UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Assessing exposure to Cryptococcus gattii Griffiths, Andrea 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-ubc_2006-0191.pdf [ 8.05MB ]
Metadata
JSON: 831-1.0092660.json
JSON-LD: 831-1.0092660-ld.json
RDF/XML (Pretty): 831-1.0092660-rdf.xml
RDF/JSON: 831-1.0092660-rdf.json
Turtle: 831-1.0092660-turtle.txt
N-Triples: 831-1.0092660-rdf-ntriples.txt
Original Record: 831-1.0092660-source.json
Full Text
831-1.0092660-fulltext.txt
Citation
831-1.0092660.ris

Full Text

Assessing Exposure to Cryptococcus gattii B y Andrea Griffiths B.Sc. Malaspina University-College A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( O C C U P A T I O N A L A N D E N V I R O N M E N T A L H Y G I E N E ) T H E U N I V E R S I T Y O F BRITISH C O L U M B I A April 2006 © Andrea Griffiths, 2006 A B S T R A C T Cryptococcus gattii is an encapsulated basidomycetous yeast that is capable of causing cryptococcosis in humans and animals. The disease caused by C. gattii frequently begins with pneumonia which in some cases can disseminate into fatal form of meningitis. Traditionally, this organism was thought to occur globally in tropical and subtropical regions, however recently it has been isolated from the Coastal Douglas Fir Zone on Vancouver Island, British Columbia (BC), a coastal temperate zone. From 1999 to present, there have been more than 130 human cases of Cryptococcosis caused by C. gattii on Vancouver Island and numerous cases in companion animals and wildlife. The aim of this study was to develop an assay to identify biomarkers of exposure to C. gattii which could be used to determine the extent of exposure of residents of B C . A Western blot analysis was developed to identify cytoplasmic antigens isolated from C. gattii which are recognized by antibodies in test sera. The sera of 77 Vancouver Island residents were tested following the 1999 outbreak of C. gattii and greater than 80% of those tested (at a 1 in 250 dilution of serum) were found to have antibodies against cytoplasmic antigens unique to the organism. Conversely, at the same dilution only 25% of those tested (n=51) from other areas of B C , the Yukon and Northwest Territories prior to the 1999 outbreak had antibodies against C. gattii antigens. The most commonly recognized antigens were 20 kDa and 50 kDa in size and their identity is presently unknown. This Western blot analysis has proven to be a useful means of suggesting potential biomarkers of exposure to C. gattii in a population. ii T A B L E O F C O N T E N T S , A B S T R A C T ii T A B L E O F C O N T E N T S . iii LIST O F T A B L E S ...v LIST O F F I G U R E S ." vi LIST O F A B B R E V I A T I O N S viii A C K N O W L E D G E M E N T S . ix C H A P T E R 1: Introduction 1 1.1 Introduction 1 1.2 Cryptococcosis 2 1.3 Cryptococcal infections in humans and other mammals 3 1.4 Cryptococcus gattii in the Environment 6 1.5 Cryptococcus gattii on Vancouver Island, British Columbia 8 1.6 Potential for Environmental Exposure to Cryptococcus gattii on Vancouver Island9 1.7 Cryptococcal Immunity. 11 1.8 Latex Agglutination Assays: Identification of Cryptococcus neoformans and C. gattii 14 1.9 Techniques Utilized to Assay Seroprevalence 15 1.9.1 Enzyme Linked Immunosorbent Assay (ELISA). . . 15 1.9.2 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) and Western Blotting 17 1.10 The Use of Seroprevalence Assays to Determine Exposure in Populations 18 1.11 Relevance of the Work Reported Here: 20 1.12 Objectives and relevant hypotheses........ 21 C H A P T E R 2: M E T H O D S A N D M A T E R I A L S . . . . 23 2.1. Preparation of Antigen from Cryptococcus neoformans and Cryptococcus gattii.23 2.2 Novel Method of Obtaining Cryptococcus Antigens for Western Blot Analysis ..25 2.3 Description of Study Population: 26 2.4 Enzyme Linked Immunosorbent Assay (ELISA) 28 2.5 Sodium Dodecyl Sulphate Polyacrylamide .Gel Electrophoresis (SDS P A G E ) 29 2.6 Staining of Membrane for Visualization of Total Protein 30 iii 2.7 Basic Western Blot Analysis Protocol 30 2.8 Development of Western Blot Analysis Conditions to Test Populations 31 2.9 Analysis of Data.. 32 C H A P T E R 3: R E S U L T S 33 3.1 Quantification of total protein from cytoplasmic antigens for development of immunoassays 33 3.2 Enzyme Linked Immunosorbent Assay (ELISA) 33 3.3 Study Population 35 3.4 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) .36 3.5 Western Blot Analysis ...37 3.6 Western Blot Analysis of Vancouver Island Residents 41 3.7 Western Blot Analysis of Miners sera collected prior to the 1999 outbreak of C. gattii on Vancouver Island '. 49 3.8 Comparing results of sera tested from Vancouver Island Residents and Miners ....51 C H A P T E R 4: DISCUSSION 54 4.1 Preparation of Antigen from Cryptococcus neoformans and Cryptococcus gattii. .54 4.2 Enzyme Linked Immunosorbent Assay (ELISA) 56 4.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) 59 4.4 Development of Western Blot Analysis 59 4.5 Western Blot Analysis of Vancouver Island Residents 62 4.6 Western Blot Analysis of Miners' sera prior to the 1999 outbreak of C. gattii 67 4.7 Comparing results of sera tested from Vancouver Island Residents and Miners ....68 4.8 Proposal for Test System Conditions 69 4.9 Potential Applications for the Test System 71 4.10 Future Directions 72 4.11 Significance of Results ....74 4.12 Strengths and. Limitations of this thesis work 75 4.13 Conclusions 75 L I T E R A T U R E C I T E D 77 Appendix A 83 Appendix B 84 iv LIST OF TABLES Table 3.1 Results from ELISA# 14 34 Table 3.2 Percent differences comparing the absorbance values of patients (1702, 1630) against pooled human serum for all three varieties 35 Table 3.3 A summary of information available about sera tested for antibodies against Cryptococcus gattii: 36 Table 3.4 The percentage sera tested with antibodies against the 20 kDa antigen reported by region following the 1999 outbreak of C. gattii 44 Table 3.5 The percentage of positive Vancouver Island residents by region for the 50 kDa antigen 45 Table 4.1 Western Blot Analysis Conditions in Seroprevalence assays 71 Table B . l Results of Vancouver Island Residents Tested for Antibodies against C. gattii by Region 84 Table B.2 Results of Vancouver Island Residents Tested for antibodies against C. gattii by Study Type 85 Table B.3 Results of Vancouver Island Residents Tested for antibodies against C. gattii by Age Group 85 Table B.4 Results of Miners from the Northwest Territories, Yukon and Central and Northern British Columbia Residents Tested for Antibodies against C. gattii 86 v L I S T O F F I G U R E S Figure 1.1 A map of Vancouver Island illustrating locations in the environment where C. gattii has been isolated 9 Figure 1.2 A schematic displaying results of an Enzyme Linked Immunosorbent Assay (ELISA) 16 Figure 1.3 A schematic displaying S D S - P A G E and Western Blotting 18 Figure 2.1 A n example of a standard curve generated from a modified Bradford Assay 25 Figure 3.1 Staining of total protein isolated from Cryptococcus cytoplasm 37 Figure 3.2 Initial Western Blot analysis image 38 Figure 3.3 Western blot analysis of sera from two B C C D C patients (A,B) at a dilution of 1 in 2000 and serum from one suspected exposed individual (C) at a dilution of 1 in 250 39 Figure 3.4 Western blot analysis of two different sources of pooled human sera from the United States and two B C C D C patients 40 Figure 3.5 Western blot analysis of sera from Vancouver Island residents 41 Figure 3.6 A graph displaying the percentage of sera tested from Vancouver Island residents with antibodies against C. gattii... 43 Figure 3.7 A graph displaying the percentage sera samples tested from Vancouver Island residents with antibodies against the 20 kDa antigen reported by study type 46 Figure 3.8 A graph displaying the percentage sera samples tested from Vancouver Island residents with antibodies against the 50 kDa antigen reported by study type 47 Figure 3.9 A graph displaying the percentage sera samples tested from Vancouver Island residents with antibodies against the 20 kDa antigen reported by age group 48 Figure 3.10 A graph displaying the percentage sera samples tested from Vancouver Island residents with antibodies against the 50 kDa antigen reported by age group : 49 v i Figure 3.11 Western blot analysis of miners' sera collected prior to the 1999 outbreak of C. gattii from the Northwest Territories, Yukon and central and Northern British Columbia 50 Figure 3.12 A graph displaying the percentage of sera tested from miners sera collected prior to the 1999 outbreak with antibodies against C.gatti 51 Figure 3.13 A graph displaying the percentage sera tested from Vancouver Island residents (post-1999) compared to miners sera tested (pre-1999) for the 20 kDa antigen 52 Figure 3.14 A graph displaying the percentage sera tested from Vancouver Island residents (post-1999) compared to miners sera tested (pre-1999) for the 50 kDa antigen..... 53 vn LIST OF ABBREVIATIONS AFLP amplified fragment length polymorphism BC CDC British Columbia Centre for Disease Control BCIP/NBT 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium CAPS ' 3-(Cyclohexylamino)-l-propanesulfonic acid CFU colony forming units CDFZ Coastal Douglas Fir Zone ELISA enzyme linked immunosorbent assay GXM glucuronoxylomannan IgG immunoglobulin G IgM immunoglobulin M kDa kiloDalton Mab monoclonal antibody NCPF National Collection of Pathogenic Fungi PCR polymerase chain reaction PBS phosphate buffered saline PBS-Tween phosphate buffered saline containing 0.5% Tween-20 PVDF poly vinyl di-fluoride RAPD random amplification of polymorphic D N A RBPP Rathtrevor Beach Provincial Park RFLP restriction fragment length polymorphism SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis viii A C K N O W L E D G E M E N T S Firstly, I would like to thank my supervisor Dr. Karen Bartlett for all of her excellent guidance and support during my time at the School of Occupational and Environmental Hygiene. In addition I would like to express gratitude to my committee members Drs. Hugh Davies and Craig Stephen for their direction during my research project. I am also indebted to the laboratory personnel at S O E H , Dr. Winnie Chu, T im M a and Tom Barnjak not only for their technical assistance, but also for making me feel so welcome in the lab. I would also like to thank Jennifer Sibley in the Department of Microbiology and Immunology for the generous lending of equipment throughout my research and Dr. Tanya Griffiths and Dr. R.T. McGillvary's laboratory for their initial assistance with developing my Western Blot assay, use of reagents and use of equipment. In addition I would like to thank Dr. Sarah Kidd for the genotyping of many of the Cryptococcus strains I used in my thesis. For moral support I thank my husband Michael and my parents. Additionally, I am grateful to the Department of Biology at Malaspina University-College for providing me with mentorship in the past. Finally, I would also like to express many thanks to all of the Vancouver Island residents who kindly donated blood for this research. Your contribution has helped reveal information regarding an emerging pathogen in British Columbia. ix CHAPTER 1: Introduction 1.1 Introduction Cryptococcus neoformans is an encapsulated basidomycetous yeast that is capable of causing cryptococcosis in humans and animals [1]. Cryptococcal meningitis has been on the rise globally because of human immunodeficiency virus (HIV), treatments such as cytotoxic chemotherapy, and organ transplantation [2]. Three different varieties of C. neoformans, variety gattii, grubii and neoformans have been described which can be differentiated based on molecular type and capsular serotype [3]. Cryptococcal isolates have been classified into five different groups: A (C. neoformans var. grubii), B and C (C. neoformans var. gattii), D (C. neoformans var. neoformans) and AD (a hybrid). These groups were based upon the serotyping of the capsular polysaccharide [4, 5]. In most cases, C. neoformans var. grubii and var. neoformans are pathogenic for individuals who are immunocompromised and C. neoformans var. gattii tends to infect people who are immunocompetent [6] [7]. C. neoformans var. grubii and var. neoformans are found globally and most often isolated from soil that contains bird excrement [8]. Evidence which supports the idea that antibodies against C. neoformans var. neoformans can be acquired from an environmental source which was published in 2001 by Goldman et al. The publication by Goldman et al demonstrated children living in New York had antibodies against C. neoformans var. neoformans because it is ubiquitous in their environment [9]. Recently, C. neoformans var. gattii has been raised to the species level based on the divergence of its genome from the other C. neoformans varieties [10]. Prior to the BC 1 outbreak, C. gattii was thought to only occur in the subtropics and tropics [7] and to be associated with eucalyptus tree debris [11]. Recent work conducted by epidemiologists at the British Columbia Centre for Disease Control (BC C D C ) has demonstrated that an outbreak of C. gattii has occurred on Vancouver Island, Canada in animals and humans. Prior to the outbreak on Vancouver Island, no outbreaks of C. gattii among humans had ever been documented. In addition, the location of this outbreak is in a temperate climate zone which would not have been anticipated given earlier understanding of C. gattii's distribution. For the most part, the human and animal cases have occurred within one biogeoclimatic zone known as the Coastal Douglas Fir Zone (CDFZ) [12]. The C D F Z covers only a small area of B C but has some of the rarest vegetation in the province. In addition, the climate of the C D F Z is the mildest in Canada and is characterized by long dry summers and wet mild winters [13] There are approximately 720,000 people residing on Vancouver Island with the majority of population residing in the C D F Z . Currently no information is available regarding the extent to which residents of British Columbia (BC) have been exposed to C. gattii. Therefore, the overall aim of this study was to develop an assay that would provide information concerning the extent of exposure to C. gattii in B C patients and residents of Vancouver Island following the 1999 outbreak. 1.2 Cryptococcosis Cryptococcosis is an infectious disease which can spread from the lungs to the brain and central nervous system, skin, urinary tract and skeletal system. The route of exposure is through inhalation of the propagules. The disease is characterized by the growth of 2 granulomatous nodules that are filled with a gelatinous material in visceral and subcutaneous tissues. Initial symptoms in humans usually include coughing or other respiratory effects as the lungs are frequently the primary site of infection. If the fungus spreads to the meninges, neurological symptoms develop, such as headache, difficulty speaking, confusion, and blurred vision [14]. A diagnosis is most often made by the identification of yeast cells that are visualized in tissue, or by culture isolated from a tissue biopsy, sputum or pus [15]. The latex agglutination test is an assay that is frequently used in the diagnosis of the disease using serum or cerebrospinal fluid (CSF) and has been reported to have sensitivity of 90% [16]. The primary drawback of current diagnostic tests is that they do not differentiate among species of Cryptococcus. 1.3 Cryptococcal infections in humans and other mammals It is well recognized that Cryptococcus neoformans is the causative agent of fungal meningitis on a global scale [17.]. The majority of cases occur in those afflicted with H I V or who are otherwise severely immunocompromised. In these infections, C. neoformans var. neoformans or C. neoformans var. grubii are the varieties of the species that are almost always responsible for the infections [17]. C gattii is also capable of causing pulmonary infection [18] and in some cases meningitis. [19]. The main route of exposure to Cryptococcus is via inhalation and it is hypothesized that without treatment, the organism eventually proliferates, ultimately affecting the central nervous system. C. gattii can cause infection in both immunocompetent and immunocompromised hosts. In 1984, Kwon-Chung and Bennett reported that approximately 15% of all clinical 3 Cryptococcus neoformans isolates from Canada, United Kingdom, Argentina and the United States (excluding Southern California) were positively identified as C. gattii. In addition, this study showed that there was an even higher occurrence of C. gattii, between 35 and 100% of all clinical isolates, in locations classified as tropical or subtropical such as Australia, Brazil, Cambodia, southern California, Paraguay, Mexico, Nepal, Vietnam, Thailand and some parts of central Africa [20]. In 1985, the first case of cryptococcosis in France caused by C. gattii was reported and was subsequently published by Dromer et al in 1992 [21]. The infection arose in a patient who was not considered immunocompromised and exhibited symptoms such as transient headaches, fever and lethargy. What was unique about this case was that the patient had resided in France for 23 years and was a former resident of an Asian country, which suggested the possibility of a latent infection. The patient underwent treatment with amphotericin B for a total of five months and was subsequently cured of the infection [21]. This was one of the first cases of C. gattii reported in an immunocompetent individual and illustrates the lengthy process of treatment for an infection. In 1987, a publication by David Ellis confirmed the environmental presence C. gattii in Australia. In this work, Ellis demonstrated that out of 31 immunocompetent patients with cryptococcal disease, 87% of the isolates were C. gattii [22]. Infections in humans are not limited to pulmonary infection and meningitis; in 1997 an immunocompetent man from Australia was admitted to hospital with a cutaneous cryptococcal infection in which C. gattii was reported as the cause [23]. Another example of an unique form of infection in an immunocompetent host arose in 1997 when a woman from Germany was diagnosed with C. gattii infection manifested as a spinal tumor [24]. 4 Exposure to Cryptococcus and infections caused by the organism are not limited to humans as a variety of animals are also exposed and afflicted with infection. A study by Malik et aim 1999 that took place in Australia investigated the levels of anti-cryptococcal antibodies in the serum of cats, dogs and koalas. The results showed that the majority of infected cats and koalas had antibodies against both C. gattii and C. neoformans var. neoformans. Additionally, the study demonstrated the prevalence of elevated anti-cryptococcal antibody levels in normal animals [25]. A report published in 2002 by Stephen et al indicated that there was an outbreak of cryptococcosis, on Vancouver Island in cats, dogs, llamas, ferrets and porpoises. The fungi isolated from two porpoises, one cat and one dog were positively identified as C. gattii [26]. Another case of cryptococcosis caused by C. gattii in a marine mammal was published in 1999, when an Atlantic bottlenose dolphin (Turisops truncates) housed in California developed a fatal cryptococcosis [27]. Recent publications by Duncan et al in 2005 [28] and 2006 [29] have focused specifically on cases of C. gattii infections in cats and dogs on Vancouver Island since the 1999 outbreak. The work by Duncan et al in 2005 demonstrated that sub-clinical infections and asymptomatic carriage of C. gattii (Serotype B) occurred in cats and dogs from Vancouver Island during the outbreak of cryptococcosis [28]. Additionally in 2006, Duncan et al identified the following as risk factors associated with the development of a C. gattii infection in cats and dogs: residing within 10 km of a logging site or other area of commercial soil disturbance, above-average level of activity of the animal, hunting by the animal, traveling of the animal on Vancouver Island and owners visiting a botanic garden or hiking [29]. 5 1.4 Cryptococcus gattii in the Environment Soil and bird excrement have been considered the major sources of Cryptococcus neoformans var. neoformans worldwide however, until the 1990's, environmental sources of C. gattii were unclear. Early work conducted by Ellis and Pfeiffer in 1990 demonstrated the association between Eucalyptus camaldulensis and C. gattii. More specifically the investigators observed that the presence of C. gattii propagules corresponded with the flowering of E. camaldulensis. The investigators proposed that C. gattii could be found many places on a global scale because the Eucalyptus tree is exported extensively [11]. This was demonstrated yet again by Ellis and Pfeiffer in 1991 when C. gattii was isolated from E . camaldulensis from an area near San Francisco [30]. In 1992, Ellis and Pfeiffer established that C. gattii was also associated with another species of eucalyptus, Eucalyptus tereticornis and that this species was globally distributed in a similar manner to E. camaldulensis [31]. Publications from 1996 [32] and 1997 [33] from Australia hypothesized that the above two eucalyptus species may not be the only environmental sources of C. gattii based on evidence gathered from genetic testing using D N A Random Amplification of Polymorphic (RAPD) profiles. The use of D N A R A P D analysis was used to link clinical infections in Australia to the source of infection, the eucalypt, by examining R A P D profiles. Additionally, Chen, in 1997 identified two other species of eucalyptus from which C. gattii could be cultured from (E. rudis, E. gomphocephala) and concluded, based on R A P D analysis, that other environmental sources must exist [33]. The D N A R A P D technique amplifies a random sequence of D N A using a primer designed by the investigator and distinguishes among isolates by examining reproducible banding patterns. The use of R A P D analysis was the initial means to distinguish between 6 environmental sources of Cryptococcus by correlating molecular types to serotypes. For example, by means of R A P D analysis C neoformans var. neoformans (Serotype D) was typed as V N I V and C. neoformans var. grubii (Serotype A) could be typed as either V N I or VNII. Additionally, R A P D analysis was used to type C. gattii into four molecular types (VGI, VGII, VGIII and V G I V ) corresponding to C. gattii Serotypes B and C [34], Additionally, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) can be used to identify genetic subtypes within each molecular sub-type of C. gattii [35]. For example, C. gattii that has been typed as VGII can be further genotyped into V G I I A and VGIIB [35]. A n investigation by Lazera et al in 2000 revealed that C. gattii could be isolated from a number of trees including Cassia grandis (pink shower tree), Moquilea tomentosa (pottery tree) and a fig tree from the Northeastern part of Brazil [36]. In 2001, C. gattii was isolated from decaying wood in the hollow of a tree (Guettarda acreana) in a remote area of the Brazilian Amazon rainforest [37]. A more recent publication from 2003, indicated that two different strains of C. gattii were isolated from insect frass collected from E. tereticornis from a tree in Australia [38]. Eucalyptus trees in India (E. tereticornis ) have also had C. gattii isolated from the tree bark [39], but additionally isolates have also come from Syzygium cumini (Java plum tree) [40]. Therefore, by examining the above information, it can be concluded that multiple environmental sources of C. gattii exist within tropical and subtropical locations. 7 1.5 Cryptococcus gattii on Vancouver Island, British Columbia Over the last five years, there have been multiple cases of cryptococcosis on Vancouver Island in humans and animals. From 1999 to present, there have been more than 139 human B C C D C confirmed cases of cryptococcosis caused by C. gattii, four of which were fatal (Laura MacDougall, B C C D C field epidemiologist, pers. comm.). In addition, there have been more than 250 laboratory diagnosed cases of C. gattii in animals reported in British Columbia with fatalities in two horses, three llamas and six porpoises [26]. The majority of the companion animals diagnosed with a C. gattii infection were either euthanized or died as a result of the infection. Almost all of these cases surfaced in an area known as the Coastal Douglas Fir Biogeoclimatic Zone, which has a temperate climate. These cases were unexpected for two reasons; firstly C. gattii (Serogroup B) has not been identified in Canada. Most C. gattii environmental isolates have been isolated from tropical and subtropical regions. Secondly, the incidence of all cryptococcal disease, including those caused by C. neoformans, is about three to five cases per million population per year [41] in British Columbia, and thus one would only expect three cases per year of cryptococcal disease in total on Vancouver Island where the population is roughly 750, 000. However, the incidence of C. gattii infections has been between 8.5 and 37 cases/million per year on Vancouver Island from 1999 to 2003. Interestingly, the incidence of C. gattii infections in Australia is typically 0.94 cases/million per year where the organism is endemic [42]. 8 1.6 Potential for Environmental Exposure to Cryptococcus gattii on Vancouver Island The environmental niches of this organism on Vancouver Island are unlike Australia in that it is not limited to one genera of tree. Environmental sampling that has been conducted on Vancouver Island in numerous locations. A map of Vancouver Island that illustrates locations of where C. gattii has been isolated from as well as human and animal cases of infection are shown in Figure 1.1. Figure 1.1 A map of Vancouver Island illustrating locations in the environment where C. gattii has been isolated. Additionally, the map shows human and animal cases of disease. Source: Sunny Mak, 2005, British Columbia Centre for Disease Control 9 Environmental samples obtained from Rathtrevor Beach Provincial Park (RBPP) up to June 2002 resulted in the successful isolation of C. gattii from a variety of trees, and from soil, woody debris, water and air. For example, there were 57 isolates of C. gattii obtained from 24 different trees in RBPP. The number of air samples taken from RBPP positive for C. gattii were three out of 11 and one isolate out of 87 soil/tree debris collected were positive [42]. More recent information has estimated that 32% of trees tested in Parksville, B C and 19% of trees tested in Port Alberni, B C have been positive for C. gattii. Additionally, soil collected on Vancouver Island had mean values of 0 C F U gram of soil (Campbell River) to 4.7 C F U per gram of soil (Parksville) of C. gattii reported [43]. Therefore although not all samples tested were positive for C. gattii, there are a number of potential environmental sources of propagules. The presence of environmental sources of C. gattii propagules on Vancouver Island tends to support the notion that people living on the Island can reasonably be assumed to be exposed, even in the absence of a diagnosed Cryptococcus illness. It has been proposed that infection with C. gattii is a result of inhalation of air-borne propagules [44]. Residents participating in recreational activities such as gardening and hiking could be exposed to C. gattii propagules, and the mild climate on Vancouver Island makes it possible to partake in these activities year-round. The climate on Vancouver Island is by far one of the mildest in Canada with the average temperature in winter being 2.7 °C [45]. This temperate environment makes it possible for people to work outside year-round. Some occupations on Vancouver Island that necessitate working in the outdoors include forestry workers, landscapers, arborists, construction workers and those involved in 10 ecotourism. Those individuals involved in tasks that require the manipulation of soil, trees and other vegetation could be exposed to C. gattii propagules. 1.7 Crvptococcal Immunity It is generally understood that cell-mediated immunity and non-specific cellular immunity are the key defenses against fungi. However, the subject of protective immunity in fungal infections is a controversial topic. When considering the significance of antibody immunity against a pathogen there are multiple factors which will come into play. Factors to consider are: whether the presence of antibody correlates with defense against infection, whether the administration of an antibody prevents or modifies the course of infection, and if there is an association of susceptibility to infections with antibody deficiencies [46]. C. neoformans is a facultative intracellular pathogen [47] that produces a polysaccharide capsule composed mainly of glucuronoxylomannan ( G X M ) which acts as an antiphagocytic coat. This feature enables C. neoformans to survive in harsh environmental conditions and within host phagocytic cells [48]. Interestingly, it is hypothesized that the ability for intracellular parasitism evolved for protection against the predation by amoebae and slime molds [49]. There has been research that failed to support the importance of antibody immunity and protection against Cryptococcus neoformans infections. In 1967, Goren conducted a study that demonstrated vaccines containing capsular polysaccharide and protein administrated to mice were not effective in protecting mice in spite of bringing about high antibody titers [50]. A study in 1979 by Dromer et al using B cell depleted mice and normal control mice infected with Cryptococcus neoformans failed to show any difference 11 in mortality between groups or the number of colony forming units (CFU) recovered from organs post infection [51]. Finally, Diamond et al point out that post infection antibody is not necessary for phagocytosis because the complement system can provide opsonins which are necessary to carry out phagocytosis [52]. Conversely, studies have also supported the hypothesis that antibody immunity is important for protection against infections caused by C. neoformans. For example, a study by Hobbs et al in 1990 [53] demonstrated that in rabbits, a reduction in C F U s found in brain tissue was correlated with the presence of antibody in cerebrospinal fluid. Additionally, in rats the transfer from an extracellular location to an intracellular infection of C. neoformans corresponded to the appearance of serum antibody [54]. A review written by Casadevall and Pirofski in 1995 cited five different murine studies that showed that the administration of antibodies generated against G X M was effective in extending survival and reducing levels of C F U s found in organs. According to this review, the most effective vaccine was one which was a hybrid of G X M isolated from a C. neoformans (serotype A ) strain that was conjugated to tetanus toxoid [46]. Work conducted by Dromer et al in 1990 showed that mouse antibodies generated against G X M were effective in enhancing the activity of anti-fungal agents such as amphotericin B [55] in experiments where mice were infected with C. neoformans and in in vitro macrophage assays. Similar findings were reported by Mukherjee et al in 1995 with the activity of the anti-fungal agents 5-flucytosine and flucanozole [56]. There is also evidence that supports the role of antibody protection in C. neoformans infections in humans. For example, a study by Diamond and Bennett in 1972 examined prognostic factors in cryptococcal meningitis and showed that the presence of serum 12 antibody was related to a favorable outcome in those with the disease [52]. Additionally, it has been shown that the appearance of specific antibodies isolated from cerebrospinal fluid can accompany recovery [57]. The protective effectiveness of monoclonal antibodies (MAbs) to C. neoformans has been shown to be dependent on three different factors: the specificity [58, 59], the concentration [60] and the isotype [59, 61] For example, a study in male D B A / 2 mice showed that a minimum of 10 |lg dose of vaccine per mouse containing anti-Cryptococcus neoformans capsular polysaccharide M A b was required to prolong survival in mice, thus demonstrating the importance of concentration [60]. The significance of specificity for protection was demonstrated in a study that examined two IgM M A b s derived from one B cell that both agglutinated C. neoformans and bound G X M . The outcome was that one M A b prolonged survival in lethally infected B A L B / c mice and the other did not [58]. The significance of isotype in the role of protection was established when an IgG3 antibody was converted from a non-protective antibody to a protective antibody by "isotype switching" to IgGi [61]. Overall in murine studies it has been demonstrated that among the IgG subclasses there were differences in efficiency for protection and it appeared that the variability in experimental infection results were due to varying proportions of protective and non-protective antibodies [46]. Studies by Mukherjee et al in the 1990's have provided evidence of antibody-mediated enhancement of fungal infection [58, 59, 61]. Mice administered IgG3 M A b s of murine origin when infected with C. neoformans reduced survival [59, 61]. Furthermore, mice with high levels of G X M in serum given GXM-binding M A b s were lethal which may have been due to shock from antigen-antibody complex formation. This occurrence was 13 limited to only one specific strain of mice (female, Swiss-Webster) and it should be noted that administering GXM-binding M A b s to rats [62], humans [63] and other strains of mice [64] has been well tolerated. Some fungi, including C. neoformans and Candida albicans seem to have mechanisms that allow them to elude antibody immunity. The thick cells walls of C. neoformans and C. albicans appear to be resistant to complement mediated lysis and C. neoformans produces proteases [65] which may degrade immunoglobulin. Furthermore, during C. neoformans infection there are abundant quantities of capsular polysaccharide produced which could be responsible for antibody unresponsiveness [66, 67]. Polysaccharide antigen present in tissue could bind antibody and ultimately diminish the effectiveness of antibodies [46]. 1.8 Latex Agglutination Assays: Identification of Cryptococcus neoformans and C. sattii The way in which all fungal infections are diagnosed is by exclusion and the diagnosis of cryptococcosis is best accomplished through clinical findings and the detection of antigens in body fluids [68]. In addition to this information, the definitive diagnosis depends on the isolation of the organism from the site of infection and identification of the causative organism. There are multiple methods that have been used to identify C. neoformans that are based on biochemical and morphological characteristics of the organism, but unfortunately these techniques are often time consuming. C. neoformans varieties and C. gattii cannot be distinguished on the basis of biochemical utilization assays or by cellular morphology. The various serotypes of C. neoformans can further complicate identification [69]. 14 In 1982, Ikeda et al established eight antigenic factors for the five serotypes of C. neoformans and utilized these to develop a slide agglutination test [69]. The commercially available test kit " C R Y P T O C H E C K I A T R O N " involves growing the organism in pure culture and mixing a small amount of the resulting growth with a drop of physiological saline and factor serum. Following this step, the slide is carefully rotated and the observer examines the slide for agglutination. This test allows C. neoformans to be identified by serotype and is faster than other biochemical and morphological methods [70]. 1.9 Techniques Utilized to Assay Seroprevalence There are several techniques which can be used to search for the presence of antibodies in sera. In the subsequent sub-sections, the two techniques commonly used in seroprevalence assays will be described. Following these descriptions, examples of studies that have used the methods to look for antibodies within populations will be cited, including studies that have specifically looked for antibodies against C. neoformans and C. gattii. 1.9.1 Enzyme Linked Immunosorbent Assay (ELISA) In 1971, Engvall and Perlman published two papers describing enzyme linked immunosorbent assay (ELISA) [71, 72]. This technique involves coating 96-well microtitre plates with an appropriate antigen and then incubating the plates with a source of antibodies. Next, an anti-human antibody conjugated with an enzyme is applied. Finally an appropriate substrate is applied that releases a chromophore as a result of the enzyme-substrate reaction and generates a colorimetric reaction. The amount of chromophore released is directly correlated with the concentration of antibody bound to the antigen. The colorimetric 15 reaction is measured using an automated plate reader at a specific wavelength and the numeric absorbance values are compared to positive and negative controls. The E L I S A was one of the first diagnostic tests to detect antibodies against HIV in human serum and is still used today. The development of E L I S A eliminated the use of toxic radioactive substances previously used to gather the same information by radio-immunosorbant assay (RIA). A figure displaying an example of a positive result and a negative result for an E L I S A is shown in Figure 1.2. Positive Result Negative Result = antigen = serum antibody = enzyme-linked secondary antibody Figure 1.2 A modified schematic displaying examples of both a positive and negative result in an Enzyme Linked Immunosorbent Assay. Source: http://www.biology.arizona.edu/inmiunology/activities/elisa/teclmique.html 16 1.9.2 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) and Western Blotting Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) was first described by Laemmli in 1970 and this technique involves the separation of proteins in a gel matrix based on size through an electric field. Approximately ten years later, Burnette described the technique of "Western blotting" as a means of detecting specific proteins by probing with antibodies [73]. Briefly, proteins are initially denatured by treatment with heat and detergent and then electrophoresed across a polyacrylamide gel. Following the electrophoresis step, the proteins are transferred to a membrane where they are incubated with antibodies. The last step visualizes the bound antibodies either colorimetrically or by chemiluminescence. A figure displaying a schematic of SDS P A G E and Western blot results is shown in Figure 1.3. 17 P r o t e i n B l o t o n N i t r o c e l l u l o s e S D S P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s *********** = = L a b e l w i t h S p e c i f i c A n t i b o d y I)<Jte<:t A n t i b o d y — f * — — R e v e a l s P r o t e i n o f Interest i - = proteins = antibody Figure 1.3 A modified schematic displaying Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) and Western blotting. Source: http://www.bio.davidson.edu/courses/Molbio/western.GIF 1.10 The Use of Seroprevalence Assays to Determine Exposure in Populations Seroepidemiology is frequently utilized to gain information regarding the extent of exposure to a particular organism within a population. Given that the outbreak of cryptococcosis has occurred primarily within the population residing on Vancouver Island, gaining information regarding the extent of exposure to the organism would yield valuable information. A recent example of seroepidemiology study using E L I S A took place in Peru in 2004 to investigate the extent of human exposure to Leptospira. Leptospira is a zoonotic disease transmitted to humans through exposure to rodent urine. The principal aim of the 18 investigators was to determine the environmental context related to exposure to Leptospira. [74]. Another investigation carried out in Vietnam examined the seroprevalence of Helicobacter pylori within rural and urban populations. Infections with H. pylori cause gastritis, peptic ulcers and the organism is also suspected of being associated with the development of gastric cancer. The investigators utilized an in-house developed E L I S A and were able to determine which populations, by location, were at a higher risk of infection with the organism [75]. Additionally, seroprevalence assays have been conducted within occupational settings to determine if exposure to pathogens constitutes a need for vaccination. For example, a study in 2003 determined the extent of exposure to hepatitis A and B in wastewater treatment workers. The results of the study indicated that exposure to hepatitis A was 3.5 times higher than in the control population. Furthermore, evidence of past hepatitis B infections was found in 35% of the wastewater treatment workers compared to only 5% in the control population. The outcome of this study supported the policy that workers in Greece should be vaccinated against the two viruses [76]. In 1996 Seaton et al designed an E L I S A to look for immunoglobulin G (IgG) antibody against non-capsular C. gattii antigens in a population of patients and non-patients from Papua New Guinea where the organism is endemic. The outcome of this study was that investigators were able to demonstrate that patients had higher antibody levels than non-patients, and that residents of Papua New Guinea had higher antibody levels than did a non-exposed control group from the United Kingdom. Additionally, it was shown that adults 19 had significantly higher antibody levels than children and men had significantly higher antibody levels than women [77]. In 1997, Hamilton described a SDS P A G E and Western blot that investigated the humoral response of patients infected with C. neoformans var. neoformans and C. gattii. The results indicated that in sera from patients infected with C. gattii, they recognized multiple bands ranging from 2 to 19 kilodaltons (kDa). No single protein band was recognized in 100% of all sera, but there were a number of protein bands that predominated C. gattii antibody recognition representing: 37, 55, 65, 74, 94 and 115 kDa [78]. 1.11 Relevance of the Work Reported Here: Overall, there are questions that remain unanswered regarding the extent of exposure to the organism for residents of Vancouver Island, where the recent C. gattii outbreak occurred. Seroprevalence assays are an effective tool to examine exposure in a population and previous work has been conducted by other investigators involving non-capsular antigens of Cryptococcus neoformans. However, there are multiple differences existing between previous experiments and this thesis work. Firstly, the study conducted by Seaton et al in 1996 examined seroprevalence of antibodies (IgG) against C. gattii within a population from Papua New Guinea where the organism is endemic. However, the study did not address the possibility of a similar antigenic reaction to Cryptococcus neoformans var. neoformans, C. neoformans var. grubii and C. gattii. Secondly, the 1997 study by Hamilton et al using Western blotting used both non-capsular antigens from C. neoformans var. neoformans and C. gattii which allowed visualization of antibody reactions to specific sized antigen, but no antigens were ever 20 shown assayed side by side. Thus, a direct comparison of antigenic response to both C. neoformans var. neoformans and C. gattii was never investigated. Furthermore, all sera tested were from patients and thus the possibility of an antigenic response in a normal healthy population was never addressed. The first goal of this project was to develop an assay to measure specific antibodies against C. gattii. The second goal was to pilot test the assay using sera collected from residents of B C . The third goal was to use the pilot data to further define the parameters of the assay. The final goal was to propose promising proteins isolated from C. gattii protein preparations to be chosen for future study. Each of these goals was achieved and this work is presented in this thesis. 1.12 Objectives and relevant hypotheses 1) To develop a test system to detect specific antibodies to Cryptococcus gattii in human serum. 2) To pilot test the test system using sera from: • residents of Vancouver Island collected after 1999 outbreak, with the hypothesis • Ho: there is no difference between the antibody responses to non-capsular C. gattii antigens of patients sera compared to non-patient sera. • Hi: a difference exists between the antibody responses to non-capsular C. gattii antigens of patients sera compared to non-patient sera 21 miners working in B C & other Northern locations collected before 1999 outbreak, with the hypothesis • Ho: there is no difference between the antibody responses to non-capsular C. gattii antigens of Vancouver Island residents' sera and non-Vancouver Island residents' sera. • Hi: a difference exists between the antibody responses to non-capsular C. gattii antigens of Vancouver Island residents' sera and non -Vancouver Island residents sera. 22 C H A P T E R 2: M E T H O D S A N D M A T E R I A L S 2.1. Preparation of Antigen from Cryptococcus neoformans and Cryptococcus gattii The initial preparation of cryptococcal antigen was based on work done by Hamilton et al in 1992 [79] with some modifications. The method described in this section was used to obtain antigen for use in developing the E L I S A and initial Western blot analysis. A capsule deficient strain of Cryptococcus gattii from the National Collection of Pathogenic Fungi (NCPF) 3746 was grown overnight on brain heart infusion agar slants (BD Diagnostic Systems Cat #299069) at 30 °C. A representative colony was inoculated in 100 mis of brain heart infusion broth supplemented with 1-cysteine and grown overnight in a 30 °C shaking incubator. Cells were centrifuged at 2000X G in a refrigerated (4°C) centrifuge and the resulting pellet was washed with 0.01 m M phosphate buffered saline (PBS) (pH 7.4). Cells were re-suspended in a volume of 20 mis of buffer containing the 'following protease inhibit6TsT~leupepfin" (TO ~\iM 'Sigma Cat # L2884), aprotinin (~15|iM Sigma Cat # A l 153, and phenylmefhane sulfonyl fluoride (5 m M , Sigma Cat # P7626). The proportions of protease inhibitors were not described in the work done by Hamilton et al in 1992, and were determined experimentally. Cells were disrupted for two minutes in an ice-jacketed glass ball ballotini (Bead beater, Biospec Inc.) containing 0.5 mm glass beads (BioSpec Products Inc. Cat # 11079105) a total of 10 times with 5 minute gaps between pulses. The homogenized mixture was decanted and centrifuged at 6000X G and the supernatant was collected containing the cellular contents. The resulting supernatant was dialyzed overnight against distilled water at 4 °C with a total of three changes of distilled water. The protein was then concentrated utilizing the ammonium sulfate precipitation method [80]. In brief, solid ammonium sulfate was added to the supernatant and centrifuged 23 at 6000 X G for one hour at 4 °C. Following this step, the supernatant was decanted and the resulting protein pellet was re-suspended in PBS. This was then dialyzed overnight against fresh PBS with a total of three changes. The total protein was quantified using a modified version of a Bradford Assay [81] (Pierce Cat # 23236). A range of standards (0 p:g/ml to 2000 \ig /ml) were prepared as per directions using dilutions of bovine serum albumin (Pierce Cat # 23209). Aliquots of 10 pi were pipetted in triplicate into a 96-well microtitre plate (Corning Incorporated Costar Cat # 3595). Following this step, 10 ul of unknown protein were added in triplicate to the 96-well microtitre plate. A total of 300 pi of Coomassie Plus reagent was added to each well and the plate was mixed with a plate shaker for 30 seconds. The plate was incubated at room temperature for 10 minutes and the absorbance was measured at 595 nm using a plate reader (Molecular Devices Spectra M A X 190). The absorbance values of the standards were graphed and the resulting four-parameter curve was used to determine the approximate concentrate of total protein from the Cryptococcus homogenate. A figure displaying a typical four-parameter curve generated from the standards may be viewed below in Figure 2.1. 24 1.4 0 500 1000 1500 2000 2500. Concentration (u.g/mL) Figure 2.1 A n example of a standard curve generated from a modified Bradford Assay to determine the concentration of total protein from an antigen preparation. The equation of the line was used to determine the resulting protein concentration from antigen preparation 2.2 Novel Method of Obtaining Cryptococcus Antigens for Western Blot Analysis The method described in this section was developed in-house specifically for development of the Western blot analysis. Sample cultures of Cryptococcus neoformans var. neoformans (# 1211 B C Clinical Isolate) and C. neoformans var. grubii (# 977 B C Environmental Isolate) and C. gattii (NCPF 3746, # 1451, B C Environmental Isolate, # 2042, B C Environmental Isolate and # 2065, B C Clinical Isolate) were selected for isolation of cytoplasmic antigens. Each culture was initially streaked for isolation on bird seed agar containing the following: 5.20 m M creatinine, 4.2 m M glucose, 5.5 m M potassium phosphate, 13 g/L agar dissolved in'750 m L aqueous extract of 105 g/L ground niger seed. One m L of a 0.12 m M solution of chloramphenicol dissolved in absolute ethanol was added 25 to 750 mis of media prior to pouring plates. A l l cultures were grown at 30 °C for a period of 48 hours. Individual colonies were selected and resuspended in 100 pi of sterile water and spread plated on bird seed agar. Plates were incubated at 30 °C for 96 hours. The resulting growth was scraped off the plates and resuspended in 10 mis of sterile phosphate buffered saline (0.01M, p H 7.4) with the same protease inhibitors listed in section 2.1. A total of ten 1ml aliquots of the cell suspension were placed in 2.0 ml tubes containing 0.5 mm glass beads (BioSpec Products Inc. Cat # 11079105). The tubes were placed in a mechanical bead beater (Fastprep Cell Disrupter, FP120 Qbiogene, Inc. Carlsbad, C A , U S A ) for 45 seconds intervals with five minute gaps a total of nine times. The homogenate was centrifuged at 1400X G for a total of five minutes and the resulting supernatant was collected. The supernatant was dialyzed overnight against distilled water at 4 °C with a total of three changes. The total amount of protein was then assayed using a modified Bradford-Assay (PierceCat # 23236) as described above. 2.3 Description of Study Population Sera from the following groups were tested for antibodies against non-capsular C. gattii antigens: Vancouver Island Residents residing in the C D F Z (post-1999 outbreak, n=77), clinical cases during the Vancouver Island outbreak obtained from the British Columbia Centre for Disease Control and Victoria General Hospital (# 11630, and #'s 1700-1702) and miners from various locations in British Colombia and Northern Canada (pre-1999 outbreak, n=51). The primary reason for testing patient sera obtained from B C ' Serum #1630 was a B C C D C sample, non-confirmed case o f C. gattii 2 Sera 1700, 1701, 1702 was a sample from Victor ia General Hospital of a patient with culture-confirmed infections with C. gattii 26 C D C and Victoria General Hospital was for use during the developmental stages of the assay. Sera samples 1700 through 1702 came directly from the Victoria General Hospital. Within the group of Vancouver Island residents tested, there were a select number which were further categorized by the following study types: outdoor workers (those employed as B C Parks workers or working in the Greater Victoria watershed), animal owners (people whose pet was diagnosed with a C. gattii infection), cases (those diagnosed by the B C C D C ) and controls (those selected from the same geographic region as cases and from the same doctor's office, where cryptococcal infection was ruled out by chest X-ray). The above four groups were categorized by the B C C D C and additional participants were added to the outdoor workers group from the Greater Victoria watershed in conjunction with the thesis work of Adrian Hingston under the supervision of Dr. Karen Bartlett at the University of British Columbia. Ethical consent for collection and testing of sera was obtained from the University of British Colombia's Clinical Research Board (Appendix A , Certificate # C04-0042). The miners' sera were collected from various mining operations in B C , the Northwest Territories and the Yukon between 1982 and 1984. These sera were drawn as part of a longitudinal study of lung cancer in employees working in mines. The reasoning for the selection of this particular group was to determine if differences could be observed in time (prior to the 1999 outbreak on Vancouver Island) and location (non-residents of Vancouver Island). The age, sex, disease status and origin of each of the participant's serum remained undisclosed to the person conducting the serological analyses. In addition to those tested from specific groups, pooled human sera of United States (US) origin from two different 27 sources (MP Biomedicals Inc, US Biological) were used to control for non-specific reactions to C. gattii. This commercially purchased sera was from anonymous donors residing in the US where the prevalence of C. gattii in the environmental is very low with the exception of Southern California [20]. 2.4 Enzyme Linked Immunosorbent Assay (ELISA) The procedure for the E L I S A was based on the method described by Seaton et al in 1996 [77]. Non-capsular antigens obtained from the protein preparations were used to coat 96-well microtitre plates at a concentration of 0.1 |lg/|il with a total of 100 ul added per well. The antigens used in the E L I S A applications were obtained using the method described by Hamilton et al in 1992. The following are the specific cultures used for E L I S A applications: Cryptococcus neoformans var. neoformans (# 1750 B C Clinical Isolate) Cryptococcus neoformans var. grubii ( A T C C 34870) and Cryptococcus gattii (NCPF 3746). The antigen-coated plates were incubated at 4 °C overnight and following this were washed three times with PBS (Fisher Cat # BP 337). Non-specific binding sites were blocked using bovine serum albumin (Sigma Aldrich Cat # A 3350) in PBS-Tween for 30 minutes at 37 °C. Plates were subsequently washed a total of five times using PBS-Tween. Dilutions of sera from patients 1702 and 1630 were prepared at the following dilutions: 1:250, 1:500, and 1:1000. Multiple dilutions of sera were used are on the same plate to determine the optimal conditions of the E L I S A . The various dilutions of sera were added at a volume of 100 | i l per well and incubated at 37 °C. A commercially available pooled human serum (US Biologicals Cat # S 1010-52.) was used to control for background reactions. Following incubation, plates were washed five times in PBS-Tween, and dilutions (1:2500, 1:1000) of 28 a goat anti-human horseradish peroxidase IgG antibody (Sigma Aldrich Cat # A 0170) were added at a volume of 100 (0,1 to each well. Plates were incubated at 37 °C and then washed a total of four times using PBS-Tween and one final five minute wash with PBS. Sodium citrate buffer containing ortho-phenyl diamine (0.2 mg/ml) (Sigma-Aldrich Cat # P 7288) and 0.5% hydrogen peroxide (Sigma Aldrich Cat # H 325100) was used to develop the assay. Following a five minute incubation at room temperature, the colorimetric reaction was terminated using 0.1 M H2SO4 (100 | i L per well) and absorbance was measured at 492 nm using an automated plate reader (Molecular Devices, SpectraMAX 190). Based on the protocol by Seaton et al in 1996, it was expected that the absorbance values for patients would be approximately three times higher than pooled human sera obtained from an area where C. gattii is not endemic. 2.5 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) A l l sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using established protocols [82]. A n 11% sodium dodecyl sulfate (SDS) polyacrylamide gel (PAGE) was utilized to separate cytoplasmic antigens. The following are the specific cultures used for S D S - P A G E and Western blot analysis applications: Cryptococcus neoformans var. neoformans (# 1211 B C Clinical Isolate) Cryptococcus neoformans var. grubii (# 977 B C Environmental Isolate) and Cryptococcus gattii ( N C P F 3746, # 1451, B C Environmental Isolate # 2042, B C Environmental Isolate # 2065, B C Clinical Isolate). The genotyping of all Cryptococcus isolates obtained from B C environmental and clinical sources was conducted by Dr. Sarah Kidd, School of Occupational and Environmental 29 Hygiene, Michael Smith Laboratories at U B C . A total of 6 p:g of total protein was loaded into each lane containing SDS loading buffer. Protein was electrophoresed for a total of 25 minutes at 200 volts in buffer containing SDS, Tris base and glycine. The proteins were transferred to a polyvinyl di-fluoride (PVDF) membrane (Biorad Cat # 162-0177) in 10 m M C A P S (Sigma Cat # C 2632) buffer containing 10% methanol at 100 volts for a total of one hour. 2.6 Staining of Membrane for Visualization of Total Protein Separated, electrophoresed proteins were visualized by use of the GelCode Blue Stain reagent (Pierce Cat # 24590). The P V D F membrane containing transferred proteins was placed in a clean container with distilled water and rinsed for two minutes. Approximately 20 mis of GelCode Blue was added to the membrane following the rinse step and placed on an orbital shaker for five minutes. The membrane was destained in a solution containing 50% methanol, and 1% acetic acid for ten minutes with a total of three changes [83]. 2.7 Basic Western Blot Analysis Protocol A l l Western blot analysis was performed using established protocols [82]. Non specific reactive sites on the P V D F blot were blocked overnight at 4 °C in 1% casein (MP Biomedicals Cat # 904798 ) diluted in Tris Buffered Saline (TBS) (NaCl, Tris) containing 0.05% Tween-20. The blots were then incubated with the human serum being tested at serial dilutions of 1:250, 1:500, 1:1000 and 1:2000 in 0.5% casein in T B S containing 0.05% Tween-20 (TBS-Tween) at room temperature for two hours with gentle agitation. Multiple 30 dilutions of sera and secondary antibody were initially utilized to determine optimal test conditions for Western blot analysis. Additionally, serial dilutions of the test serum can be used in a semi-quantitative fashion to estimate the strength of the response in a Western blot analysis. The blot was washed for 15 minutes with TBS-Tween with a total of three changes. A n alkaline phosphatase goat anti-human IgG Fc secondary antibody (1:5000, Sigma-Aldrich, A 9544) diluted in 0.5% casein in TBS-Tween was then applied to the membrane for one hour at room temperature with shaking. Following this incubation, the blot was washed for 15 minutes with TBS-Tween with a total of three changes. The results were visualized using BCIP/NBT-purple liquid substrate (Sigma-Aldrich, B 3679) and the reaction was terminated using distilled water after three minutes. The approximate sizes of the bands were estimated using a pre-stained protein ladder. The pre-stained ladder contained ten different pre-stained proteins in the following sizes: 11, 17, 26, 34, 43, 56, 72, 95, 130 and 170 kDa (Fermentas Cat # SM0671). 2.8 Development of Western Blot Analysis Conditions to Test Populations Initial Western blot analysis took place utilizing the same method of antigen preparation used in ELISAs but, the newly developed in-house protocol described in section 2.2 was used for testing sera from Vancouver Island residents and miners. A l l developmental work was conducted using patient sera (1702) as a positive control and commercially purchased pooled human sera as a negative control. The same secondary antibody that was used in E L I S A was also used in initial Western blot analysis, which was a goat anti-human horseradish peroxidase IgG antibody. The blots were developed using an E C L substrate (Amersham Bioscience Cat # R P N 2106) and the chemiluminescent signal 31 was captured using film and an X-ray developer. Many initial blots conducted high levels of background signal making banding difficult to visualize. Thus, for ease of result interpretation, all Western blot analysis was performed as per the protocol in section 2.7 using an alkaline phosphatase goat anti-human IgG Fc secondary antibody. 2.9 Analysis of Data Once all Western blot analysis was completed the most commonly bound antigens were determined. Following this step, the results were stratified according to age, study type, place of residence, sex, and whether the sera were collected pre-1999 or post-1999. The Chi Square test was performed on data to look for differences among the groups of sera which were pilot tested. The results obtained from patient sera used for developmental work were not included in any statistical analyses. This non-parametric statistical analysis was performed using SPSS 13.0 for Windows. A non-parametric statistical test was selected because the variables are categorical. 32 C H A P T E R 3: R E S U L T S 3.1 Quantification of total protein from cytoplasmic antigens for development of immunoassays Two different methods for the extraction of cytoplasmic antigen were described in the methods section; one was based on work by Hamilton et al and was utilized for ELISAs and initial Western blot analysis and the other was a novel method developed during this study for all subsequent Western blots. The modified method developed during the course of the study resulted in a substantial increase in the total amount of protein obtained when considering the protein preparation of the C. gattii culture using N C P F 3746. The protein yield increased from 0.1 ug /u\l to 3.3 ug /p.1 without the use of an ammonium sulfate step to concentrate protein which had been essential during the early stages of assay development in this study to obtain an adequate amount of protein. 3.2 Enzyme Linked Immunosorbent Assay (ELISA) Fifteen ELISAs were conducted using the Hamilton antigen preparation, isolated from C. gattii (NCPF 3647) (Hamilton et al 1992). Samples of sera (1630, 1700, 1701, & 1702) were utilized as a positive control in the assay and commercially available pooled normal serum was used as a negative control. The difference between the two values was calculated as a percent difference using the following formula: absorbance of serum assayed at 492nm (1630. 1700. 1701 & 1702) - absorbance of pooled human serum at492nm X 100 absorbance of serum assayed at 492nm 33 Secondary antibody was at a dilution of 1 in 1000 in the assay. A n example of the results from an E L I S A using three different samples of sera obtained from the B C C D C is shown in Table 3.1. Table 3.1 Results from E L I S A #14, September 2004 comparing the absorbance values at 492 nm of pooled human serum against samples patient sera of cultured confirmed (C. gattii) sera samples (denoted by sample numbers 1700, 1701, and 1702). Primary Antibody Dilution Pooled 1700 % Difference 1701 % Difference 1702 % Difference Secondary Antibody 1 in 250 0.0563 0.139 59 0.143 61 0.161 65 1 in 2500 1 in 500 0.0480 0.106 54 0.116 59 0.116 59 1 in 1000 0.0460 0.082 44 0.079 42 0.0920 50 1 in 250 0.0510 0.147 65 0.149 66 0.170 70 1 in 1000 1 in 500 0.0467 0.113 58 0.128 63 0.128 64 1 in 1000 0,0490 0.0920 46 0.0810 40 0.0970 49 To investigate the specificity of the E L I S A , cytoplasmic antigens were obtained from Cryptococcus neoformans var. neoformans (# 1750) and C neoformans var. grubii ( A T C C 34870). In this assay, pooled human serum and samples of sera (1630, 1702) were utilized to compare reactions against C. neoformans var. neoformans, C neoformans var. grubii and C. gattii (NCPF 3746). As in the above assay, percent differences were calculated between absorbance values obtained against each organism for each serum. This assay was repeated on six different occasions at various primary antibody concentrations. A table summarizing the percent differences between pooled human serum and patient sera is shown in Table 3.2. The patient sera (1702) and suspected patient sera (1630) reacted to all 34 three serotypes of Cryptococcus. However, the patient (1702) with an active, confirmed C. gattii infection had a higher titre than suspected case (1630) hence the higher values for percent difference. The cross reactivity to all three serotypes of Cryptococcus prevented further development of the E L I S A . Table 3.2 A summary of the percent differences when comparing the absorbance values at 492 nm of patients (1702, 1630) against pooled human serum for all three varieties Primary Antibody Concentrations Serum C. gattii C. neoformans var. grubii C. neoformans var. neoformans 1 in 500 Non-confirmed patient (1630) 35% 41% 40% 1 in 500 Patient (1702) 66% 81% 75% 3.3 Study Populat ion A summary of the information available regarding samples of sera following the completion of the seroprevalence assays may be found in Table 3.3. 35 Table 3.3 A summary of information available about sera tested for antibodies against Cryptococcus gattii Group Time Sex No. of subjects Region No. of subjects Study Type No. of subjects Age No. of subjects Vancouver Island Residents n=77 Post 1999 outbreak (2004) Male 48 Female 29 Victoria 26 Duncan 12 Nanaimo 17 Parksville 5 Courtenay 5 Case 10 Control 11 Animal Owner 8 Outdoor Worker 27 <40 14 40-49 16 50-59 32 >60 14 me a n= 50.5 + 11.8 Miners from B C and other Northern locations n=51 Pre 1999 outbreak (1982 to 1984) Male 50 Female 1 Yukon 12 N.W.T 10 BC Interior 20 BC Northern 9 Miners from various pit and underground operations <40 3 40-49 23 50-59 18 >60 7 mean= 49.9 + 8.7 3.4 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) A n example of a membrane stained to visualize total protein is shown in Figure 3.1. This image showing stained total protein demonstrates three key results: 1) it shows that the S D S - P A G E effectively separated the antigens 2) it illustrates that proteins were transferred in an efficient manner to the P V D F membrane and 3) helps to show that antibody-antigen reactions were specific (i.e. antibodies were not binding to all proteins being electrophoresed) 36 kDa 1 100-72-33-24-17-Figure 3.1. Staining of total protein isolated from cytoplasm. A total of 6 |lg of protein isolated from C. gattii (#2042, lane 1), Cryptococcus neoformans var. neoformans (#1211, lane 2) and C neoformans var. grubii (#977, lane 3) was electrophoresed on an 11% SDS gel and protein was transferred to a P V D F membrane. Following electrophoresis, the total protein was stained using Gel Code. The protein bands ranged in size from 20 to 100 kDa. 3.5 Western Blot Analysis Figure 3.2 shows an example of an initial Western blot analysis using the antigen preparation described by Hamilton et al in 1992 and probed with serum from a culture-confirmed patient (1702) with a C. gattii infection. Goat anti-human horseradish peroxidase IgG antibody was used as the secondary antiserum. A l l developmental work was conducted using patient sera (1702) as a positive control and commercially purchased pooled human sera as a negative control. 37 Figure 3.2 The results of an initial Western Blot analysis utilizing antigens from C. gattii (NCPF 3746, lane 1) and C. neoformans var. grubii (# 970, B C environmental isolate, lane 2) and probing with serum from a patient with a culture-confirmed case of C. gattii. The results of the above method were difficult to interpret due to excess background signal and lacks of clear bands. Thus, in all subsequent blots an alkaline phosphatase conjugated secondary antibody was used. The Western blots shown below (Figure 3.3) illustrate the combined outcome of using a new secondary antibody, an alkaline phosphatase IgG Fc, to detect bound human antibody and the novel method of antigen preparation. Three antigens believed to be specific to C. gattii (20, 30 and 50 kDa) were recognized by patient sera (A,B) and the 50 kDa antigen was recognized by sera obtained from a suspected exposed individual (C). 38 A B C Figure 3.3 Western blot analysis of sera from two B C C D C patients (A,B) at a dilution of 1 in 2000 and serum from one suspected exposed individual (C) at a dilution of 1 in 250. Serum was assayed antibodies against cytoplasmic antigens from C. neoformans var. grubii (lane 1), C. neoformans var. neoformans (lane 2), and C . gattii initial procedure for protein extraction (lane 3), and C. gattii modified protein extraction procedure (lane 4). During the process of developing the Western blot analysis protocol, it was necessary to examine additional sources of sera to ensure that the antibody-antigen reactions such as those displayed in Figure 3.3 were distinctive. The images of a Western blot analysis displaying the reaction of pooled human sera from two sources of origin are shown below along with examples of B C C D C patient serum (Figure 3.4). In addition to the antigens (20, 30, and 50 kDa) shown in Figure 3.3, a reaction to a 40 kDa antigen was also observed in patient serum (Figure 3.4, sample C). 39 k D a **** ____________ 55- 55-A 1 2 k D a B 1 2 -40 -30 -20 -50 -30 -20 Figure 3.4 Western blot analysis of two different sources of pooled human sera from the United States (A, B) at a dilution of 1 in 250, and two samples of patient serum obtained from the B C C D C (C,D) at a dilution of 1 in 2000. Serum was assayed for antibodies against cytoplasmic antigens from C. neoformans var. neoformans (lane 1) and C. gattii (lane 2). The results of the above blots indicated that in samples of patient sera one or more of the antigens isolated from C. gattii ranging in size between 20 and 50 kDa were recognized. The results of the pooled human sera (Figure 3.4 samples A and B) showed no specific reaction to antigen isolated from C. gattii in this size range. 40 3.6 Western Blot Analysis of Vancouver Island Residents Once the Western blot analysis conditions were established using patient sera provided by the BC CDC as positive controls and two different sources of pooled human serum from the United States to control for background exposure, the screening of residents from Vancouver Island commenced. An example of three different Western blots testing sera from residents of Vancouver Island may be viewed in Figure 3.5. Figure 3.5 Western blot analysis of sera from Vancouver Island residents (A,B,C). Sera ( 1 in 500) was assayed for antibodies against cytoplasmic antigens from C. neoformans var. neoformans (lane 1) and C. gattii (lane 2). 41 A total of 77 samples of sera from Vancouver Island residents were evaluated using Western blot analysis. It was possible to group the results of these residents together because high airborne C. gattii concentrations were demonstrated in areas of Vancouver Island and thus any resident was potentially exposed to the propagules regardless of disease status. The results obtained from patient sera (1702, 1630) used in the development of the assay were not included in any statistical analyses. The following are the approximate sizes of the cytoplasmic antigens that were most commonly recognized by sera: 20 kDa, 30 kDa, 40 kDa and 50 kDa. Among these proteins, the 20 kDa and the 50 kDa antigens were most commonly recognized among Vancouver Island residents tested (Figure 3.6). When examining the results by sex, no significant differences existed for the number of sera samples reacting to the 20 kDa or the 50 kDa antigens at any of the four dilutions tested. 42 90 0 _1 in 1000 • 1 in 2000 H1 in 500 • 1 in 250 20 kDa 30 kDa 40 kDa 50 kDa Sizes of Antigens (kDa) Figure 3.6 A graph displaying the percentage of sera tested from Vancouver Island residents (n=77) with antibodies against C. gattii. The most commonly recognized antigens are displayed along with the four different dilutions at which they were tested (1 in 250, 1 in 500, 1 in 1000 and 1 in 2000). Information regarding the geographic origin of 69 samples of sera was available for residents of Vancouver Island and Vancouver. The total percentage residents tested with antibodies against the 20 and 50 kDa antigens isolated from C. gattii are reported in Table 3.4 and Table 3.5 respectively. The greatest percentage of samples with antibodies against the 20 kDa antigen at a dilution of 1 in 500 was from residents of Nanaimo (70.6 %) and Victoria (69.2 %). Chi Square analysis of the data stratified by region revealed no significant differences for the 20 kDa antigen for Vancouver Island sera at any dilution tested. No analyses were conducted on any of the results of the less commonly recognized antigens (i.e. 30 kDa and 40 kDa). 43 Table 3.4 The percentage of sera tested with antibodies against the 20 kDa antigen reported by region following the 1999 outbreak of C. gattii. Primary Antibody 20 kDa Antigen Region Concentration (% of Positive samples) Courtenay (n=5) 1 in 500 60.0 1 in 2000 0.0 Parksville (n=5) 1 in 500 40.0 1 in 2000 0.0 Nanaimo (n=17) 1 in 500 70.6 1 in 2000 41.2 Duncan(n=12) 1 in 500 58.3 1 in 2000 8.3 Victoria (n=26) 1 in 500 69.2 1 in 2000 11.5 Vancouver(n=4) 1 in 500 50.0 1 in 2000 0.0 The highest percentage of sera samples tested for antibodies against the 50 kDa antigen at the 1 in 500 dilution were residents tested from Nanaimo (47.1%) and followed by Duncan (41.7%), Parksville (40%) and Courtenay (40%) (Table 3.5). It should be noted that there were some samples of sera from Vancouver with antibodies against the 50 kDa antigen at the 1 in 250 dilution (Appendix B, Table B.l). No significant differences were found when results were stratified by region for the 50 kDa antigen at any dilution. The results all of antigens at each of the four dilutions of sera as reported by region are appended (Appendix B, Table B.l). 44 Table 3.5 The percentage sera tested with antibodies against the 50 kDa antigen reported by region following the 1999 outbreak of C. gattii. Region Primary Antibody 50 kDa Antigen Concentration (% of Positive samples) Courtenay (n=5) 1 in 500 40.0 1 in 2000 20.0 Parksvile (n=5) 1 in 500 40.0 1 in 2000 . 0.0 Nanaimo (n=17) 1 in 500 47.1 1 in 2000 11.8 Duncan(n=12) 1 in 500 41.7 1 in 2000 33.3 Victoria (n=26) 1 in 500 26.9 1 in 2000 15.4 Vancouver(n=4) 1 in 500 0.0 1 in 2000 0.0 In addition to information available on the region where the sera was collected, it was also possible to stratify the results based on study type. Samples originated from outdoors workers, cases of cryptococcosis, controls from the case-control study and animal owners (owner of pets with C. gattii infections). The greatest percentage of samples of sera with antibodies against the 20 kDa antigen at a 1 in 500 dilution was the outdoor workers group (81%); samples categorized as cases had the lowest percentage o f positive samples (30%>). Ch i Square analysis of the data revealed a significant difference at the 1 in 500 dilution (p < 0.025) for the 20 kDa antigen (Figure 3.7). 45 1 in 500 Outdoor Workers (n=27) 1 in 2000 Animal Owners (n=8) Figure 3.7 Percentage sera samples tested from Vancouver Island residents with antibodies against the 20 kDa antigen reported by study type at 1 in 500 and 1 in 2000 dilutions of sera. The greatest percentage of samples of sera with antibodies against the 50 kDa antigen as reported by study type were sera samples categorized as cases (50%) and those classified as outdoor workers that the lowest percentage samples (22%) with antibodies against this sized antigen at the 1 in 500 dilution (Figure 3.8). No significant differences were found for antibodies against the 50 kDa antigen when the results were stratified by study type. The results of the sera for all of antigens at each of the four dilutions tested as reported by study type may be viewed in Appendix B, Table B.2. 46 ._ 100 J o 9 0 -Figure 3.8 Percentage sera samples tested from Vancouver Island residents with antibodies against the 50 kDa antigen reported by study type at 1 in 500 and 1 in 2000 dilutions of sera. The results for 76 of the residents of Vancouver Island were stratified by age. The greatest percentage of sera samples with antibodies to the 20 kDa antigen at the 1 in 500 dilution were those falling into the age category of 50 to 59 years of age (72%) where as those less than the age of 40 had the lowest percentage sera samples with antibodies against this antigen (43%) (Figure 3.9). No significant differences were found for antibodies against the 20 kDa antigen when the results were stratified by age. 47 40 to 49 50 to 59 1 in 500 40 to 49 j 50 to 59 1 in 2000 Figure 3.9 Percentage sera samples tested from Vancouver Island residents with antibodies against the 20 kDa antigen reported by age group at 1 in 500 and 1 in 2000 dilutions of sera. The greatest percentage of sera samples with antibodies against the 50 kDa antigen when stratified by age at the 1 in 500 dilution were those falling into the age category of 50 to 59 years of age (44%) where as those between the age of 40 and 49 had the lowest percentage sera samples with antibodies against this sized antigen (13%) (Figure 3.10). Chi Square analysis revealed significant differences at both the 1 in 250 dilution (p < 0.031) and the 1 in 1000 dilution (p < 0.047). The results all of antigens at each of the four dilutions of sera as reported by age group may be viewed in Appendix B, Table B.3. 48 (A 100 90 + 80 70 i S 60 + "So 50 40 4-o ro (A in a> — co — a> en — ro ro co 30 20 10 0 <40 40 to 49 50 to 59 >60 1 in 500 <40 I 40 to 49 | 50 to 59 I >60 1 in 2000 Figure 3.10 Percentage sera samples tested from Vancouver Island residents with antibodies against the 50 kDa antigen reported by age group at 1 in 500 and 1 in 2000 dilutions of sera 3.7 Western Blot Analysis of Miners sera collected prior to the 1999 outbreak of C. gattii on Vancouver Island Pilot sera from a collection obtained in the early 1980's from various locations in the Northwest Territories, Yukon, as well as the interior and Northern British Columbia was assayed for antibodies against C. gattii. These sera were drawn as part of a longitudinal study of lung cancer in employees working in mines. A n example of a serum that did not react to C. gattii cytoplasmic antigens and two examples of reactive sera for this group may be viewed below (Figure 3.11). 49 1 2 kDa -50 -20 Figure 3.11 Western blot analysis of miners' sera (1 in 250) collected prior to the 1999 outbreak of C. gattii from the Northwest Territories, Yukon and central and Northern British Columbia. This figure displays examples of a negative result (A) and two positive results (B,C). Sera were assayed for antibodies against cytoplasmic antigens from C. neoformans var. neoformans (lane 1) and C. gattii (lane 2). A total of 51 samples of sera from miners were evaluated using Western blot analysis for antibodies against C. gattii antigens. The following are the approximate sizes of the cytoplasmic antigens that were most commonly recognized by sera were: 20 kDa, 35 kDa, 40 kDa and 50 kDa. Among these antigens, the 20 kDa and the 50 kDa were most commonly recognized among miners tested at all dilutions tested. No analyses were conducted on the 35 kDa and 40 kDa antigens (Figure 3.12). 50 13 <U 100.0 90.0 80.0 70.0 60.0 52 <° £ o 50.0 40.0 30.0 20.0 10.0 0.0 11 in 250 H 1 in 500 H i in 1000 • 1 in 2000 20 kDa 35 kDa 40 kDa Sizes of Antigens (kDa) 50 kDa Figure 3.12 Percentage of miners' sera tested (n=51) with antibodies against C. gattii. The most commonly recognized antigens are displayed at four tested dilutions (1 in 250, 1 in 500, 1 in 1000 & 1 in 2000). 3.8 Comparing results of sera tested from Vancouver Island Residents and Miners The results of residents tested from Vancouver Island after the 1999 outbreak and miners from other locations collected before the 1999 outbreak were compared. When examining the 1 in 500 dilution for the 20 kDa antigen, 64.9 % of residents tested had antibodies against C. gattii compared to only 17.6 % of all miners tested (Figure 3.13). 51 100.0 90.0 • - « 80.0 5 o i o>0 « 2 </>'ro 50.0 o> O l | ~ 30.0 CO X J 20.0 1 in 250 • Residents • Miners 1 in 500 1 in 1000 Dilutions of Sera 1 in 2000 Figure 3.13 Percentage of sera tested from Vancouver Island residents (post-1999) compared to miners sera tested (pre-1999) with antibodies against C. gattii. This figure displays results for a reaction to the 20 kDa antigen at various dilutions of sera. The results for the 50 kDa at the 1 in 500 dilution showed that 32.5 % of residents tested had antibodies against this size protein compared to 7.8 % of miners tested (Figure 3.14). A Chi Square test comparing the results of miners to residents revealed significant differences (p=< 0.001) for the following dilutions for the 20 kDa antigen: 1 in 250, 1 in 500 and 1 in 1000. A significant difference also existed at the 1 in 2000 dilution for the 20 kDa antigen (p=< 0.031). Significant differences were also revealed when comparing miners to residents for antibodies against the 50 kDa antigens at the following dilutions: 1 in 250 (p= < 0.001), 1 in 500 (p='< 0.002), 1 in 1000 (p= < 0.002) and 1 in 2000 (p= < 0.045). 52 The results all of antigens at each of the four dilutions of sera for miners may be viewed in Appendix B, Table B.4. • Residents • Miners 1 in 250 1 in 500 1 in 1000 Diutionsof Sera 1 in 2000 Figure 3.14 Percentage of sera tested from Vancouver Island residents (post-1999) compared to miners sera tested (pre-1999) with antibodies against C. gattii. This figure displays results for a reaction to the 50 kDa antigen at four serum dilutions. 53 C H A P T E R 4: DISCUSSION 4.1 Preparation of Antigen from Cryptococcus neoformans and Cryptococcus gattii The isolation of cytoplasmic antigens from Cryptococcus neoformans var. neoformans, C neoformans var. grubii and C. gattii proved to be a difficult experimental task. The primary challenge was obtaining an adequate amount of total protein to conduct immunoassays. The antigen preparation method based on the work of Hamilton et al in 1992 was followed for use in for the E L I S A and initial Western blot development. This preparation was neither sensitive nor specific enough to discern proteins unique to C. gattii. A n in-house method was developed specifically for this study with the aim of increasing the quantity and quality of protein for Western blot analysis. A comparison of the protein yield for the method described by Hamilton et al in 1992 and the novel method developed for this study revealed a large difference in protein yields. For example, the difference in total protein yield increased from 0.1 ug /ul to 3.3 ug /ul, which is equivalent to a 33-fold increase when using C. gattii N C P F 3746. The initial method adapted from Hamilton et al, 1992 utilized a large volume bead beater with a blade. The major disadvantage associated with this method was that it necessitated the use of a large volume of buffer (200 mis) for the beater to operate. One consequence of using the large volume of buffer was that the homogenate was not cooled effectively between the one-minute pulses potentially resulting in damage to proteins. Another consequence of using this large volume of buffer was that the proteins became diluted in this large volume, thus requiring treatment with ammonium sulfate to concentrate the protein. The use of the ammonium sulfate method to concentrate the protein also was fraught with technical difficulties. For example, it was frequently difficult to pellet 54 the protein as the large volume resulted in protein being dispersed throughout the solution. Additionally, the presence of the ammonium sulfate made it necessary to dialyze the protein against PBS overnight to reduce the salt content of the homogenate which ultimately resulted in a more dilute protein sample because of the osmotic nature of the protein pellet. The novel method developed for this study used small volumes of cell suspension for homogenizing cells to obtain cytoplasmic antigens. The smallest volume that could be used for homogenizing was 1 ml compared the 200 mis required for the method described by Hamilton et al in 1992. Using this smaller volume allowed for adequate cooling between pulses in the mechanical bead beater and lessened the dilution factor of the protein. Another major difference in the two antigen preparation methods was the way in which the cells were grown and harvested. The method described by Hamilton et al in 1992 used brain-heart liquid medium for growth of C. gattii. One drawback associated with growth in this liquid medium was that to obtain adequate numbers of cells, it often took five to seven days. Bacteria grow very easily in brain-heart medium, and it was difficult to avoid contamination of the slower growing cryptococci over the prolonged incubation period, as the cell density within the flasks had to be monitored. The method developed for this study used a bird seed agar which after initial selection of colonies only required 48 hours to obtain adequate growth of cells, reducing the chance and effects of contamination. Cells were harvested using the method developed for this study by scraping the lawn growth from the solid bird seed agar into the buffer containing the protease inhibitors thus resulting in the loss of very few cells. In contrast, the method described by Hamilton et aim 1992 required that the cells growing in liquid culture be centrifuged, washed several times 55 and then introduced to the buffer containing the protease inhibitors resulting in the loss of cells due to the number of times the cells were manipulated. The development of the novel antigen preparation for this study was paramount for the outcome of this research. The change in the antigen preparation resulted in the ability to visualize a large range of protein sizes which was not possible when the Hamilton et al, 1992 protocol was utilized. Specifically, it was not possible to visualize proteins smaller than 50 kDa when the Hamilton et al, 1992 method was used. This is because the transfer of smaller proteins to the P V D F membrane is inefficient when these proteins are in low concentration. Once the overall yield of protein had increased, it was possible to visualize antibody-antigen reactions to a large range of proteins. 4.2 Enzyme Linked Immunosorbent Assay (ELISA) It can be concluded that the use of E L I S A was not an effective means to determine exposure to C. gattii for residents of Vancouver Island due to a lack of specificity using Seaton's protocol. The results of ELISAs performed using commercially purchased pooled human serum and culture-confirmed patient serum demonstrated a difference in absorbance values when using C gattii (NCPF 3746) as an antigenic substrate. The calculated percent difference between the absorbance values of pooled human serum and patient serum (1702) was as high as 70% under conditions that examined exposure to C. gattii (NCPF 3746) cytoplasmic antigens. Therefore, it can be concluded that it is possible to distinguish between the sera of patients with Cryptococcus infections and pooled human serum. The percent difference in absorbance values was even higher in C. neoformans var. neoformans (# 1750 at 75%) and C. neoformans var. grubii ( A T C C 34870, at 81%) for this 56 patient's serum (1702), which indicated a lack of antibody specificity.to these antigens. A similar occurrence was seen when testing the patient serum sample 1630, with calculated percent differences of 40% for C. neoformans var. neoformans (# 1750), 41% for C. neoformans var. grubii ( A T C C 34870) and 35% for C. gattii (NCPF 3746). This finding was an additional piece of information that indicated a lack of antibody specificity to these antigens. The similar absorbance values obtained from ELISA's indicate that antibodies in sera tested will bind equally to antigens and thus using making it impossible to use absorbance values as a means to determine specific antigen reactions to C. gattii. It is noteworthy that serum sample 1630 was a resident of Vancouver Island and not a patient as initially thought which explains the lower absorbance values and calculated percent difference compared to patient 1702. Some possible explanations for antibodies in sera binding to the multiple Cryptococcus antigens are: 1) cross reactivity of the individual's antibodies with similar antigens among the varieties, 2) the individual has been exposed either within his or her environment to these varieties, 3) the individual may have presently or in the past had an infection with these other varieties of Cryptococcus. Evidence of literature which supports the idea that antibodies against C. neoformans var. neoformans can be acquired from an environmental source was published in 2001 by Goldman et al. The publication by Goldman et al demonstrated children living in New York had antibodies against C. neoformans var. neoformans because it is ubiquitous in their environment [9]. A n alternative explanation for the antigen-antibody reaction to all three varieties was the possibility of capsule being present in the antigen preparation, which would result in efficient binding to all varieties. A capsule deficient strain of C. gattii (NCPF 3746) that 57 was used in the E L I S A as it was thought to reduce cross reactivity. To resolve the potential issue of capsule contamination, antigen preparations were repeated and extra steps were taken to reduce the possibility of contamination. Once these preparations were completed, the ELISAs were repeated and the outcome remained the same. Additionally, the strain of C. gattii (NCPF 3746) used in ELISAs was a culture from Papua New Guinea (PNG) and there is- the possibility that there was sufficient genetic divergence existed between the P N G stain and the strain associated with the B C outbreak to result in loss of sensitivity for the assay. Antibody-antigen specificity was never addressed in the publication by Seaton et al in 1996. The C. gattii (NCPF 3746) used in the study by Seaton et al was isolated from Papua New Guinea, where C. gattii is endemic. The E L I S A protocol described in the publication by Seaton et al used C. gattii (NCPF 3746) cytoplasmic antigens. This strain of C. gattiiwe used was purportedly identical to one the utilized by Seaton's study and the method used to isolate the antigens was based on the protocol outlined the publication by Seaton et al. The study by Seaton et al adequately addressed the issue of sera controls (unexposed population was residents of London, England), but the study failed to control for the possibility of antibody-antigen reactions to the C. neoformans var. neoformans and C. neoformans var. grubii. Antibody-antigen specificity was addressed in this thesis work and the evidence gathered here showed that serum from patient 1702 and serum sample 1630 bound all three varieties of Cryptococcus assayed in the E L I S A . The lack of specificity in the E L I S A resulted in the need to develop the Western blot analysis instead. 5 8 4 . 3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) One method to examine specific antigen-antibody reactions is to separate proteins by size which can be accomplished using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS P A G E ) . Different percentages of acrylamide were used to determine the ideal conditions to separate the cytoplasmic antigens. In general, the lower the percentage of acrylamide in the gel, the faster the proteins will migrate and as the acrylamide percentage increases, proteins migrate more slowly through the matrix resulting in greater resolution. Through experimental trial and error it was concluded that an 11% acrylamide gel was the ideal concentration for the separation of cytoplasmic antigens from C. gattii, C. neoformans var. neoformans and C. neoformans var. grubii. In general, the percentage of acrylamide used in SDS P A G E procedures ranges between 10% and 12% and the optimal percentage is based on qualitative observations made by the investigator. The visualization of these proteins once they had been transferred to a P V D F membrane was accomplished using GelCode Blue. The staining of this membrane revealed the presence of numerous protein bands in all of the Cryptococcus tested. Staining the proteins was an important step as it aided in visualizing the potential sites of antibody binding for each different variety of Cryptococcus. 4 . 4 Development of Western Blot Analysis Initially Western blots were conducted using the same secondary antibody used in the ELISAs which was a goat anti-human horseradish peroxidase secondary antibody. These initial blots took place using patient serum and cytoplasmic antigens from C. gattii and C. neoformans var. grubii. The bound antibodies were detected using 59 chemiluminescence captured on X-ray film developer. The primary problem encountered when using this method of detection was that the signal was difficult to distinguish due to excessive background signal. Multiple attempts were made to diminish the background signal by increasing wash steps and modification of blocking protocols, but eventually this antibody was replaced with an alkaline phosphatase goat anti-human conjugated serum. The use of the alkaline phosphate conjugated secondary antibody in place of the horseradish peroxidase conjugated antibody improved the visualization of antibody-antigen reactions. The technique for cytoplasmic antigen extraction devised specifically for this study yielded a higher quantity of protein compared to the method described by Hamilton et al in 1992. During the developmental stages of Western blot analysis, it was possible to assay the antigens obtained from using the method based on the work by Hamilton et al, 1992and compare it to the technique developed for this study. The results of this particular exercise were imperative as this is when antibody-antigen reactions to proteins less than 70 kDa were able to be visualized. The side-by-side Western blot analysis showed sera from Vancouver Island residents and patients binding to C. gattii cytoplasmic antigens less than 70 kDa using the antigens from the in-house developed method where as this was not the case with the antigens obtained using the method developed by Hamilton et al, 1992. Cytoplasmic antigens from C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii obtained using the newly developed method were assayed side by-side in a Western blot analysis. By carrying out this exercise, it was now possible to visualize antibody-antigen reactions C. neoformans var. neoformans, C. neoformans var. grubii and C. gattii in parallel. It was observed that antibodies were binding to antigens of all three of the Cryptococcus tested in sera from patients and residents which agrees completely with the 60 results obtained from the ELISAs. Furthermore, the parallel Western blot analyses showed in some cases that residents' antibodies were binding to similarly sized antigens in the different varieties, strengthening the evidence of cross-reactivity among species. In conclusion, assaying the varieties in parallel with different samples of sera from residents made it possible to identify antibody-antigen reactions unique to C. gattii by subtracting reactions that were common to the other cryptococcal preparations. These results were encouraging, but to ensure the results were not due to non-specific antigen-antibody reactions, pooled human sera from two different US sources were assayed beside patient sera. The observations from this assay demonstrated that the pooled human serum had distinct binding to C. neoformans var. neoformans (55 kDa) cytoplasmic antigens as expected because this organism is commonly found in the environment and only faint binding to the C. gattii cytoplasmic antigens. Conversely, the patient sera obtained from the B C C D C demonstrated distinct binding to antigens from both organisms, but the sizes of at least three different antigens being recognized in the C. gattii lane were unlike those in C. neoformans var. neoformans lane. Western blot analysis conducted by Hamilton et al in 1997 investigated the humoral response of patients infected with C. neoformans var. neoformans and C. gattii. The work by Hamilton et al did not show Western blot analysis with C. neoformans var. neoformans and C. gattii antigens assayed in parallel. The weakness in conducting an assay in this manner is that if similar antibody-antigen interactions are taking place in both species, it would be difficult to differentiate. This is because when carrying out an S D S - P A G E , the size of a protein is always an estimate and therefore to clearly identify unique antibody-antigen interactions they must be shown side-by-side in the same matrix. 61 In summary, the key factors in developing the informative Western blot analysis were: 1) obtaining an adequate quantity of protein by devising an effective technique, 2) using a secondary antibody that enabled clear visualization of antibody-antigen interactions, 3) using a source of pooled human serum to control for non-specific binding of proteins and 4) assaying antigens in parallel to control for similar antigenic reactions among species. The above information has demonstrated that it was possible to achieve the goal of developing an assay to measure specific antibodies against C. gattii. 4.5 Western Blot Analysis of Vancouver Island Residents Once Western blot analysis had been completed on Vancouver Island residents' sera (n=77), it was possible to examine the results in a number of ways. The results obtained from patient sera (1702, 1630) used in the development of the assay were not included in any statistical analyses. Overall, the most commonly recognized antigens were 20 kDa (82%) and 50 kDa (48%) at the 1 in 250 dilution. Work by Hamilton et al in 1997 examined the response of patient IgG antibodies to cytoplasmic antigens of C. gattii and found that the antigens predominately reacted to were: 37, 55, 65, 74, 94 and 115 kDa. Differences in results could have several explanations. Firstly, Hamilton et al, 1997 was only examining the antigenic response in patients with C. gattii infections. There is no information contained in the publication by Hamilton et al, 1997 indicating that samples of pooled human serum or sera of non-patients was examined. The problem with not assaying sera from a pooled source and/or sera from non-patients is that this method fails to control for background exposure to C. gattii antigens. The Western blot analysis carried out in this thesis work controlled for background exposure to C. gattii antigens by using pooled human 62 serum and also examined the antibody response in a group where the majority of participants were not infected. Hamilton et al, 1997 used cytoplasmic antigens isolated from N C P F 3169 and N C P F 3170, strains of C. gattii, which are different than the strain used in the work by Seaton et al, who used N C P F 3746. Hamilton et al, 1997 combined the techniques of bead beating followed by iso-electric focusing to obtain antigens. The C. gattii strains used in this thesis . were isolated from environmental and clinical sources in B C and the antigens were obtained using a newly developed method. The C. gattii isolates used in this thesis work were Serogroup B, and belonged to the VGII molecular group (# 1451, VGIIb, #2042, VGIIa, and #2065, VGIIa ) Kidd reported that 95% of isolates collected in B C from 1999 to 2002 belonged to the VGII molecular group [35]. The genotypes of N C P F 3169 and N C P F 3170 are not published. The work by Hamilton et al, in 1997 reported antibody responses to antigens 65, 74, 94 and 115 kDa in size. In this thesis work, no antibody responses were reported to antigens larger than 55 kDa as the resolution of the larger sized antigens was difficult to differentiate. Additionally, when a similar antigen response was observed across the Cryptococcus species it was in antigens greater than 55 kDa in size. Therefore, although antibody responses to antigens greater than 55 kDa were observed, it could not be stated with absolute certainty that this was a specific response to C. gattii antigens. When the results were stratified by sex, no significant differences were found among residents of Vancouver Island. This finding is inconsistent with the results of a study conducted in Papua New Guinea in 1996, where C. gattii is endemic, and which utilized 63 E L I S A to assay for IgG antibodies against cytoplasmic antigens. In that study, it was demonstrated that males had higher absorbance values compared to females and that a greater proportion of males had positive test results. This difference in results may be due in part to cultural differences between gender roles in Canada compared to Papua New Guinea. According to Seaton et al, females in Papua New Guinea still tend to stay close to home and tend to their families where as males must leave their home environment to seek employment [77]. Conversely, both men and women in Canada frequently leave their residence for employment. In addition, environmental testing conducted around residential dwellings in Papua New Guinea failed to show the presence of C. gattii propagules and thus the lack of evidence for exposure among females. One explanation for the difference in findings of this thesis work compared to that done by Seaton with respect to sex is likely related to differences in cultural norms. When results were examined by region, no significant difference existed at any dilution for the 20 kDa antigen. It is interesting to note that at the 1 in 2000 dilution for the 20 kDa protein, 41% of Nanaimo residents tested had antibodies recognizing this antigen with the next highest value being residents tested from Victoria at 11%. A potential explanation for could be that residents of Nanaimo live in an area where C. gattii is endemic and frequently travel to recreational areas where C. gattii is also endemic, such as Parksville, Englishman River and Little Qualicum Falls. When examining the results for the 50 kDa antigen, no significant differences existed among the various dilutions but 33% of residents in Duncan tested had antibodies at the lowest dilution 1 in 2000 which was approximately twice the value of those residing in Victoria. After examining the results by region, despite 64 no significant differences being found, more samples of sera should be tested to gain a better understanding the number of individuals who have antibodies against C. gattii antigens. The results were examined by age and no significant differences were found at any dilution for the 20 kDa antigen. Conversely, the results for the 50 kDa antigen showed significant differences for the 1 in 250 dilution (p< 0.031) and the 1 in 1000 (p< 0.047) dilution. When examining the 1 in 250 dilution, 64% of those greater than 60 years of age had antibodies against the 50 kDa antigen compared to only 21% of those less than 40 years of age. When considering all dilutions, excluding 1 in 2000, it was calculated that on average those tested who were less than 50 years of age had a smaller percentage of positive results (14%) compared to those tested who were greater than 50 years of age (40%). According to a publication by Hoang et al in 2004, the median age of individuals diagnosed with cryptococcosis (HIV-negative patients) between 1997 and 2002 through the Vancouver General Hospital was 67 years of age with range of 47 to 75 years of age. The information gathered by Hoang et al was specifically examining the cases of cryptococcal infection on Vancouver Island [84]. A possible explanation for a higher percentage of those tested with antibodies against C. gattii antigens in those greater than 50 years of age could be related to the time spent outdoors engaging in activities such as yard work which frequently requires the manipulation of soil and pruning of trees or other recreational activities. The results for those assayed were stratified based on different study types (i.e. outdoor workers, cases, controls and pet owners) no significant differences were found for the 50 kDa antigen at any dilution. Conversely, a significant difference was observed at the 1 in 500 dilution (p< 0.025) for the 20 kDa antigen. The highest percentage of positive samples for this antigen were among those in the outdoor workers group (81%) and those 65 owning pets with C. gattii infections (75%) compared to. those classified as cases (30%). The primary explanation for outdoor workers having a high percentage of participants with antibodies against the 20 kDa antigen is' elevated exposure to C. gattii propagules due to engaging in outdoor tasks such as planting, pruning, cutting and/or tree removal. There are two potential explanations for why pet owners tested have a high percentage of participants with antibodies against the 20 kDa protein. One possibility could be due to the presence of C. gattii in the soil or trees surrounding the residence of the pet. Another explanation could be that many pet owners take their animals to local recreational areas and are thus exposed when they visit parks. The explanation for why there is a greater percentage of outdoor workers and pet owners with antibodies against the 20 kDa antigen compared to cases is unknown, however one possible explanation for this is variability in how a hosts immune system processes antigens. For example, when antibodies are formed to protein or protein-conjugates they may be variously expressed in the host. This type of result has been seen in work by Kakeya et al in 1997 when mice infected with C. neoformans var. neoformans expressed antibodies against a variety of antigens which were later identified as belonging to a family of proteins called "heat shock proteins". Not all mice infected with C. neoformans var. neoformans had antibodies against the same antigens, but there were some antigens which were classified as "immunodominant" [85]. This demonstrates that although a host may be infected with the same organism, it may not express antibodies against all the same antigens. A study by Burt et al in 2003 demonstrated that a commercially purchased anti-heat shock protein antibody (anti-HSP-90) raised in an undisclosed mammal recognized a variety of antigens (47, 72 and 82 kDa) isolated from Candida albicans [86]. This study is an example of how 66 an antibody specifically designed to recognize one type of antigen can recognize multiple antigens. Further work is necessary to determine the identity of the 20 kDa and 50 kDa proteins, but from evidence we have thus far from Kidd et al [35, 42] we know there are two molecular subspecies of C. gattii in the environment and in B C clinical cases. The subtle differences between the molecular species can only be determined by comparing the genomes of the B C isolates to the recently completed Cryptococcal genome sequences (http://www.bcgsc.ca/about/news/crypto_public), and thus lead to the identify of the expressed proteins 20 kDa and 50 kDa. These antigens (20 and 50 kDa) are unique to C. gattii which is evident from examining the results from the Western blot analyses assaying antigens from C. gattii and C. neoformans var. neoformans in parallel with sera being tested. Despite the uniqueness of these proteins in C. gattii, they may not be equally processed by the host which accounts for differences among sera samples. The above information has demonstrated that it was possible to achieve the goal of pilot testing the assay using sera collected from residents of B C . Additionally, the information gathered from pilot testing was useful for proposing promising proteins isolated from C. gattii protein preparations to be chosen for future study. 4.6 Western Blot Analysis of Miners' sera prior to the 1999 outbreak of C. gattii Sera obtained from a study of miners collected in the early 1980's from various locations in Northern and Central B C , Yukon and Northwest Territories was examined for evidence of antibodies against C. gattii antigens. One aspect that-had to be carefully controlled when examining this collection of sera was whether or not the antibodies would 67 still be reactive after being stored for greater than 20 years. However, antibody-antigen reactions were observed against C. neoformans var. neoformans cytoplasmic antigens, thus validating quality of these sera. Approximately 25% of miners tested had antibodies against the 20 kDa antigen and 18% had antibodies against the 50 kDa antigen at the 1 in 250 dilution. However, when examining the results for the 50 kDa antigen at the 1 in 250 dilution, 10% or less of those tested residing in the Yukon or Central and Northern B C had antibodies where as 50% of those tested from the Northwest Territories had antibodies against this antigen. In this case, at this dilution, the values were significant in a Chi Square analysis. The explanation for these results is limited as there is no information regarding the travel history of the miners. It is possible that those working in these Northern locations may have been recruited from an area where the organism is endemic (i.e. Southern California). Alternatively, it could be argued that Cryptococcus[gattii has been present in the Pacific Northwest for longer than previously thought. 4.7 Comparing results of sera tested from Vancouver Island Residents and Miners When examining the results for 1 in 250 dilution for the 20 kDa antigen, 81.1% of residents sera tested had antibodies against C . gattii antigens compared to only 25.5% of all miners sera tested. The results for the 50 kDa at the 1 in 250 dilution showed that 48.1% of residents tested had antibodies against this size antigen compared to 17.6% of miners tested. Chi Square analysis revealed significant differences between Vancouver Island residents tested after the 1999 outbreak and non-Vancouver Island residents tested prior to the 1999 outbreak for all dilutions for the 20 kDa (p< 0.001) and the 50 kDa (p< 0.002) antigens. 68 Therefore, the null hypothesis: a difference does not exist between the antibody responses to non-capsular C. gattii antigens of Vancouver Island residents' sera and non -Vancouver Island residents sera is rejected. These results provide important information about the number of individuals with antibodies against C. gattii prior to the 1999 outbreak. This suggests that prior to the 1999 outbreak, non-residents of Vancouver Island were not widely exposed to C. gattii propagules. To find out additional information about exposure to C. gattii for those residing on Vancouver Island prior to the 1999 outbreak, it would be beneficial to test additional sources of sera from Vancouver Island collected prior to 1999. Comparing the results of sera tested from miners to sera from Vancouver Island residents has an important component in validating Western blotting analysis as a technique for examining antibody responses to C. gattii antigens. These results showed that the cytoplasmic antigens recognized by the IgG antibodies of Vancouver Island residents tested were not a result of "sticky" non-specific binding to the proteins because i f this were the case then no differences would have been observed between those tested prior to the outbreak and after the outbreak. 4.8 Proposal for Test System Conditions Currently, it is not possible to utilize an Enzyme Linked Immunosorbent Assay (ELISA) as a test system to examine exposure to C. gattii within a population due to a lack of specificity with the assay. The best strategy to look for antibodies against C. gattii antigens within a population is to utilize a Western blot analysis. 69 Work published by Hamilton et al in 1997 examined patient sera for IgG antibody interactions with C. gattii using Western blot Analysis. However, it was not possible to follow the same conditions described by Hamilton et al, 1997 for a number of reasons to be discussed below. Firstly, the means employed by Hamilton et al, 1997 to detect antibody-antigen reactions resulted in a background signal and thus made it impossible to interpret results. Secondly, the work by Hamilton et al, 1997 did not assay C. gattii in parallel with C. neoformans antigens thus failing to control for similar antibody-antigen interactions between the Cryptococcus species. Finally, work by Hamilton et al, 1997 failed to consider background exposure to Cryptococcus antigens within populations by utilizing a source of pooled human sera or non-patient sera. The methods employed in this thesis work made it possible to examine sera for antibodies against C. gattii antigens within a population while controlling for background exposure and controlling for the possibility of similar antibody-antigen reactions among Cryptococcus species. Seroprevalence studies have been carried out in various test groups to examine exposure to different pathogens utilizing Western blot analysis. A summary of Western blot analysis conditions that have been used to investigate seroprevalence in patients and populations may be seen in Table 4.1. By examining the information in this table, it is possible to compare this thesis work to conditions that have been used for other seroprevalence assays. The pilot data gathered in this study was useful to define the parameters of the assay. One aspect to take into consideration when defining the parameters of an assay is the sensitivity of the detection for antibody-antigen interactions. To test sera for antibodies against C. gattii antigens in a population it is not possible to use sera being tested at a 70 dilution factor greater than 1 in 250, as under this condition, banding patterns become overwhelmed with background signal. However, it should be noted that once the primary antibody concentration progresses from 1 in 1000 to 1 in 2000 for Vancouver Island residents, one-half of the positive samples are lost. Therefore, it can be concluded that 1 in 1000 should be the most dilute concentration of sera when looking for antibodies against to C. gattii antigens within the Vancouver Island population. Table 4.1 Western Blot Analysis Conditions for other Seroprevalence Studies Study Exposure to Organism Test Group Primary Antibody Concentration Secondary Antibody Concentration Hamilton et al C.gattii patients 1 in 100 1 in 500 1997 [78] C.neoformans var. neoformans 1 in 200 1 in 1000 Cox et al Cryptosporidium Brazilian 1 in 50 1 in 500 2005 [87] population Woo et al SARS patients 1 in 1000 N/A 2004 [88] Bartlett & C.gattii B C 1 in 250 1 in 5000 Griffiths C.neoformans population & 1 in 500 (unpublished) 2006 var. neoformans patients 1 in 1000 1 in 2000 4.9 Potential Applications for the Test System Seroepidemiology is a useful tool to identify where and to whom exposure to an organism is occurring by examining populations. This assay was developed to look for antibodies against C. gattii specific antigens. One example of an occupational hygiene application of this test to determine where exposure is occurring would be to compare sera from arborists and forestry workers on Vancouver Island to a similar group of workers from another region of Canada where the organism has not been isolated (i.e. Ontario or Quebec). Another example of an occupational hygiene application would be to determine which 71 groups of workers are being exposed to C. gattii by comparing workers from different industries (i.e. comparing arborists and forestry workers to miners on Vancouver Island) A n example of an environmental hygiene application would be to test cord blood from infants in B C samples as well as randomly selected sera from different age groups from B C . Then, compare these sera to samples collected from other regions in Canada to ascertain i f exposure is related to length of time living in B C or Vancouver Island. Overall, this test system may help to understand exposure to different populations that can be stratified by occupation, age or geographical region. 4.10 Future Directions Overall, further testing of sera is need from locations across British Columbia to aid in the validation of these preliminary results and ultimately gain more insight about exposure to C. gattii propagules. In addition, testing sera from Vancouver Island prior to the 1999 outbreak is important as this could contribute to understanding of how long C. gattii has been present in our environment. As differences were revealed for various age groups and study groups for the 20 kDa and 50 kDa antigen, isolating and/or identifying these particular antigens would be beneficial. A n attempt was made at one point during this work to isolate the antigens based on size using size exclusion chromatography, but unfortunately the resolution of the column was not sufficient. A n alternative method to isolate these antigens of interest is immunoprecipitation. This technique involves binding IgG antibodies from human sera to protein A agarose and subsequently incubating these IgG-conjugated beads with cytoplasmic antigens from C. gattii. Following this incubation step, a series of washes removes unbound antigen. Finally, 72 the IgG antibodies and their cognate C. gattii antigens are eluted from the Protein A matrix with a change in pH/salt concentration or with a gentle elution buffer (available from Pierce) and separated by S D S - P A G E . The antigens are transferred to a P V D F membrane and stained with a Coomassie blue/40% M e O H mixture to determine the approximate molecular masses and facilitate excision of interesting bands from the P V D F . Excised bands can be sent to the Genome B C Proteomics Centre at the University of Victoria for Edman degradation and subsequent N-terminal sequence determination with the end goal of identifying the antigens of interest. If identification of the antigens is successful, it would then be possible to express them recombinantly and use the purified proteins as a substrate for E L I S A The possibility of using E L I S A to examine exposure to C. gattii in a population would be advantageous for the following reasons: 1) the observations would be quantitative rather than qualitative because the automated plate reader discerns differences in optical density which is correlated with concentration; 2) by having quantitative results, establishing end points for an assay would be possible; 3) would allow the screening of large populations which are necessary to characterize the seroprevalence. Alternatively, a semi-quantitative Western blot analysis method could be developed using the 20 kDa and 50 kDa antigens. What this would involved would be using different dilutions of sera and use digital imaging to estimate band intensity among various samples and compare them to positive controls. Understanding the identity of the antigens would also be beneficial in understanding if the antibodies generated against the antigens are due solely to exposure or i f they are related to infection. A recent publication by Lee et al in 2006 has isolated and expressed a 73 liver stage antigen (LSA) from Plasmodium falciparum, the organism responsible for causing malaria. The purified L S A was utilized in both ELISAs and Western blot analysis for an early sero-diagnosis of malaria in a population residing in malaria-endemic areas of Mandalay, Myanmar (Burma)[89]. Quinn et al in 2002 developed an E L I S A to examine exposure to test for antibodies to Bacillus anthracis (anthrax) protective antigen (PA) in human serum. The overall aim of the publication by Quinn et al was to develop a rapid means of determining i f an individual had been exposed to P A , one of the virulence factors associated with B. anthracis [90]. Overall further information about antibody-antigen interactions for C. gattii will help contribute to the knowledge base of Cryptococcal immunity which at this time is still contentious. 4.11 Significance of Results A Western blot analysis was developed to examine exposure to C. gattii propagules in a population on Vancouver Island where there have been greater than 130 cases of cryptococcosis caused by C. gattii since 1999. This work describes the cross reactivity to antigens from Cryptococcus neoformans var. neoformans and C. neoformans var. grubii which had not been considered in previous studies. The results of this work indicate that a significant difference exists between residents tested from Vancouver Island since the 1999 outbreak and those tested from other areas of British Columbia, the Yukon and Northwest Territories and prior to the 1999 outbreak. The high number of samples that showed an IgG antibody response to the 20 kDa and 50 kDa antigens serve as a starting point to learn more about the immune response to C. gattii. 74 4.12 Strengths and Limitations of this thesis work The strength of this thesis work is that the assay developed can be specifically utilized to address exposure to C gattii in residents of British Columbia. The assay was designed to control for cross-reactivity to two other varieties of Cryptococcus which has not been done in previous investigations that examined antibody-antigen interactions. Finally, this thesis work has provided preliminary findings on exposure to C. gattii antigens in residents on Vancouver Island. A limitation is this work is that it is not possible to control for the possibility of previous exposure to C. gattii in residents of Vancouver Island because we do not know what the travel history is of the participants. Additionally we do not know the travel history or place of residence is of the miners whose sera were tested to examine exposure prior to the 1999 outbreak. 4.13 Conclusions Using a full complement of cytoplasmic antigens to test for exposure to C. gattii using E L I S A is not possible, due to lack of specificity with this method. Western blot analysis provided a qualitative means to pilot test sera for antibodies against C. gattii antigens among residents of Vancouver Island. Significant differences were found between the number of residents with antibodies against C. gattii cytoplasmic antigens when stratifying the data according to different parameters (i.e. study type, age) and different sized antigens. Testing of more Vancouver Island residents and non Vancouver Island residents should be conducted in order to have more robust statistical analysis. 75 A significant difference was found between those participants tested prior to the 1999 outbreak and after the 1999 outbreak. This suggests that fewer non-Vancouver Island residents tested have been exposed to C. gattii propagules prior to the 1999 outbreak compared to Vancouver Island residents tested after the 1999 outbreak. 76 LITERATURE CITED 1. Campbell, G.D. , Primary pulmonary cryptococcosis. A m Rev Respir Dis, 1966. 94(2): p. 236-43. 2. D'Souza, C . A . , et al., Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals. Curr Genet, 2004. 46(2): p. 92-102. 3. Franzot, S.P., L F . Salkin, and A . Casadevall, Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates. J Clin Microbiol, 1999. 37(3): p. 838-40. 4. Evans, E . E . , The antigenic composition of Cryptococcus neoformans. I. A serologic classification by means of the capsular and agglutination reactions. J Immunol, 1950. 64(5): p. 423-30. 5. Wilson, D . E . , J .E. Bennett, and J.W. Bailey, Serologic grouping of Cryptococcus neoformans. Proc Soc Exp Biol Med, 1968. 127(3): p. 820-3. 6. Chen, S., et al., Epidemiology and host- and variety-dependent characteristics of infection due to Cryptococcus neoformans in Australia and New Zealand. Australasian Cryptococcal Study Group. Clin Infect Dis, 2000. 31(2): p. 499-508. 7. Sorrell, T . C , Cryptococcus neoformans variety gattii. Med Mycol , 2001. 39(2): p. 155-68. 8. Hajjeh, R . A . , M . E . Brandt, and R.W. Pinner, Emergence of cryptococcal disease: epidemiologic perspectives 100 years after its discovery. Epidemiol Rev, 1995. 17(2): p. 303-20. 9. Goldman, D . L . , et al., Serologic evidence for Cryptococcus neoformans infection in early childhood. Pediatrics, 2001. 107(5): p. E66. 10. Kwon-Chung, K . J . , Boekhout, T. , Fell, J.W., and Diaz, M . , Proposal to Conservethe name Cryptococcus gattii against C.hondurianus and Cbacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae). Taxon, 2002. 51(4): p. 804-806. 11. Ellis, D . H . and T.J. Pfeiffer, Natural habitat of Cryptococcus neoformans var. gattii. J Clin Microbiol, 1990. 28(7): p. 1642-4. 12. Fyfe, M . , MacDougall,L., Bartlett, K . Romney, M . , Kidd,S., Stephen, C , Starr, M . , Pearce, M . , Mak,S., Boekhout,T., Meyer,W., and Kibsey, P., Outbreak of Cryptococcus neoformans var. gattii Infections in Humans, Terrestrial and Marine Mammals: Emergence of a Tropical Fungus in a. Termperate Environment. Submitted, 2004. 13. Egan, B. The Ecology of the Coastal Douglas-fir Zone. Ecosystems of British Columbia 1999 [cited 2006. 14. Mosby's, Cryptococcosis, in Mosby's Medical, Nursing, & Allied Health Dictionary 1994, Mosby's: St Louis. 15. Andersen, L . E . , Cryptococcosis, in Mosby's Medical, Nursing and Allied Health, K . N . Andersen, Editor. 1994, Mosby: St. Louis, p. 418. 16. Heelan, J.S., L . Corpus, and N . Kessimian, False-positive reactions in the latex agglutination test for Cryptococcus neoformans antigen. J Clin Microbiol, 1991. 29(6): p. 1260-1. 77 .17. Kwon-Chung, K .J . , Cryptococcosis, in Medical mycology, K .J .B . Kwon-Chung, J.E. , Editor. 1992, Lea & Febiger: Philadelphia, p. 297-446. 18. Torda, A . , R .K. Kumar, and P.D. Jones, The pathology of human and murine pulmonary infection with Cryptococcus neoformans var. gattii. Pathology, 2001. 33(4): p. 475-8. 19. Hu l l , C M . and J . Heitman, Genetics of cryptococcus neoformans. Annu Rev Genet, 2002. 36: p. 557-615. 20. Kwon-Chung, K .J . and J.E. Bennett, Epidemiologic differences between the two varieties of Cryptococcus neoformans. A m J Epidemiol, 1984. 120(1): p. 123-30. 21. Dromer, F., O. Ronin, and B. Dupont, Isolation of Cryptococcus neoformans var. gattii from an Asian patient in France: evidence for dormant infection in healthy subjects. J Med Vet Myco l , 1992. 30(5): p. 395-7. 22. El l is , D.H. , Cryptococcus neoformans var. gattii in Australia. J C l i n Microbio l , 1987. 25(2): p. 430-1. 23. Hamann, I.D., R.J. Gil lespie, and J.K. Ferguson, Primary cryptococcal cellulitis caused by Cryptococcus neoformans var. gattii in an immunocompetent host. Australas J Dermatol, 1997. 38(1): p. 29-32. 24. Grosse, P., et al., Encephalomyelitis due to Cryptococcus neoformans var gattii presenting as spinal tumour: case report and review of the literature. J Neurol Neurosurg Psychiatry, 2001. 70(1): p. 113-6. 25. Mal ik , R., et al., Serum antibody response to Cryptococcus neoformans in cats, dogs and koalas with and without active infection. Med Myco l , 1999. 37(1): p. 43-51. 26. Stephen, C , et al., Multispecies outbreak of cryptococcosis on southern Vancouver Island, British Columbia. Can Vet J , 2002. 43(10): p. 792-4. 27. Mi l ler , W .G . , et al., Cryptococcosis in a bottlenose dolphin (Tursiops truncatus) caused by Cryptococcus neoformans var. gattii. J C l in Microbio l , 2002. 40(2): p. 721-4. 28. Duncan, C , et al., Sub-clinical infection and asymptomatic carriage of Cryptococcus gattii in dogs and cats during an outbreak of cryptococcosis. Med Myco l , 2005. 43(6): p. 511-6. 29. Duncan, C .G . , C. Stephen, and J . Campbell, Evaluation of risk factors for Cryptococcus gattii infection in dogs and cats. J A m Vet Med Assoc, 2006. 228(3): . p. 377-82. 30. Pfeiffer, T. and D. El l is , Environmental isolation of Cryptococcus neoformans gattii from California. J Infect Dis, 1991. 163(4): p. 929-30. 31. Pfeiffer, T.J. and D.H. El l is , Environmental isolation of Cryptococcus neoformans var. gattii from Eucalyptus tereticornis. J Med Vet Myco l , 1992. 30(5): p. 407-8. 32. Sorrell, T .C. , et al., Natural environmental sources of Cryptococcus neoformans var. gattii. J C l in Microbiol , 1996. 34(5): p. 1261-3. 33. Chen, S.C., et al., Cryptococcus neoformans var. gattii infection in northern Australia: existence of an environmental source other than known host eucalypts. Trans R Soc Trop M e d Hyg, 1997. 91(5): p. 547-50. 34. Sorrell, T .C. , et al., Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting. J C l in Microbiol , 1996. 34(5): p. 1253-60. 78 35. Kidd, S.E., et al., Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas. Eukaryot Cell, 2005. 4(10): p. 1629-38. 36. Lazera, M.S . , et al., Possible primary ecological niche of Cryptococcus neoformans. Med Mycol , 2000. 38(5): p. 379-83. 37. Fortes, S.T., et al., First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest. Mycoses, 2001. 44(5): p. 137-40. 38. Kidd, S.E., T . C . Sorrell, and W. Meyer, Isolation of two molecular types of Cryptococcus neoformans var. gattii from insect frass. Med Mycol , 2003. 41(2): p. 171-6. 39. Gugnani, H . C . , et al., Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. Med Mycol , 2005. 43(6): p. 565-9. 40. Randhawa, H.S., T. Kowshik, and Z . U . Khan, Efficacy of swabbing versus a conventional technique for isolation of Cryptococcus neoformans from decayed wood in tree trunk hollows. Med Mycol , 2005. 43(1): p. 67-71. 41. Kwon-Chung, K . J . , et al., Virulence, serotype, and molecular characteristics of environmental strains of Cryptococcus neoformans var. gattii. Infect Immun, 1992. 60(5): p. 1869-74. 42. Kidd, S.E., Hagen, F.,Tscharke,R.L., Huynh,M., Bartlett, K . H . , Fyfe, M . , L . MacDougall, T. , Boekhout, Kwon-Chung, K . J . , and Meyer, W . . A rare genotype of Cryptococcus gattii caused by the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). 2004. 43. Bartlett, K . H . Emerging Pathogen: Cryptococcus gattii - Environmental surveillance during an outbreak, in University of Washington School of Public Health & Community Medicine 2006. 44. Ellis, D . H . and T.J. Pfeiffer, Ecology, life cycle, and infectious propagule of Cryptococcus neoformans. Lancet, 1990. 336(8720): p. 923-5. 45. Island, T . V . Weather Conditions and Climate. 2000 [cited; Available from: http://www.islands.bc.ca/general/climate.html. 46. Casadevall, A . , Antibody immunity and invasive fungal infections. Infect Immun, 1995. 63(11): p. 4211-8. 47. Feldmesser, M . , et al., Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect Immun, 2000. 68(7): p. 4225-37. 48. Casadevall, A . and L . Pirofski, Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans. Curr M o l Med, 2005. 5(4): p. 421-33. 49. Steenbergen, J.N. and A . Casadevall, The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans. Microbes Infect, 2003. 5(7): p. 667-75. 50. Goren, M . B . , Experimental murine cryptococcosis: effect of hyper immunization to capsular polysaccharide. J Immunol, 1967. 98(5): p. 914-22. 51. Monga, D.P., et al., Experimental cryptococcosis in normal and B-cell-deficient mice. Infect Immun, 1979. 26(1): p. 1-3. 79 52. Diamond, R.D. and J.E. Bennett, Prognostic factors in cryptococcal meningitis. A study in 111 cases. Ann Intern Med, 1974. 80(2): p. 176-81. 53. Hobbs, M . M . , et al., Opsonic activity of cerebrospinal fluid in experimental cryptococcal meningitis. Infect Immun, 1990. 58(7): p. 2115-9. 54. Goldman, D. , S.C. Lee, and A . Casadevall, Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat. Infect Immun, 1994. 62(11): p. 4755-61. 55. Dromer, F. , et al., Improvement of amphotericin B activity during experimental cryptococcosis by incorporation into specific immunoliposomes. Antimicrob Agents Chemother, 1990. 34(11): p. 2055-60. 56. Mukherjee, J. , et al., Monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan enhance fluconazole efficacy. Antimicrob Agents Chemother, 1995. 39(7): p. 1398-405. 57. La Mantia, L . , et al., Cryptococcal meningoencephalitis: intrathecal immunological response. J Neurol, 1986. 233(6): p. 362-6. 58. Mukherjee, J . , et al., Protective and nonprotective monoclonal antibodies to Cryptococcus neoformans originating from one B cell. J Exp Med, 1995. 181(1): p. 405-9. 59. Mukherjee, J . , M . D . Scharff, and A . Casadevall, Protective murine monoclonal antibodies to Cryptococcus neoformans. Infect Immun, 1992. 60(11): p. 4534-41. 60. Dromer, F., et al., Protection of mice against experimental cryptococcosis by anti-Cryptococcus neoformans monoclonal antibody. Infect Immun, 1987. 55(3): p. 749-52. 61. Yuan, R., et al., Isotype switching from IgG3 to IgGl converts a nonprotective murine antibody to Cryptococcus neoformans into a protective antibody. J Immunol, 1995. 154(4): p. 1810-6.. 62. Goldman, D . L . , S.C. Lee, and A . Casadevall, Tissue localization of Cryptococcus neoformans glucuronoxylomannan in the presence and absence of specific antibody. Infect Immun, 1995. 63(9): p. 3448-53. 63. Gordon, M . A . and A . Casadevall, Serum therapy for Cryptococcal meningitis. Clin Infect Dis, 1995. 21(6): p. 1477-9. 64. Mukherjee, J. , et al., Therapeutic efficacy of monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin B. Antimicrob Agents Chemother, 1994. 38(3): p. 580-7. 65. Brueske, C . H . , Proteolytic activity of a clinical isolate of Cryptococcus neoformans. J Clin Microbiol, 1986. 23(3): p. 631-3. 66. Cherniak, R. and J.B. Sundstrom, Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect Immun, 1994. 62(5): p. 1507-12. 67. Kozel, T.R. , W . F . Gulley, and J. Cazin, Jr., Immune response to Cryptococcus neoformans soluble polysaccharide: immunological unresponsiveness. Infect Immun, 1977.18(3): p. 701-7. 68. Kaufmann, L . , Reiss, E . , Serodiagnosis of fungal diseases, in Manual of Clinical Laboratory Immunology, N.R. Rose, Fahey, J .L. , Editor. 1986, American Society of Microbiology: Washington, D C . p. 446-466. 69. Ikeda, R., et al., Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol, 1982. 16(1): p. 22-9. 80 70. Crypto Check Iatron - Instructions for Use, Iatron Laboratories: Tokyo, Japan, p. 2-6. 71. Engvall, E . and P. Perlman, Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry, 1971. 8(9): p. 871-4. 72. Engvall, E . , K . Jonsson, and P. Perlmann, Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta, 1971. 251(3): p. 427-34. 73. Burnette, W . N . , "Western Blotting": Electrophoretic Transfer of Proteins from Sodium Dodecyl Sulfate-Polyacrylamde Gels to Unmodified Nitrocellulose and Radiographic Detection with Anibody and Radioiodinated Protein A. Anal Biochem, 1981. 112:p.195-203. 74. Johnson, M . A . , et al., Environmental exposure and leptospirosis, Peru. Emerg Infect Dis, 2004. 10(6): p. 1016-22. 75. Hoang, T . T . , et al., Seroprevalence of Helicobacter pylori infection in urban and ruraTVietnam. Clin Diagn Lab Immunol, 2005. 12(1): p. 81-5. 76. Arvanitidou, M . , P. Mamassi, and A . Vayona, Epidemiological evidence for vaccinating wastewater treatment plant workers against hepatitis A and hepatitis B virus. Eur J Epidemiol, 2004. 19(3): p. 259-62. 77. Seaton, R . A . , et al., Exposure to Cryptococcus neoformans var. gattii—a seroepidemiological study. Trans R Soc Trop Med Hyg, 1996. 90(5): p. 508-12. 78. Hamilton, A . J . , Ignacio Figueroa, J. , Jeavons, L . , and R . A . Seaton, Recognition of cytoplasmic yeast antigens of Cryptococcus neoformansvar. neoformans and Cryptococcus neoformans var. gattii by immune human sera, d Medical Microbiology, 1997. 17: p. 111-119. 79. Hamilton, A . J . , et al., A 34- to 38-kilodalton Cryptococcus neoformans glycoprotein produced as an exoantigen bearing a glycosylated species-specific epitope. Infect Immun, 1992. 60(1): p. 143-9. 80. Pierce. Ammonium Sulfate Precipitation of Protein. 2005 [cited 2005 March 1]. 81. Bradford, M . M . , A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the the principle of protein-dye binding Anal Biochem, 1976. 72: p. 248-254. 82. Sambrook, J. , and Russell, D. W , Molecular cloning : a laboratory manual. 3rd ed. 2001, Cold Spring Harbor, N .Y . : Cold Spring Harbor Laboratory Press. 83. Pierce. Instructions Gel Code Blue Reagent. 2005 [cited 2005 January 6]. 84. Hoang, L . M . , et al., Cryptococcus neoformans infections at Vancouver Hospital and Health Sciences Centre (1997-2002): epidemiology, microbiology and histopathology. J Med Microbiol, 2004. 53(Pt 9): p. 935-40. '85. Kakeya, H . , et al., A 77-kilodalton protein of Cryptococcus neoformans, a member of the heat shock protein 70 family, is a major antigen detected in the sera of mice with pulmonary cryptococcosis. Infect Immun, 1997. 65(5): p. 1653-8. 86. Burt, E . T . , et al., Isolation and partial characterization of Hsp90 from Candida albicans. Ann Clin Lab Sci, 2003. 33(1): p. 86-93. 87. Cox, M . J . , et al., Age-specific seroprevalence to an immunodominant Cryptosporidium sporozoite antigen in a Brazilian population. Epidemiol Infect, 2005. 133(5): p. 951-6. 81 88. Woo, P.C. , et al., Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet, 2004. 363(9412): p. 841-5. 89. Lee, H.W., et al., Usefulness of the recombinant liver stage antigen-3 for an early serodiagnosis of Plasmodium falciparum infection. Korean J Parasitol, 2006. 44(1): p. 49-54. 90. Quinn, C P . , et al., Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerg Infect Dis, 2002. 8(10): p. 1103-10. 82 Appendix B Table B . l Results of Vancouver Island Residents Tested for Antibodies against C. gattii by Region 20 kDa 25 kDa 30 kDa 35 kDa 40 kDa 45 kDa 50 kDa 55 kDa Courtenay 1 in 250 100.0 20.0 40.0 0.0 0.0 0.0 40.0 0.0 n=5 1 in 500 60.0 0.0 0.0 0.0 0.0 0.0 40.0 0.0 1 in 1000 40.0 0.0 0.0 0.0 0.0 0.0 20.0 0.0 1 in 2000 0.0 0.0 0.0 0.0 0.0 0.0 20.0 0.0 Parksville 1 in 250 40.0 20.0 40.0 0.0 20.0 20.0 40.0 0.0 n=5 1 in 500 40.0 0.0 20.0 0.0 0.0 0.0 40.0 0.0 1 in 1000 20.0 0.0 0.0 0.0 0.0 0.0 40.0 0.0 1 in 2000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Nanaimo 1 in 250 82.4 0.0 11.8 5.9 23.5 11.8 52.9 0.0 n=17 1 in 500 70.6 0.0 5.9 5.9 17.6 11.8 47.1 0.0 1 in 1000 52.9 0.0 5.9 0.0 11.8 11.8 47.1 0.0 1 in 2000. 41.2 0.0 0.0 0.0 5.9 0.0 11.8 0.0 Duncan 1 in 250 75.0 0.0 16.7 0.0 25.0 8.3 58.3 0.0 n=12 1 in 500 58.3 0.0 8.3 8.3 16.7 8.3 41.7 0.0 1 in 1000 50.0 0.0 8.3 0.0 16.7 8.3 41.7 0.0 1 in 2000 8.3 0.0 8.3 0.0 16.7 0.0 33.3 0.0 Victoria 1 in 250 84.6 0.0 19.2 3.8 30.8 7.7 42.3 3.8 n=26 1 in 500 69.2 0.0 11.5 3.8 23.1 3.8 26.9 3.8 1 in 1000 46.2 0.0 7.7 3.8 7.7 3.8 15.4 3.8 1 in 2000 11.5 0.0 0.0 0.0 0.0 0.0 15.4 0.0 Vancouver 1 in 250. 50.0 0.0 0.0. 0.0 25.0 0.0 25.0 0.0 n=4 1 in 500 50.0 0.0 0.0 0,0 0.0 0.0 0.0 0.0 1 in 1000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1 in 2000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Values are expressed as a percent 84 Table B.2 Results of Vancouver Island Residents Tested for Antibodies against C. gattii by Study Type 20 kDa 25 kDa 30 kDa 35 kDa 40 kDa 45 kDa 50 kDa 55 kDa Animal Owners 1 in 250 88 0 . 38 0 13 13 38 0 Animal Owners 1 in 500 75 0 13 0 0 13 25 0 Animal Owners 1 in 1000 38 0 0 0 0 13 25 0 Animal Owners 1 in 2000 0 0 0 0 0 0 13 0 BC Parks 1 in 250 100 0 0 0 0 17 •50 0 BC Parks 1 in 500 83 0 0 0 17 17 17 0 BC Parks 1 in 1000 33 0 0 0 0 0 33 0 BC Parks 1 in 2000 17 0 0 0 0 0 17 .0 Case 1 in 250 70 0 0 10 10 20 70 10 Case 1 in 500 30 0 0 10 10 10 50 10 Case 1 in 1000 20 0 0 10 0 10 40 10 Case 1 in 2000 10 0 0 0 0 0 30 0 Control 1 in 250 92 9 27 0 18 0 55 0 Control 1 in 500 64 0 0 9 9 0 36 0 Control . 1 in 1000 64 0 0 0 9 0 27 0 Control 1 in 2000 9 0 0 0 9 0 18 0 * Values are expressed as a percent Table B.3 Results of Vancouver Island Residents Tested for Antibodies against C . gattii by Age Group 20 kDa 25 kDa 30 kDa 35 kDa 40 kDa 45 kDa 50 kDa 55 kDa <40 1 in 250 64 0 . 21 7 21 0 21 0 n=14 1 in 500 43 0 14 7 14 0 14 0 1 in 1000 21 0 7 0 0 0 7 0 1 in 2000 0 0 0 0 0 0 7 0 40 to 49 1 in 250 88 0 13 0 13 6 31 0 n=16 1 in 500 69 0 0 6 6 6 13 0 1 in 1000 44 0 0 0 6 6 13 0 1 in 2000 13 0 0 0 6 0 13 0 50 to 59 1 in 250 81 3 16 3 34 16 59 3 n=32 1 in 500 72 0 6 3 22 13 44 3 1 in 1000 50 0 3 3 9 9 41 0 1 in 2000 28 0 3 0 3 0 19 0 >60 1 in 250 79 7 21 0 7 7 64 0 n=14 1 in 500 57 0 14 0 7 0 36 0 1 in 1000 36 0 7 0 7 0 36 0 1 in 2000 7 0 0 0 7 0 21 0 Values are expressed as a percent 8 5 Table B.4 Results of Miners from the Northwest Territories, Yukon and Central and Northern British Columbia Residents Tested for Antibodies against C. gattii Primary Antibody 20 kDa 25 kDa 30 kDa 35 kDa 40 kDa 45 kDa 50 kDa 55 kDa 1 in 250 25.5 0.0 0.0 3.9 9.8 2.0 17.6 5.9 1 in 500 17.6 0.0 0.0 2.0 5.9 2.0 7.8 0.0 1 in 1000 3.9 0.0 0.0 2.0 2.0 0.0 5.9 0.0 1 in 2000 3.9 0.0 0.0 2.0 2.0 0.0 3.9 0.0 Values are expressed as a percent 86 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0092660/manifest

Comment

Related Items