobase Kilodalton Low Ca++ restricted Li popol ysaccharide Mucosa! immune system Morbid mortality weekly report Molecular weight marker 11 MQ 0/N PCR PTH PY pYV RO RP slgA SIS SDS SDS-PAGE TNF TE Tris WWII Yops Millipore filtered - type 1 laboratory reagent grade water Overnight Polymerase chain reaction Post transfusion hepatitis Predictive value Yersinia virulence plasmid Reverse osmosis - type 3 laboratory reagent grade water Released protein Secretary immunoglobulin A Systemic immune system Sodium dodecyl sulphate Sodium dodecyl sulphate polyacrylamide electrophoresis Tumour necrosis factor TrisEDTA Tris-(hydroxymethyl) aminomethane World war II Yersinia outer membrane protein 12 LIST OF APPENDICES Appendix 1 Application for Ethical approval to Manawatu-Whanganui Ethics Committee. Appendix 2 Yersinia IgA antibody assay development - proposed schedule for clinical trial. Appendix 3 Blood donor information sheet & consent form Yersinia research - stage 1. Appendix 4 Blood donor information sheet & consent form Yersinia research - stage 2. Appendix 5 Y. enterocolitica IgA antibody assay trial. Results of serum stool and unit culture on blood donors from stage 2. Appendix 6 Blood donor enrollment forms. Appendix 7 Nucleotide sequence for ail gene in Y. enterocolitica. Appendix 8 Coding sequences of Y. enterocolitica plasmid p YVe227. 13 PAGE 88 89 91 93 95 108 110 112