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Efficacy of composting to decontaminate Cryptococcus gattii-colonized plant waste Ge, Bin 2011

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Efficacy of Composting to Decontaminate Cryptococcus gattii-colonized Plant Waste by BIN GE B.Sc. The University of British Columbia, 2006  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Occupational and Environmental Hygiene)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER) August, 2011 ©Bin Ge, 2011  Abstract Cryptococcus gattii is a human fungal pathogen that emerged on Vancouver Island, BC in 1999. This study aimed to investigate C. gattii survival in composting systems and occupational exposure to C. gattii in work tasks associated with composting.  The presence of C. gattii was monitored in composting feedstock and product for one calendar year in a municipal composting facility in Cumberland, BC. Additionally, the survival of an environmental C. gattii isolate was tested in a composting experiment conducted with customdesigned composters that simulated in-vessel composting and home backyard composting. Potential inhalation occupational exposure to C. gattii while performing composting-related tasks were measured during residential yard waste chipping by city workers in Parksville, BC and during the composting experiment where C. gattii contaminated feedstock was composted.  C. gattii persisted through three out of five in-vessel composting trials despite high composting temperatures (mean > 60ºC, peak 85ºC) which were achieved evenly throughout the composting material for long periods (> 65 hours). C. gattii was also detected in one out of two yard composting trials after 60 days of composting. The year-long composting feedstock and compost monitoring for C. gattii returned no positive samples. Air sampling during composting-related work tasks found no detectable level of C. gattii.  Current composting standard and practice in BC are unlikely to be adequate in eliminating C. gattii from contaminated composting feedstock. Based on the results of this study, the risks of occupational exposure to C. gattii during residential yard waste chipping and composting of contaminated material are low.  ii  Preface  A Biohazard Approval Certificate (Protocol number B09-0149) was granted by the UBC Biosafety Committee for experiments conducted in the thesis research.  iii  Table of contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of contents ..................................................................................................................... iv List of tables ........................................................................................................................... vii List of figures ........................................................................................................................... ix List of abbreviations ............................................................................................................... xi Acknowledgements ................................................................................................................. xii Dedication .............................................................................................................................. xiii 1  Introduction .......................................................................................................................1  1.1 Cryptococcus gattii................................................................................................................................... 2 1.1.1 Taxonomy ............................................................................................................................................ 2 1.1.2 Cryptococcosis ..................................................................................................................................... 2 1.1.3 Laboratory identification ...................................................................................................................... 3 1.2 Ecology ........................................................................................................................................................ 4 1.2.1 Geographical distribution ..................................................................................................................... 4 1.2.2 Environmental niche ............................................................................................................................ 5 1.3 Emergence on Vancouver Island, British Columbia and nearby regions ................................................. 6 1.3.1 Epidemiology of C. gattii cryptococcosis in BC and the Pacific Northwest of the US ............................ 6 1.3.2 Environmental sampling of C. gattii on Vancouver Island, BC and nearby regions ................................ 6 1.3.3 Occupational exposure to C. gattii on Vancouver Island........................................................................ 8 1.4 Composting ............................................................................................................................................. 9 1.4.1 Optimal composting parameters ......................................................................................................... 10 1.4.2 Composting methods .......................................................................................................................... 12 1.4.3 Composting acts and regulations in BC ............................................................................................... 12 1.4.4 Current gap in composting regulations regarding C. gattii elimination ................................................. 14 1.4.5 Composting on Vancouver Island ....................................................................................................... 15 1.5  Study rationale ...................................................................................................................................... 15  iv  1.6  Research questions .................................................................................................................................... 16  2  Pilot studies ...................................................................................................................... 17  2.1  Pilot studies: introduction..................................................................................................................... 17  2.2 Pilot studies: methods ........................................................................................................................... 17 2.2.1 Laboratory cell characteristic and survival experiments ....................................................................... 17 2.3 Pilot studies: results................................................................................................................................... 25 2.3.1 Growth rate and maximum growth concentration comparison between laboratory minimal culture and soil medium ..................................................................................................................................................... 25 2.3.2 Cell size comparison between C. gattii cells grown in 1/16th strength MEB and SEB ........................... 29 2.3.3 Melanization of C. gattii cells in MEB and different soil extract media ............................................... 30 2.3.4 Resistance to UV irradiation in melanized and non-melanized C. gattii cells ....................................... 34 2.3.5 C. gattii heat survival in contaminated endemic soil samples and seeded composting feedstock ........... 38 2.4  Pilot studies: discussion ............................................................................................................................. 39  3  Methods ........................................................................................................................... 43  3.1 C. gattii concentrations in composting feedstock and compost product from the Comox Valley Biosolids Composting Facility............................................................................................................................................ 43 3.1.1 Study site: Comox Valley Biosolids Composting Facility.................................................................... 43 3.2  Air sampling for C. gattii during wood waste chipping in Parksville, BC ................................................ 44  3.3 C. gattii composting simulation ................................................................................................................. 45 3.3.1 In-vessel composting simulation ......................................................................................................... 45 3.3.2 Yard composting experiment .............................................................................................................. 51 3.4  Air sampling during the composting of C. gattii contaminated material.................................................. 52  4  Results .............................................................................................................................. 54  4.1 C. gattii concentrations in composting feedstock and compost product from the Comox Valley Biosolids Composting Facility............................................................................................................................................ 54 4.2  Air sampling for C. gattii during wood waste chipping in Parksville, BC ................................................ 56  4.3 C. gattii composting simulation ................................................................................................................. 58 4.3.1 In-vessel composting simulation ......................................................................................................... 58 4.3.2 Yard composting experiment .............................................................................................................. 64 4.4  Air sampling during the composting of C. gattii contaminated material.................................................. 67  v  5  Discussion ........................................................................................................................ 69  5.1 Was C. gattii present in compost feedstock and product in Comox Valley Biosolids Composting Centre between August 2008 and July 2009?................................................................................................................. 69 5.2 If present in the compost feedstock, does C. gattii persist through the composting process of a composting system meeting BC provincial regulatory composting requirements in Canada? ......................... 70 5.3 If present in the composting feedstock, does C. gattii aerosolize and become detectable in air during the composting of contaminated feedstock, leading to potential occupational exposure?....................................... 75 5.4 Was C. gattii present in air when city workers in Parksville, BC were chipping garden waste from residential homes in fall 2008? ........................................................................................................................... 76 5.5 Strengths and limitations .......................................................................................................................... 80 5.5.1 Strengths ............................................................................................................................................ 80 5.5.2 Limitations......................................................................................................................................... 81  6  Conclusions and recommendations................................................................................. 82  References................................................................................................................................ 83 Appendix A: Supplemental Figures ....................................................................................... 89  vi  List of tables Table 1. Maximum acceptable trace elements concentrations in Class A compost (49). ............ 14 Table 2. Initial C. gattii concentrations in soil samples used in heat survival experiment. .......... 23 Table 3. Initial pathogen concentrations for screened compost heat survival experiment............ 24 Table 4. Doubling times and maximum cell concentrations for C. gattii strains in various culture media. ....................................................................................................................................... 26 Table 5. Average C. gattii cell sizes in when cultured in MEB and SEB. ................................... 30 Table 6. Ultraviolet irradiation survival proportions of C. gattii (VGIIa) cells grown in different media. ....................................................................................................................................... 35 Table 7. Ultraviolet irradiation survival proportions of C. gattii (VGIIb) cells grown in different media. ....................................................................................................................................... 36 Table 8. Ultraviolet irradiation survival proportions of C. gattii (VGI) cells grown in different media. ....................................................................................................................................... 37 Table 9. C. gattii concentrations in positive soil samples incubated for various times at 55°C/65°C................................................................................................................................. 38 Table 10. Calculated and actual starting C. gattii and E. coli concentrations for compost heat survival experiment. .................................................................................................................. 39 Table 11. C. gattii concentrations in Comox Valley Biosolids Composting Facility feedstock and compost samples. ...................................................................................................................... 55 Table 12. Airborne C. gattii concentrations in locations where garden waste chipping took place. ................................................................................................................................................. 57 Table 13. Heating time and average temperature during the heating phase of in-vessel simulation. ................................................................................................................................................. 58 Table 14. pH values for composting material at the beginning (Day 0) and end (Day 60) of the in-vessel composting simulation. ............................................................................................... 60 Table 15. Moisture content values for in-vessel simulation composting material at different sampling time. ........................................................................................................................... 61 Table 16. Coliform concentrations for composting material in in-vessel simulation measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 37ºC. ..................................................................................................................... 62 Table 17. Thermo-tolerant coliform concentrations for composting material in in-vessel simulation measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 45ºC. .......................................................................................... 63 Table 18. Average C. gattii concentrations in in-vessel composting simulation samples at different times. .......................................................................................................................... 64 Table 19. Average yard composter temperatures and ambient temperatures. .............................. 64 Table 20. pH values of composting feedstock and finished compost for yard composters. ......... 65 vii  Table 21. Moisture content of composting feedstock and finished compost for yard composters. ................................................................................................................................................. 65 Table 22. Coliform concentrations for composting material in yard composters measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 37ºC. ......................................................................................................................................... 66 Table 23. Thermo-tolerant coliform concentrations for composting material in yard composters measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 45ºC. .............................................................................................................. 66 Table 24. Air C. gattii concentrations on different dates and during different activities in simulation composting trials. ..................................................................................................... 68  viii  List of figures Figure 1. Distribution of human and animal cryptococcosis and isolation of C. gattii from the environment on Vancouver Island and in neighbouring regions. Figure adapted from Kidd et al. (8), by permission from the American Society for Microbiology. ................................................8 Figure 2. C. gattii VGIIa strain in 1/16th strength MEB. 48 hour sample showing calibrated graticule for cell size measurement. ........................................................................................... 20 Figure 3. Concentrations of VGIIa cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB. .......... 27 Figure 4. Concentrations of VGIIb cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB. .......... 28 Figure 5. Concentrations of VGI cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB. ............. 29 Figure 6. C. gattii (VGIIa) colonies on1/16th strength MEB agar. .............................................. 31 Figure 7. C. gattii (VGIIa) colonies on endemic SEB agar......................................................... 32 Figure 8. C. gattii (VGIIb) colonies on UBC SEB1 agar............................................................ 33 Figure 9. C. gattii (VGIIa) colonies on UBC SEB2 agar. ........................................................... 34 Figure 10. Cement mixer and enclosure setup for in-vessel composting experiment. ................. 46 Figure 11. ACR Smart Button temperature datalogger (right) and protective PVC piping. ......... 48 Figure 12. Garden waste chipping sampling locations (n=128) in Parksville, BC. ...................... 56 Figure 13. Average temperature (ºC ) in composter and ambient temperature during heating period for Trial 2. ...................................................................................................................... 59 Figure 14. Locations where positive C. gattii had been found in Parksville BC from 2000 – 2011 2000 (S. Mak, 2011, personal communication). ......................................................................... 78 Figure 15. Garden waste chipping locations overlaid above positive historical sampling locations in Parksville, BC. ...................................................................................................................... 79 Figure 16. Concentrations of VGIIa cells in 1/16th strength MEB. ............................................ 89 Figure 17. Concentrations of VGIIa cells in 1/32nd strengh MEB. ............................................ 90 Figure 18. Concentrations of VGIIa cells in 1/64th strengh MEB. ............................................. 91 Figure 19. Concentrations of VGIIa cells in SEB ...................................................................... 92 Figure 20. Concentrations of VGIIb cells in 1/16th strengh MEB. ............................................. 93 Figure 21. Concentrations of VGIIb cells in 1/32nd strength MEB. ........................................... 94 Figure 22. Concentrations of VGIIb cells in 1/64th strength MEB. ............................................ 95 Figure 23. Concentrations of VGIIb cells in SEB. ..................................................................... 96 Figure 24. Concentrations of VGI cells in 1/16th strength MEB. ............................................... 97 Figure 25. Concentrations of VGI cells in 1/32nd strengh MEB. ............................................... 98 Figure 26. Concentrations of VGI cells in 1/64th MEB. ............................................................ 99 Figure 27. Concentrations of VGI cells in SEB........................................................................ 100 Figure 28. Average temperature in composter and ambient temperature during heating period for Trial 1. .................................................................................................................................... 101 Figure 29. Average temperature in composter and ambient temperature during heating period for Trial 3. .................................................................................................................................... 102 ix  Figure 30. Average temperature in composter and ambient temperature during heating period for Trial 4. .................................................................................................................................... 103 Figure 31. Average temperature in composter and ambient temperature during heating period for Trial 5. .................................................................................................................................... 104 Figure 32. Average temperature in composter and ambient temperature during heating period for Trial 6. .................................................................................................................................... 105 Figure 33. Average temperature in yard composting bin and ambient temperature for home composting Trial 1. ................................................................................................................. 106 Figure 34. Average temperature in yard composting bin and ambient temperature for home composting Trial 2. ................................................................................................................. 107  x  List of abbreviations BC  British Columbia  CDC  Centre for Disease Control  CFU  Colony forming unit  C. gattii  Cryptococcus gattii  C. neoformans  Cryptococcus neoformans  dw  Dry weight  kg  Kilogram  L  Litre  LoD  Limit of detection  LT  Lauryl-trypose  m3  Cubic metre  MEA  Malt extract agar  MEB  Malt extract broth  MPN  Most probable number  mg  Milligram  MUG  4-Methylumbelliferyl-p-D-glucuronide  SEA  Soil extract agar  SEB  Soil extract broth  TMECC  Test Methods for the Examination of Composting and Compost  UBC  University of British Columbia  US  United States  UV  Ultraviolet  ww  Wet weight  ºC  Degrees Celsius xi  Acknowledgements I would like to first express my most sincere gratitude to my research supervisor, Karen Bartlett. Karen selflessly shared her research knowledge and experience, gave constructive criticism and provided ongoing academic and moral support throughout my studies at SOEH. I thoroughly enjoyed working with her and am grateful to have her as my thesis supervisor. I also thank the rest of my thesis committee, Jim Atwater and Ray Copes. They each provided unique expertise and perspectives to my thesis research. For all the staff and students in the program, I thank you for all your research and technical support. Most importantly, memories and friendships were made here that will last a lifetime. I thank everyone at the Parksville Public Works Yard, Comox Valley Biosolids Composting Facility and Vancouver Landfill for their help and support during my research project. Special thanks to Peter Crawshaw at the Parksville Public Works Yard, who has provided tremendous support for the project right from its inception. I thank the UBC Department of Environmental Engineering for accommodating my pilot experiments. I also thank Sunny Mak from the BC Centre for Disease Control for generating the C. gattii positive sampling map of Parksville, which is included as Figure 14. I also thank the Bridge Program in UBC and WorkSafe BC for providing personal funding during my studies.  xii  Dedication  To my partner and best friend, Viola Lam, and my family. Without your support and encouragement this thesis would have never been possible, and I would not be the person I am today.  xiii  1  Introduction  Cryptococcus gattii has emerged on Vancouver Island, British Columbia (BC) as a primary fungal pathogen. The yeast is found on many native tree species on the Island. At the same time, much of the plant and wood waste are composted on Vancouver Island to reduce the amount of solid waste being sent to the landfill. Plant waste colonized by C. gattii on Vancouver Island is very likely to enter composting facilities as feedstock. The composting of plant material potentially contaminated with C. gattii is a unique problem on Vancouver Island. Possible exposures to C. gattii could occur during the processing of contaminated plant waste, composting, and, if the pathogen is not removed by the composting process, the use of compost products. Further, if C. gattii is not eliminated by the composting process, the pathogen could migrate to uncolonized areas via the transport of contaminated compost products. However, no studies have examined the efficacy of composting in eliminating C. gattii from contaminated plant waste. Similarly, occupational exposure to C. gattii in plant waste processing and composting has received little research attention. This research aimed to determine whether composting is effective in eliminating C. gattii from colonized plant waste and whether wood waste processing and composting expose workers and other compost users to aerosolized C. gattii. The specific goals of the project were: 1) to monitor compost feedstock and product in a biosolids composting facility on Vancouver Island for the presence of C. gattii, 2) to investigate whether C. gattii could survive the composting process in composting systems meeting provincial composting standards; 3) to investigate whether C. gattii was present in air during the composing of C. gattii contaminated material, leading to potential occupational exposure; and 4) to investigate whether C. gattii was present in air while city workers were chipping garden waste in the City of Parksville on Vancouver Island in fall 2008.  1  1.1  Cryptococcus gattii  1.1.1 Taxonomy Literally meaning “hidden seed,” Cryptococcus is a genus of fungus classified within the Basidiomycota phylum. There are at least 39 separate species within the Cryptococcus genus (1). Only two species, Cryptococcus neoformans and Cryptococcus gattii, are consistently infectious to humans due to their ability to grow at physiologic temperature of 37oC (1). C. gattii was first identified by Gatti and Eeckels in 1970 (2,3). The fungus grows asexually as an encapsulated yeast both clinically and in culture. In 1975, Kwon-Chung discovered the perfect, or sexual, form of Cryptococcus and placed the basidomycete in the new genus Filobasidiella (4). C. gattii is closely related to another common fungal pathogen, C. neoformans. C. gattii was previously classified as a variety of C. neoformans. In 2002, the two C. gattii serotypes, B and C, were separated from three C. neoformans serotypes, A, D and hybrid AD, to form a new species classification (5). The two species of Cryptococcus display differences in mating as well as phenotypic and genotypic traits. A comprehensive review of the differences between the two species was published by Sorrell in 2001 (6). Extensive DNA-typing experiments have divided different environmental and clinical C. gattii isolates into four molecular types (VGI, VGII, VGIII and VGIV) based on the DNA sequence inside the URA5 gene (7). Further divisions among the molecular types, such as the division of VGII strain into VGIIa and VGIIb, have been identified (7,8). Recently, the genomes of VGI and VGIIa strains were sequenced and compared (9). The comparison revealed considerable differences between the genome sequences in VGI and VGIIa strains, with an overall sequence divergence of 7.6% between the two strains, suggesting a potential for further speciation within the current C. gattii group (9).  1.1.2 Cryptococcosis In humans, both C. neoformans and C. gattii cause infections called cryptococcosis. A key distinction between the pathogenesis of the two species is host immune status. C. neoformans is 2  an opportunistic yeast pathogen that typically causes infections in immunocompromised individuals, whereas C. gattii is a primary pathogen capable of causing serious and potentially fatal infections in immunocompetent hosts (1,6). In addition to humans, cryptococcosis affects a number of mammals such as dogs, cats, horses, mouse, sheep and dolphins (10). Cryptococcosis is most commonly acquired by inhalation of infectious cryptococcal propagules (2). It is currently unknown whether the infectious propagules are basidiospores or small, desiccated yeast cells (1,6). In humans, C. gattii-associated cryptococcosis has a number of possible clinical presentations. The most typical clinical manifestation is a localized primary pulmonary infection characterized by lung lesions called cryptococcomas (11,12). C. gattii could also form cryptococcomas in the central nervous system (CNS), or cause disseminated infections in the CNS and skin (12,13). The median incubation period for the fungal disease was estimated to be 6-7 months (14). Symptoms for cryptococcosis include coughing, haemoptysis, shortness of breath and fever (11). Diagnosis of the disease is usually made through chest x-rays and cryptococcal antigen detection in blood or cerebrospinal fluid cultures (11,12). Medical treatment for cryptococcosis is available but usually involves a lengthy treatment period (12). Typical treatment regiments include combinations of amphotericin B, flucytosine, and/or fluconazole for at least 6 to 10 weeks (12).  1.1.3 Laboratory identification In environmental laboratories, a two-stage approach is used to identify C. gattii in specimens. The first step is to culture the specimen on a selective and differential medium called Staib agar (15), on which Cryptococcus species appear as round brown yeast colonies after 2 to 7 days of incubation at 30oC. The browning effect seen in Cryptococcus colonies is caused by the production of melanin from phenolic substrates inside the medium by an enzyme called phenoloxidase (1). In environmental samples that are likely to be contaminated by bacteria, the addition of an antibiotic to the Staib agar recipe suppresses bacterial growth which may otherwise interfere with Cryptococcus identification. 3  To differentiate between C. neoformans and C. gattii colonies, brown colonies on Staib agar are transferred to another selective and differential medium containing L-canavanine, glycine, and bromthymol blue (CGB agar) (16) and incubated for 48 hours at 30oC. Growth of C. gattii colonies, which are resistant to canavanine, makes the medium environment more basic, causing the pH indicator bromthymol blue to produce a deep blue colour in the medium (16). C. neoformans is susceptible to canavanine and thus unable to grow on CGB agar, and the medium remain yellow in colour (16).  1.2  Ecology  1.2.1 Geographical distribution In contrast to C. neoformans’ ubiquitous distribution around the world, C. gattii colonization was mostly limited to tropical and subtropical regions of the world. Since the first environmental C. gattii isolate was described by Ellis and Pfeiffer in 1989 in Southern Australia (17), a number of extensive environmental sampling studies have been conducted in an attempt to characterize the pattern of C. gattii colonization around the world. Environmental isolates have been found from Southern California in the United States (18), Colombia (19-21), Argentina (22), Brazil (23-25), India (26,27), Egypt (28) and Italy (29). Reported human C. gattii infections were also mostly from the tropic and subtropics. A review of 96 clinical isolates by Kwon-Chung and Bennet in 1984 reported C. gattii infections in Australia, Brazil, Cambodia, Hawaii, Southern California, Mexico, Paraguay, Thailand, Vietnam, Nepal and Central Africa (30). Subsequent clinical reports on C. gattii continued to report the appearance of the disease in tropical or subtropical regions such as Paupa New Guinea (31), South Africa (32), Singapore (33), Greece (34), Zaire (35) and Argentina (22). Although rare incidences of C. gattii infections had been reported in temperate regions, the source of these infections were not well understood (1). It had been hypothesized that all C. gattii infections in temperate regions were previously acquired in tropical or subtropical regions of the world (1). However, in 1999, the emergence of C. gattii on Vancouver Island clearly 4  showed that the pathogen is able to colonize temperate zones and may be present in other temperate regions of the world.  1.2.2 Environmental niche Attempts to uncover environmental sources of C. gattii have taken place soon after the first environmental isolates were discovered by Ellis and Pfeiffer in 1989 (17). After obtaining C. gattii isolates from Eucalyptus camaldulensis (river red gum) and Eucalyptus tereticornis (forest red gum), Ellis and Pfeiffer hypothesized that C. gattii was specifically associated with these two species of eucalypt (17). E. camaldulensis commonly grows along rivers and streams throughout Australia, whereas E. tereticornis grows in a wide area from southern Papua New Guinea to south-eastern Victoria State in Australia (36). Both eucalypts are native trees in Australia and are commercially exported to other regions of the world. Environmental sampling of various tree species in different countries following Ellis and Pfeiffer’s report found C. gattii on a number of eucalyptus trees as well as on other tree species. Countries with C. gattii colonized eucalypts include: United States (California), Colombia, Argentina, Brazil, India and Egypt (18-22,24,26-28). Contrary to Ellis and Pfeiffer’s hypothesis, C. gattii isolates were also found in Colombia on Cupressus lusitanica (Mexican Cypress), Pinus radiate (Monterey pine), Acacia decurrens (black wattle), and Terminalia catappa (almond tree) (19,21), as well as in Brazil on Moquilea tomentosa (pottery tree), Cassia grandis (pink shower tree) and Guettarda acreana (quina-quina tree) (23,25). The evidence is now clear that C. gattii is not exclusively associated with any particular tree species in the wild. It is still unclear what role tree species play in the life cycle of C. gattii (6). In fact, C. gattii may not be associated exclusively with trees. In environmental samples collected on Vancouver Island, C. gattii specimens were isolated from soil, seawater, freshwater, air, as well as a number of native BC tree species (8).  5  1.3  Emergence on Vancouver Island, British Columbia and nearby  regions 1.3.1 Epidemiology of C. gattii cryptococcosis in BC and the Pacific Northwest of the US In August 2001, an unusual cluster of cryptococcosis in humans and animals were reported on Vancouver Island, British Columbia (37). Epidemiological and microbiological investigations of the disease cluster identified an outbreak of C. gattii infections on Vancouver Island starting from 1999 (38). By the end of 2010, 281 cases (E. Galanis, 2011, personal communication) of human C. gattii cryptococcosis were documented in BC, with at least 19 cases dying from or with the infection (38-40). The mean disease incidence of C. gattii infections on Vancouver Island from 1999 – 2007 is 25.1 cases per 1,000,000 persons per year, which is one of the highest reported disease rates for C. gattii cryptococcosis worldwide (8,38). Beginning in September 2004, cases of C. gattii cryptococcosis were identified in mainland BC residents without recent travel history to Vancouver Island or other endemic areas, indicating an expansion of the C. gattii outbreak (41). Shortly after in December 2004, a case of C. gattii cryptococcosis was reported in an Oregon resident (42). By the end of July 2010, a total of 60 cases of C. gattii cryptococcosis were reported to the US CDC, with 43 cases from Oregon, 15 from Washington, one from California and one from Idaho (42). Among the 60 reported cases, 9 died from C. gattii cryptococcosis and 6 more died with the infection (42). Travel history was known for 52 of the 60 cases; 46 confirmed that they had not travelled to Vancouver Island or any other endemic areas (42), suggesting that these were locally acquired infections.  1.3.2 Environmental sampling of C. gattii on Vancouver Island, BC and nearby regions Following the cryptococcosis disease outbreak, extensive C. gattii environmental sampling was carried out both on Vancouver Island and nearby regions including mainland BC and Washington State in the US. From 2001 to 2006, more than 5,700 tree swabs, soil, air and water samples were collected on Vancouver Island; 519 were positive for C. gattii (8). Overall, 8% of 6  tree swabs and 10% of soil samples collected were positive for C. gattii (8). Positive samples were obtained on a number of native tree species, such as Quercus garryana (Garry oak), Alnus spp. (alder), Pseudotsuga spp (Douglas fir), Arbutus spp. (arbutus), as well as in shrubs, on stumps and cut logs (8). The range of areas colonized by C. gattii on the island extends from the southern border at Victoria north to Campbell River, and west to Port Alberni, covering an area of approximately 13,000 square kilometres. From October 2001 to December 2005, more than 2,000 additional environmental samples were collected from locations off Vancouver Island (41). Approximately 3% of the samples (n=60) were positive for C. gattii (41). Most of the positive samples (n=35) came from the Gulf Islands neighbouring Vancouver Island, while the remaining samples came from BC mainland (n=5) and Washington State in the US (n=2) (41). Using environmental sampling results and animal and human surveillance data, Kidd and colleagues mapped the distribution of C. gattii within BC (Figure 1) (8).  7  Figure 1. Distribution of human and animal cryptococcosis and isolation of C. gattii from the environment on Vancouver Island and in neighbouring regions. Figure adapted from Kidd et al. (8), by permission from the American Society for Microbiology.  C. gattii’s mechanism of migration is not well understood. Current hypotheses for the pathogen’s spreading mechanism include migration via the movement of contaminated vehicles, soil, wood products and plants.  1.3.3 Occupational exposure to C. gattii on Vancouver Island While large-scale environmental sampling has found a number of C. gattii “hotspots” following the cryptococcal disease outbreak on Vancouver Island, little is known about occupational exposure to C. gattii on the Island. Because of C. gattii’s close relationship with plants and soil, 8  workplace exposure may occur for a number of outdoor occupations, particularly those working with plants and soil, such as arborists, landscapers and other city workers. In one study evaluating occupational exposure of arborists and city workers to C. gattii on Vancouver Island, 30 out of 398 air samples were positive for C. gattii (44). The geometric mean C. gattii concentration for the positive samples was 296 CFU/m3 (44). In addition, positive occupational air samples had C. gattii concentrations that were, on average, 6 times higher compared to non-occupational positive air samples, suggesting that exposure during work activities may be significantly higher than environmental exposure (44).  1.4  Composting  Composting is the purposeful biological degradation of organic waste material. The compost product can be beneficially applied to land and is used in gardening, landscaping, agriculture and horticulture. Types of commonly composted materials or feedstock are: food waste, yard waste, biosolids, manure and by-products or product residuals in the agriculture and forestry industry (45). Aerobic composting is the decomposition of organic substrate in the presence of oxygen. An aerobic composting system will go through three general phases (46): 1) Mesophilic phase in which readily degradable material is decomposed and the composting material begins to produce heat; 2) Thermophilic phase during which high microbial activity increases the temperature of compost material to up to 75oC (47) and the majority of biodegradable substrate is decomposed; 3) Curing or maturation phase during which microbial activity slows down, allowing the compost material to cool and become biologically stable (48). In each composting phase, a different flora of microbes becomes established within the composting material. Compost, a stabilized soil-like material free of pathogens and plant seeds, is produced after the organic material in composting material is heated to an adequate temperature and oxidized by different microorganisms (45,46). 9  Anaerobic composting occurs when the organic substrate is decomposed in the absence of oxygen. Anaerobic composting systems produce methane, carbon dioxide and other organic acids and alcohols (45). Because anaerobic composing is, in general, less efficient and more odourous, almost all engineered composting systems are aerobic (45). This thesis will focus on the more efficient aerobic composting process. Unless otherwise specified, all composting systems mentioned refer to the aerobic approach to composting.  1.4.1 Optimal composting parameters The efficiency of the composting process and the quality of the compost product are influenced by a number of parameters, including carbon-nitrogen ratio, aeration, moisture content, pH and temperature (45,46).  1.4.1.1 Carbon-nitrogen ratio The carbon to nitrogen (C/N) ratio in the feedstock material is important for composting systems because carbon and nitrogen are two main elements required for sustaining microbial growth. The optimal C/N ratio for composting systems is in the range of 15-30: 1 (45). A C/N ratio below this range will result in losses of nitrogen through volatilization of ammonia, leading to undesirable odour production (46). Composting microbial activity in a system with high C/N ratio will be very low due to the limited nitrogen available to support microbial growth (45).  1.4.1.2 Aeration Aerobic composting systems require adequate ventilation of the compost substrate to ensure the availability of oxygen and the removal of carbon dioxide for microorganisms inside. The optimal oxygen concentration within the compost is between 5-15% (46). Aeration of compost piles is achieved through either natural ventilation or forced ventilation. Forced ventilation systems in actively aerated compost piles may be operated continuously or controlled by compost operators,  10  timers, or electronic sensors monitoring the oxygen concentration or temperature inside the system (45).  1.4.1.3 Moisture content Appropriate moisture content in composting material is important for microbial growth. During the composting process, the ideal moisture content is between 50 to 65% (46). Excessive moisture in composting system will reduce aeration efficiency and have a negative effect on oxygen concentration. In contrast, low moisture content will significantly reduce microbial activity in a composting system (45).  1.4.1.4 pH The pH range of a composting system varies significantly during the composting process (46). In the thermophilic phase where carbon dioxide is produced in large quantities, the composting material becomes more acidic, with pH values ranging from 5 to 6. During the curing phase, the pH may increase to as much as 8.5 due to the low production of carbon dioxide and its continual release, as well as the degradation of proteins from inactive thermophilic cells (46). For maximum composting efficiency, pH value should be kept between 6 and 7.5 (46).  1.4.1.5 Temperature Composting temperature is one of the most important factors in determining the process’ efficiency and quality of the product. During the initial mesophilic phase, the organic substrate begins to heat up and readily biodegradable material is decomposed. In the following thermophilic phase, temperature of the composting material could reach temperatures up to 75 oC (47). Thermophilic organisms replace mesophilic organisms in this heating phase and microbial breakdown in the composting system reaches a maximum rate (46). The high temperature achieved is also the primary mode of pathogen inactivation during the composting process (48). After the thermophilic phase, a decline in composting material temperature indicates that the rate 11  of organic oxidation is slowing down, and the material is allowed to cure and becomes biologically and chemically stable (45). The optimal temperature during the thermophilic phase is believed to be in the range of 45 oC to 60oC (45,46). However, for sufficient reduction and elimination of pathogenic organism, a temperature of 55-60oC must be sustained by the composting system for at least 2-3 days (46).  1.4.2 Composting methods A number of different methods are used to produce compost. The most basic method is the static pile, where compost feedstock is arranged in large piles or windrows which may be turned occasionally. Aeration and temperature control in static piles are largely unmanaged and depend mostly on natural diffusion and convection. In a similar method called the aerated static pile, air is forced into the composting windrows through either positive or negative pressure. Forced aeration increases the availability of oxygen to organisms inside the windrows, resulting in a more efficient degradation process. The composting method with the most process control is called in-vessel composting, in which the compost feedstock is introduced into an enclosed system where aeration and temperature are monitored and controlled by fans (45,46). Depending on the type of in-vessel system, mechanical mixing may also be applied continuously or periodically to the composting material (45).  1.4.3 Composting acts and regulations in BC In BC, compost that is produced and used inside the province is regulated by the Organic Matter Recycling Regulation under the Waste Management Act and Public Health Act (49). The Canadian Council of Ministers of the Environment (CCME) has also created an extensive composting guideline designed to assist provincial policy makers in regulating composting and compost quality (50).  12  The BC Organic Matter Recycling Regulation and the CCME Guidelines for Compost Quality provide very similar criteria for compost product safety and quality (49,50). Both the BC Regulation and CCME Guidelines define two classes of compost. Class A compost may be used in all types of applications such as in agriculture, horticulture and residential gardens with no volume restriction (49). Class B compost can only be applied with an approved application plan and specific land application methodologies (49). This thesis will focus on Class A compost because compost produced in almost all municipal and commercial facilities satisfies the Class A requirements whereas Class B compost is often produced and used only in an agricultural setting. To be considered Class A according to both the BC Organic Matter Recycling Regulation and the CCME Guidelines for Compost Quality, the compost must meet the following requirements (49,50): 1. Physical contaminants: compost should be free of sharp objects greater than 3 mm per 500 litre of compost; 2. Chemical contaminants: the concentrations of specific heavy metals must be lower than the maximum acceptable cumulative metal additions to soil (shown in Table 1); 3. Biological contaminants: to ensure the elimination of pathogens, one of the following criteria must be met if the compost is not produced solely from yard waste: a) using in-vessel composting method with mechanical aeration and controlled environmental conditions, the material shall be maintained at 55oC or greater for at least three days; b) using the windrow method, the material shall be maintained at 55 oC or greater for at least 15 days, with at least 5 turns during the thermophilic phase; c) using the aerated static pile method, the material shall be maintained at 55 oC or greater for at least three consecutive days. In addition, Salmonella must be absent and the level of thermo-tolerant coliforms must be lower than 1000 MPN per gram of total oven-dried solid; 13  4. Maturity: compost must be treated in an aerobic process for at least 14 days and cured for 21 days. The C/N ratio of the finished product must be between 15-35: 1. The compost must not re-heat to greater than 8oC above ambient temperature. Table 1. Maximum acceptable trace elements concentrations in Class A compost (49).  Element  Concentration (mg/kg dry weight)  Arsenic Cadmium Chromium Cobalt Copper Lead Mercury Molybdenum Nickel Selenium Zinc  13 3 100 34 400 150 0.8 5 62 2 500  1.4.4 Current gap in composting regulations regarding C. gattii elimination The current requirements in composting regulations and guidelines aim to remove the majority of bacterial pathogens (such as E. coli and Salmonella), viruses and parasites from composting feedstock material. Elimination of C. gattii and other fungal environmental pathogens in the composting process was not specifically considered in developing these composting requirements. However, C. gattii survival in composting systems meeting the regulatory requirements may be possible due to a number of factors. C. gattii is an environmental microorganism; it is well adapted to surviving in environmental substrates such as soil and plant waste compared to enteric bacterial pathogens. Environmental organisms like C. gattii are also more likely to be able to tolerate much wider temperature, humidity and pH ranges than enteric bacterial pathogens adapted to living within animal hosts. Current regulatory requirements for composting may not be adequate for eliminating C. gattii from composting feedstock material. In this thesis, elimination or complete inactivation of C. gattii is defined as having a composting product that is free of detectable levels of C. gattii with a detection limit of 2.0 CFU/g of compost on a dry weight basis. 14  1.4.5 Composting on Vancouver Island In Canada, composting is an increasingly popular method of solid waste treatment and recycling. In general, composting diverts significant amount of organic waste from landfills and produces a product that has beneficial properties when applied to land and soil. On Vancouver Island, due to the lack of available space for landfills, composting is widely promoted and practiced as a “green” option for the treatment of organic waste. Currently, there are 32 composting facilities on Vancouver Island that are authorized to operate by the BC Ministry of Environment (M. Bell, 2010, personal communication). In many communities, composting is the only method used to treat green waste such as yard trimmings and leaves. Regions such as the Capital Regional District, Cowichan Valley Regional District, and Regional District of Nanaimo have bylaws that mandate yard trimmings to be composted (51-53). Other solid wastes such as food waste and biosolids, which are dewatered sewage sludge from municipal sewage treatment plants, are also composted along with the plant waste in various regions on Vancouver Island. Both the windrow and in-vessel composting methods are used in industrial composting facilities on Vancouver Island. Home composting is also strongly promoted and encouraged in communities on Vancouver Island.  1.5  Study rationale  There is clear ecologic evidence that C. gattii has successfully colonized native plant species and soil in many populated areas on Vancouver Island and that the range of the organism is actively expanding. In almost perfect accordance, the epidemiology of human C. gattii cryptococcosis in BC and the Pacific Northwest in the US shows the same pattern of expansion of the endemic regions. Recent ecological niche modelling work by Mak and colleagues concluded that Vancouver Lower Mainland in BC, the San Juan Islands and Puget Trough in Washington, and the Willamette Valley in Oregon are all areas with optimal conditions for C. gattii colonization (43). Because these regions contain densely populated cities such as Vancouver and Seattle, there is an increasing potential for higher exposure to C. gattii and higher burden of C. gattii cryptococcosis in both BC and the Pacific Northwest in the US if C. gattii fully colonizes the area. 15  However, relatively little is known about how C. gattii disseminates in the environment and what human activities are associated with high levels of C. gattii exposure. Composting, which is widely practiced using plant waste on Vancouver Island, may have a role in human exposure to C. gattii and the pathogen’s dissemination. With the high prevalence of C. gattii colonization of plant species on Vancouver Island, plant waste colonized with C. gattii is likely to be incorporated in the composting of various waste materials in local municipalities. Occupational exposure to C. gattii may occur during the handling of such contaminated composting feedstock. If C. gattii is not effectively eliminated by the composting process, additional exposure may occur during transport and application of contaminated compost. Finally, the use of contaminated compost in uncolonized areas with appropriate growth conditions for C. gattii may aid its migration in BC and the Pacific Northwest in the US.  1.6  Research questions  1. Is C. gattii present in compost feedstock and product in Comox Valley Biosolids Composting Centre between August 2008 and July 2009? 2. If present in the compost feedstock, does C. gattii persist through the composting process of a composting system meeting provincial regulatory composting requirements in British Columbia? 3. If present in the composting feedstock, does C. gattii aerosolize and become detectable in air during the composting of contaminated feedstock, leading to potential occupational exposure? 4. Is C. gattii present in air when city workers in Parksville, BC are chipping garden waste from residential homes in fall 2008?  16  2  Pilot studies  2.1  Pilot studies: introduction  Pilot studies described below were performed with various C. gattii strains prior to the thesis project. The overall goals of the pilot studies were to: 1. Develop laboratory techniques and gain experience in working with C. gattii. 2. Investigate cell growth, cell size and cell melanization characteristics of C. gattii cells grown in different culture media. 3. Investigate whether melanization of C. gattii cells provide resistance against ultraviolet radiation. 4. Investigate C. gattii cell survival in high temperatures in soil and composting feedstock.  2.2  Pilot studies: methods  2.2.1 Laboratory cell characteristic and survival experiments Microbial growth and cell characteristics are closely linked to the growth medium or substrate environment. Cells grown in laboratory media cultures may have very different growth rates and phenotypic characteristics compared to wild type cells growing in the natural environment. Because the aim of the composting experiment was to study the survival of C. gattii cells in compost in the natural environment, it was important to grow and condition the cells in an appropriate laboratory media before use in the experiment to ensure that cell characteristics were as close to naturally grown wild type cells as possible. The cell growth and cell size experiments were designed to compare C. gattii cell growth and cell size in laboratory enrichment media in different concentrations against cells grown in media made from soil collected from endemic areas. Three strains of C. gattii cells were used for the following cell growth and UV exposure pilot experiments. The C. gattii strains were chosen from the culture collection maintained in a -80°C 17  freezer (Nuaire, Plymouth, MN) in the laboratory in the School of Environmental Health, University of British Columbia (UBC). All three isolates were originally collected on Vancouver Island. The three strains represent the three genotypes of C. gattii present on Vancouver Island: VGI, VGIIa, and VGIIb. The VGI isolate was collected in Duncan, BC in 2003 (Laboratory Storage Identification Code: 713; C. gattii isolate #2901-2); the VGIIa and VGIIb isolates were collected at Rathtrevor Beach Park in Parksville, BC in 2002 (Laboratory Storage Identification Code 94; C. gattii isolate # 152 and Laboratory Storage Identification Code 95; C. gattii isolate # RB 001, respectively).  2.2.1.1Growth rate comparison between laboratory minimal culture and soil medium To compare C. gattii growth rates in different media, the three C. gattii isolates (VGI, VGIIa, and VGIIb) were grown in different dilutions (1/16 th, 1/32nd, 1/64th) of malt extract broth or MEB (malt extract 17.0 g/L; mycological peptone 3.0 g/L. From OXOID, Cambridge, UK) and a soil extract broth (SEB) medium in a shaker at 20°C (Memmert, Schwabach, Germany) for 48 hours. The SEB was made by combining 250 g of soil from endemic regions on Vancouver Island with 600 ml of distilled water and autoclaving (Market Forge, Everett, MA) the mixture for 3 hours at 121°C. The mixture was filtered (Grade 1: 11um, Whatman, Kent, UK) and adjusted to have a pH of 7.0 and with final concentrations of 29.4mM KH2PO4 (Fisher Scientific, Waltham, MA) and 0.0016% w/v yeast extract (Fisher Scientific, Waltham, MA). Lastly, the culture media mix was sterilized in the autoclave at 121°C for 20 minutes (Recipe: B. Bandoni, 2004, personal communication). The starting concentrations of C. gattii for the growth experiment were approximately 50 cells/ml. Samples were collected from all 12 cultures (3 isolates x 4 media) every eight hours for the first 24 hours and every four hours from 24 hours onward. Appropriate dilutions of the culture samples were plated in duplicate using standard spread plating techniques on 1/8th strength malt extract agar (MEA) and grown in 30°C for 48 hours. The concentrations of C. gattii cells in cultures at different sampling times were determined by colony count.  18  Cell concentration data were analysed using S-Plus (Version 8.0). Cell concentrations were plotted on a log scale versus time for growth curves. A best-fit line for the exponential growth phase was manually applied to each growth curve and growth rate was determined from the fitted line. Two points along the fitted line were chosen and the concentrations values and time values were applied to the following equation for the generation time (generation/hour)(54). ( LogN1-LogN0) / Log2 (t1-t0) Doubling times for each C. gattii strain in different growth media were calculated by taking the reciprocal of generation time.  2.2.1.2Cell size comparison between C. gattii cells grown in 1/16th strength MEB and SEB During the aforementioned growth experiment, culture samples were collected from the 1/16 th strength MEB and SEB cultures for all three C. gattii isolates for cell size measurements. Samples for cell size measurements were collected at 32, 36, 40, 44 and 48 hours post culture inoculation. Wet-mount slides were prepared for the culture samples using lactophenol cotton blue stain (BD Diagnostics, Franklin Lakes, NJ). For cell size measurements, the mounted slides were viewed with 400X magnification using a phase-contrast microscope (Nikon, Japan) equipped with an eyepiece graticule (Model G22, Walton and Beckett, USA). The microscope set up was calibrated with a stage micrometer (Graticules, England). Digital photos of the slides were taken using a camera and 32 random C. gattii cells per isolate per medium per sampling time were measured using the projected graticule (Figure 2). In total, 960 cell size measurements were made (32 measurements x 3 isolates x 2 media x 5 sampling time).  19  Figure 2. C. gattii VGIIa strain in 1/16th strength MEB. 48 hour sample showing calibrated graticule for cell size measurement.  Statistical analysis for the cell size experiment data was performed in S-Plus (Version 8.0). Single-tail Welch Modified Two-Sample t-Test was used to compare the average cell sizes of different C. gattii strains grown in 1/16th MEB and SEB at different sampling times.  2.2.1.3Melanization of C. gattii cells in MEB and different soil extract media Melanin is a diverse class of dark, hydrophobic macromolecules produced by many different organisms (55). The specific chemical makeup of melanins varies depending on the source, but the molecules are generally composed of phenolic or indolic monomers with protein and carbohydrate complexes (55,56). In most organisms, melanins are non-essential for normal growth and function, but aids in the protection of the organisms against environmental stress (55).  20  C. gattii’s ability to produce melanin was discovered by Staib in the early 1960s (reviewed in (57)). When supplied with diphenolic compounds such as L-3,4-dihydroxyphenylalanine (or LDOPA, a precursor to neurotransmitters dopamine, norepinephrine, and epinephrine), C. gattii cells produces a dark pigment known as DOPA melanin (55,56). Most fungal cells capable of melanin production synthesize the pigment from a monomer called dihydroxynaphthalene (DHN) and have the ability to form DHN melanin without extracellular DHN substrate (55,56). In contrast, C. gattii cannot make DOPA melanin in the absence of extracellular diphenolic compounds (55,56). In clinical infections, C. gattii has demonstrated the ability to synthesize melanin from a variety of physiologic substrates available in human hosts (56). Melanin produced inside hosts are believed to help C. gattii cells become resistant to antibody-mediated phagocytosis and oxidants produced by host immune cells, thus increasing the virulence of the pathogen (55,56). The function of melanin in C. gattii’s natural environment is less well known. It has been proposed that melanin offers C. gattii cells protection against ultraviolet (UV) irradiation, lytic enzyme attacks in soil and temperature extremes (55,56). C. gattii’s ability to produce melanin was tested in four different growth media. The three C. gattii isolates, cultured in 1/16th strength MEB using methods described previously in Section 2.2.1.1, were plated on 1/16th strength MEB agar and three types of soil extract agar using standard spread plating techniques. The three different soil extract media included one created from soil in endemic region on Vancouver Island, which was identical to the SEB medium previously described and used in the cell growth and cell size experiments, and two additional soil media created from soil found on campus in UBC in Vancouver. The first UBC soil agar (UBC SEB1) was made from bedding soil used in planters on the northwest side of the School of Environmental Health building (2206 East Mall) in UBC. The second UBC soil agar (UBC SEB2) was made using soil found under a tree on the southwest side of the same building. All soil extract media were made using the method previously described in Section 2.2.1.1, except agar (Laboratory Grade, Fisher Scientific, Waltham, MA), at a final concentration of 1.5%, was added to the liquid SEB to produce solid agar media for plating.  21  The culture plates were incubated at 30°C (John Scientific, Toronto, ON) and observed daily after 48 hours of incubation for up to 7 days for brown colonies indicating melanin production.  2.2.1.4Resistance to UV irradiation in melanized and non-melanized C. gattii cells Susceptibility of melanized and non-melanized cells to UV irradiation was investigated using the three strains of C. gattii cells (previously described in Section 2.2.1.1) grown in full strength MEB, 1/16th strength MEB and the three types of soil extract broths (SEB). The different soil extract broths were created from endemic regions on Vancouver Island (SEB1), bedding soil used in planters in UBC (SEB2), and local soil in UBC (SEB3). The preparation method and origins of all three SEBs used were as previously described in Section 2.2.1.3. C. gattii cells were grown in a continuous shaker at 20°C (Memmert, Schwabach, Germany) for 48 hours. Cell concentrations in the culture were determined by cell counts under a light microscope (Nikon, Japan) with a hemocytometer (Hausser Scientific, Horsham, PA). Appropriate dilutions of the cultures were made so that approximately 100 colonies would appear on each culture plate prepared for UV irradiation. The culture dilutions were plated in triplicate on 1/8th strength MEA using standard spread plating techniques and irradiated immediately after spreading with a bactericidal UV light (254 nm) inside a laminar flow hood (Nuaire, Plymouth, MN) for 0, 15, 30, 45, and 60 seconds. The intensity of the UV light inside the flow hood was measured using a UV light radiometer (Model IL1700, International Light, Peabody, MA) and the exposures correlate to doses of approximately 30, 60, 90, and 120 mJ/cm2, respectively for different irradiation times. The irradiated plates were incubated at 30°C for 48 hours and colonies that appeared were enumerated to determine the survival rates. A total of 57 UV exposure trials were conducted with all three C. gattii isolates and five media (7 trials for 1/16th strength MEB, 2 for full strength MEB, and 4 trial each for the three soil media). Data from some trials were excluded from analysis because insufficient colonies (<15) were observed in the control plates. These trials include three trials of VGIIb in UBC planter soil SEB agar and one trial of VGI in UBC planter soil SEB agar. Statistical analysis of data from UV irradiation trials were analysed with Microsoft Excel 2010. 22  2.2.1.5C. gattii heat survival in contaminated endemic soil samples and laboratory inoculated compost 2.2.1.5.1 C. gattii heat survival in endemic soil samples Six soil samples collected from Vancouver Island in May 2009 that were positive for C. gattii were sealed in resealable Ziploc bags and incubated at 55°C (VWR Scientific, West Chester, PA) for 21 days. After 21 days at 55°C, incubation temperature was increased to 65°C, and the sample samples were incubated for another 21 days. The initial concentrations of C. gattii, analyzed immediately after collection, in the soil samples are shown in Table 2. Table 2. Initial C. gattii concentrations in soil samples used in heat survival experiment.  Soil sample  Starting concentration (CFU/g soil wet weight)  A  1100  B  4750  C  7750  D  185000  E  395000  F  725000  CFU = colony forming unit  During the 42 day incubation period, C. gattii concentrations were measured after 3, 7, 14, 21, and 42 days. To determine the concentration of C. gattii in the soil samples, five 2 g subsamples were taken from every compost feedstock and compost product sample, mixed with 10 ml of sterile distilled water in 50 ml polypropylene sterile plastic test tubes (Fisher Scientific, Hampton, NH) and vortexed (Scientific Industries, Bohemia, NY) on high setting (#9) for 30 seconds. Duplicate culture plates were made by spreading sample solution onto Staib agar (described below). Spread plating was performed with standard technique: 100 µl of sample solution was placed onto the surface of the agar and spread evenly with a sterilized bent glass rod. The plates were incubated at 30°C (Johns Scientific, Toronto, ON) for up to 7 days and brown yeast colonies matching the characteristics of C. gattii colony were identified and counted. 23  Staib agar was prepared based on media formulation published by Staib and colleagues (15). The final concentrations of ingredients per litre of agar were: ground niger seed 23.33 g; creatinine 0.78 g; glucose 1.0 g; potassium phosphate 1.0 g; agar powder 17.33 g; chloramphenicol 0.05 g; alcohol 1.33 ml; and distilled water 1000 ml.  2.2.1.5.2 C. gattii and E. coli heat survival in screened compost Another heat survival experiment was performed with C. gattii in compost. Screened compost from the Vancouver Landfill in Delta BC was used to assess C. gattii and E. coli heat survival in compost in this small-scale laboratory experiment. The C. gattii cells used in the experiment belonged to the genotype VGIIa, which was collected in Parksville, BC in 2002 (Isolate #152; Laboratory Storage Identification Code: 94). E. coli isolate #2098 (Laboratory Storage Identification Code: ATC25922) was used in the experiment. C. gattii and E. coli cells were cultured in 1/16th strength malt extract broth (OXOID, Cambridge, UK) and tryptic soy broth (BD Diagnostics, Franklin Lakes, NJ), respectively, for 76 hours in a continuous shaker (Memmert, Schwabach, Germany) at 20°C. Final cell concentrations in the cultures were determined by cell counts under a light microscope (Nikon, Japan) with a hemocytometer (Hausser Scientific, Horsham, PA). The compost samples were divided into 50 g batches, placed into 250 ml polypropylene jars (Nalgene, Penfield, NY) and sterilized by autoclaving at 121°C for 20 minutes. The compost samples were then seeded with different concentrations of C. gattii and E. coli (Table 3), and incubated at 62°C (VWR Scientific, West Chester, PA) for 3 days. C. gattii and E. coli concentrations were measured immediately after inoculation and daily during the incubation period using methods already described in Section 2.2.1.5.1, with the exception of tryptic soy agar (BD Diagnostics, Franklin Lakes, NJ) being used for plating E. coli colonies. Table 3. Initial pathogen concentrations for screened compost heat survival experiment.  Sample  Starting concentration (CFU/g compost wet 24  weight) C. gattii high  1 x 106  C. gattii low  5 x 105  E. coli high  1 x 106  E. coli low  5 x 105  CFU = colony forming unit  2.3  Pilot studies: results  2.3.1 Growth rate and maximum growth concentration comparison between laboratory minimal culture and soil medium Doubling times and maximum cell concentrations in the four culture media tested are shown in Table 4. Growth curves for the three C. gattii strains in the four growth media are shown in Figure 4-6. Doubling time and maximum C. gattii cell concentrations measured in each medium are shown in Table 4. Individual growth curves used to calculate the doubling times are included in Appendix A. Compared to growing in malt extract broth (MEB) in different strengths, all three C. gattii isolates (VGI, VGIIa and VGIIb) showed slower growth in soil extract broth (SEB). Growth rates in different dilutions of MEB were similar within individual C. gattii strains. Maximum cell concentrations in culture were highest in 1/16 th strength MEB for all three C. gattii strains. The second highest cell concentrations in culture for each strain all occurred in 1/32nd strength MEB cultures. The lowest maximum cell concentrations were found in 1/64 th strength MEB cultures for the VGIIa and VGIIb strain, and in SEB culture for the VGI strain.  25  Table 4. Doubling times and maximum cell concentrations for C. gattii strains in various culture media.  C. gattii Strain  Growth medium  Doubling time  Maximum cell  (hour)  concentration (CFU/ml)  VGIIa  VGIIb  VGI  1/16th strength MEB  1.9  1.9x107  1/32nd strength MEB  1.9  9.8x106  1/64th strength MEB  1.8  5.5x106  SEB  2.0  7.6x106  1/16th strength MEB  1.9  1.5x107  1/32nd strength MEB  2.0  1.2x107  1/64th strength MEB  1.9  5.4x106  SEB  2.4  6.0x106  1/16th strength MEB  1.7  2.0x107  1/32nd strength MEB  1.8  1.2x107  1/64th strength MEB  1.8  6.2x106  SEB  2.2  4.6x106  CFU = colony forming unit  26  Concentrations of VGIIa cells in 1/16th, 1/32th, 1/64th strength MEB and SEB  Concentration (CFU/ml)  107 1/16 MEB 1/32 MEB 1/64 MEB SEB  106  105  104  103  102  101 0  10  20  30  40  50  Time (hour) Figure 3. Concentrations of VGIIa cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB.  27  Concentrations of VGIIb cells in 1/16th, 1/32th, 1/64th strength MEB and SEB  Concentration (CFU/ml)  107  1/16 MEB 1/32 MEB 1/64 MEB SEB  106  105  104  103  102  101 0  10  20  30  40  50  Time (hour) Figure 4. Concentrations of VGIIb cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB.  28  Concentrations of VGI cells in 1/16th, 1/32th, 1/64th strength MEB and SEB  Concentration (CFU/ml)  107  1/16 MEB 1/32 MEB 1/64 MEB SEB  106  105  104  103  102  101 0  10  20  30  40  50  Time (hour) Figure 5. Concentrations of VGI cells in 1/16th, 1/32nd, 1/64th strength MEB and SEB.  2.3.2 Cell size comparison between C. gattii cells grown in 1/16th strength MEB and SEB For all sampling times, all three C. gattii strains had larger cell diameters when grown in 1/16 th strength MEB compared to SEB. Average cell sizes of the three strains of C. gattii grown in 1/16th MEB and SEB are shown in Table 5. Differences in cell diameters were statistically significant for all three strains at all five different sampling periods except for VGI cell size at 36 hours.  29  Table 5. Average C. gattii cell sizes in when cultured in MEB and SEB.  C. gattii  Time  Average cell  Average cell  Strain  (hour)  diameter in MEB  diameter in SEB  (µm) [N = 32]  (µm) [N=32]  32  3.9  3.2  <0.01  36  3.4  3.1  <0.01  40  3.4  3.0  <0.01  44  3.7  2.8  <0.01  48  3.3  2.8  <0.01  32  3.5  3.0  <0.01  36  3.5  3.0  <0.01  40  3.8  3.3  <0.01  44  3.4  3.0  <0.01  48  3.2  2.9  <0.05  32  3.3  3.0  <0.01  36  3.4  3.2  0.076  40  3.7  3.3  <0.01  44  3.5  3.3  <0.05  48  3.6  3.2  <0.01  VG IIa  VG IIb  VG I  p-value  2.3.3 Melanization of C. gattii cells in MEB and different soil extract media No melanization was observed in colonies grown on 1/16th strength MEB for any of the three C. gattii isolates (Sample picture of VGIIa in Figure 6). All three C. gattii strains produced brown melanized colonies on all three soil extract agars prepared from soil from endemic regions and from UBC campus. The darkest colonies were produced on soil agar made from endemic area soil and UBC SEB2 agar made from UBC soil (Sample pictures of VGIIa in Figure 7 & 9). Light brown colonies approximately 1 mm in diameter were produced on UBC SEB1, which was made from planter soil in UBC (Sample picture of VGIIb in Figure 8). Regardless of the media tested, all three C. gattii strains produced similarly coloured colonies. 30  Figure 6. C. gattii (VGIIa) colonies on1/16th strength MEB agar.  31  Figure 7. C. gattii (VGIIa) colonies on endemic SEB agar.  32  Figure 8. C. gattii (VGIIb) colonies on UBC SEB1 agar.  33  Figure 9. C. gattii (VGIIa) colonies on UBC SEB2 agar.  2.3.4 Resistance to UV irradiation in melanized and non-melanized C. gattii cells Ultraviolet (UV) irradiation survival proportions of the three strains of C. gattii cells grown in different culture media are shown in Table 6-8. Due to the large variance in the data from the pilot UV exposure experiments, few statistically significant results were found. In general, melanized C. gattii cells grown in three different kinds of soil extract media did not have higher survival rates after UV irradiation compared to non-melanized cells grown in standard and diluted laboratory malt extract broth (MEB) media. For all three C. gattii strains tested, survival rates for cells grown in 1/16th strength MEB were lowest for almost all UV exposure levels. Cells 34  grown in different soil media showed higher UV resistance than those grown in 1/16 th strength MEB for some UV doses tested, particularly for VGIIa cells. However, the protective effect was not observed consistently across different UV irradiation doses and C. gattii strains. For all three strains of C. gattii tested, UV resistance was the highest when the cells were grown in full strength MEB. Survival rates for cells grown in full strength MEB were particularly high compared to those observed in other media in trials involving high doses of UV irradiation. For the highest UV exposure dose (120 mJ/cm2), survival of VGIIa, VGIIb, and VGI cells grown in full strength MEB were 31%, 29%, and 34% higher than cells grown in 1/16th MEB respectively, with the difference for VGI being statistically significant. Table 6. Ultraviolet irradiation survival proportions of C. gattii (VGIIa) cells grown in different media.  Culture Media  Cell survival proportion at various ultraviolet irradiation doses (mJ/cm2)  (number of  [95% confidence interval]  trials)  0 (control)  30  60  90  120  1/16th MEB (7)  1.0  0.88  0.67  0.38  0.27  [1.0-1.0]  [0.63-1.13]  [0.41-0.93]  [0.15-0.61]  [0.09-0.46]  Full strength  1.0  1.0  0.85  0.73  0.58  MEB (2)  [1.0-1.0]  [-1.03-3.06]  [-0.56-2.25]  [-1.93-3.39]  [-2.59-3.76]  Rathtrevor  1.0  0.83  0.77  0.54  0.47  SEB agar (4)  [1.0-1.0]  [0.63-1.03]  [0.69-0.86]  [0.41-0.68]  [0.28-0.67]  UBC local  1.0  1.1  0.79  0.70  0.45  soil SEB1 (4)  [1.0-1.0]  [0.94-1.26]  [0.42-1.16]  [0.46-0.93]  [0.15-0.74]  UBC planter soil  1.0  0.92  0.69  0.43  0.28  SEB2 (4)  [1.0-1.0]  [0.72-1.11]  [0.51-0.87]  [0.38-0.48]  [0.18-0.39]  35  Table 7. Ultraviolet irradiation survival proportions of C. gattii (VGIIb) cells grown in different media.  Culture Media  Cell survival proportion at various ultraviolet irradiation doses (mJ/cm2)  (number of trials)  [95% confidence interval] 0 (control)  30  60  90  120  1.0  0.90  0.75  0.53  0.41  [1.0-1.0]  [0.78-1.02]  [0.55-0.96]  [0.27-0.79]  [0.10-0.73]  Full strength  1.0  0.87  0.87  0.75  0.70  MEB (2)  [1.0-1.0]  [-0.66-2.41]  [0.68-1.05]  [0.69-0.82]  [0.58-0.82]  Rathtrevor  1.0  0.92  0.76  0.60  0.45  SEB agar (4)  [1.0-1.0]  [0.76-1.07]  [0.51-1.01]  [0.41-0.78]  [0.18-0.72]  UBC local  1.0  0.94  0.69  0.53  0.41  soil SEB1 (4)  [1.0-1.0]  [0.62-1.26]  [0.31-1.07]  [0.26-0.79]  [0.06-0.76]  UBC planter soil  1.0  1.1  0.85  0.91  0.47  SEB2 (1)  [n/a]  [n/a]  [n/a]  [n/a]  [n/a]  1/16th MEB (6)  36  Table 8. Ultraviolet irradiation survival proportions of C. gattii (VGI) cells grown in different media.  Culture Media  Cell survival proportion at various ultraviolet irradiation doses (mJ/cm2)  (number of trials) [95% confidence interval] 0 (control)  30  60  90  120  1.0  0.85  0.60  0.37  0.20  [1.0-1.0]  [0.74-0.96]  [0.48-0.72]  [0.20-0.54]  [0.07-0.34]  Full strength  1.0  0.90  0.74  0.67  0.54  MEB (2)  [1.0-1.0]  [0.84-0.96]  [0.08-1.40]  [0.28-1.05]  [0.45-0.62]  Rathtrevor  1.0  0.96  0.79  0.52  0.23  SEB agar (4)  [1.0-1.0]  [0.61-1.30]  [0.59-0.98]  [0.30-0.73]  [0.02-0.44]  UBC local  1.0  0.91  0.76  0.42  0.22  soil SEB1 (3)  [1.0-1.0]  [0.62-1.21]  [0.59-0.93]  [0.24-0.60]  [0.14-0.29]  UBC planter soil  1.0  0.86  0.63  0.45  0.30  SEB2 (4)  [1.0-1.0]  [0.62-1.10]  [0.59-0.67]  [0.33-0.57]  [0.21-0.40]  1/16th MEB (7)  37  2.3.5 C. gattii heat survival in contaminated endemic soil samples and seeded composting feedstock 2.3.5.1C. gattii heat survival in endemic soil samples Concentrations of C. gattii in different samples incubated at 55°C for various lengths of time are shown in Table 9. Because C. gattii concentrations were not measured immediately prior to incubation, the only available initial concentrations for the experiment were from analysis performed immediately after the soil samples were collected. With the exception of Sample E, C. gattii initially present in the soil samples persisted through 21 days of incubation at 55°C. After further incubation at increased temperature (65°C), C. gattii level in one sample (Sample C) was tested to be below the limit of detection. For Sample E, no detectable levels of C. gattii were found in any testing time post incubation. Overall, concentrations of C. gattii showed a decreasing trend as incubation time/temperature increased. However, this trend was not observed for all other samples. Table 9. C. gattii concentrations in positive soil samples incubated for various times at 55°C/65°C.  C. gattii concentration (CFU/g wet weight) Soil  Immediately  55°C  55°C  55°C  55°C  65°C  sample  after sampling  Day 3  Day 7  Day 14  Day 21  Day 42  A  1100  1600  250  250  50  4200  B  4750  <LoD  300  4800  1850  450  C  7750  26200  25800  19800  18500  <LoD  D  185000  30000  16700  25900  60300  14100  E  395000  <LoD  <LoD  <LoD  <LoD  <LoD  F  725000  30000  248000  18350  81000  5800  CFU = colony forming unit <LoD = limit of detection = 50 CFU/g  38  2.3.5.2C. gattii and E. coli heat survival in screened compost Table 10 shows the concentrations of C. gattii and E. coli measured immediately after compost seeding with the pathogens prior to heat incubation at 62°C. Compared to the calculated inoculation concentrations, actual pathogen concentrations measured immediately after inoculation were lower. No detectable levels of C. gattii or E. coli were found in any postincubation samples collected. Limit of detection for both organisms was 2,500 CFU/g compost on a wet weight basis. Table 10. Calculated and actual starting C. gattii and E. coli concentrations for compost heat survival experiment.  Sample  Calculated starting  Measured starting  concentration (CFU/g  concentration (CFU/g  compost wet weight)  compost wet weight)  C. gattii high  1 x 106  7.33 x 105  C. gattii low  5 x 105  4.65 x 105  E. coli high  1 x 106  7.45 x 105  E. coli low  5 x 105  1.25 x 105  CFU = colony forming unit  2.4  Pilot studies: discussion  It is well established that the growth rate and phenotypic characteristics of microorganisms are closely linked to their growth environment. All three strains of C. gattii showed decreased growth rates in diluted laboratory culture media compared to full-strength media. Further, growth rates of C. gattii cells were the lowest when grown in culture media made from endemic region soil. Similar to growth rates, cell sizes were found to be significantly smaller for all three strains of C. gattii when grown in soil extract media compared to laboratory media. Compared to full strength laboratory media, diluted laboratory media and soil media contain only a fraction of the nutrients. The smaller cell sizes and slower growth rates observed in the growth experiments were likely to be caused by the reduced amount of available nutrients in the diluted laboratory media and soil media. When grown in these “minimal” media, C. gattii cells proliferate in a 39  more competitive environment with limited nutrients that is more similar to natural environments than standard nutrient-rich laboratory media. Therefore, growth rate and cell characteristics of C. gattii in minimal media are likely to be more similar to those in cells growing in the wild. Many studies have shown that C. gattii cells produce melanin in various growth environments (1,56). The evidence is clear that C. gattii is able to produce melanin using extracellular phenolic compounds. When grown in three different kinds of soil agar media, C. gattii formed brown colonies that indicate melanin production. Although melanization of C. gattii cells in soil or soil extract media have never been investigated by other studies, different phenolic compounds have been found in soil (58,59), providing evidence to support that substrates needed for C. gattii to form melanin are available in the organism’s natural environment. Melanization of fungal cells generally allows the cells to better cope with a number of environmental stressors such as heat and desiccation (1). The primary ecological niche of C. gattii is environmental and accidental infections in mammals play no significant role in the life cycle of C. gattii. Therefore, it is unlikely that C. gattii’s ability to produce melanin has evolved solely for increased virulence during accidental infections. Melanization of C. gattii cells in the wild may aid the organism’s survival in different natural environments, such as allowing the cells to become more resistant to ultraviolet (UV) radiation exposure, which was tested in the pilot experiments. In the UV exposure experiment, melanized cells did not consistently show higher survival after UV irradiation. In contrast, cell survival was the highest when C. gattii was grown in full strength laboratory media that did not stimulate cell melanization. Other published studies comparing UV resistance in melanized versus non-melanized Cryptococcus species have found conflicting results. Wang and Casadevall found that melanized C. neoformans cells were more resistant to UVC (254 nm wavelength) irradiation than non-melanized cells (60). However, only one strain of C. neoformans was tested in the study. Another study by Schiave and colleagues found little overall difference in UVB (300 nm wavelength) susceptibilities in 8 strains of C. neoformans and C. laurentii cells (61). It is worth noting that in both aforementioned studies, clinical isolates of Cryptococcal isolates were used and melanin production was induced by supplying the cells with l-3,4 dihydroxyphenylalanine (L-DOPA), a phenolic precursor 40  compound to dopamine found in humans and other mammals. In this pilot study, an environmental C. gattii isolate was tested and melanization was induced by growing the cells in soil extract media. Due to differences in growth media environment, Cryptococcus species tested and the origin of experimental isolates, results from this study may not be directly comparable to those found in previous studies. The heat survival experiments showed that, in most cases, C. gattii collected directly from the environment was able to survive for weeks in 55°C when the organism was kept in its original environmental substrate – soil. After the 21 day incubation at 55°C, most samples tested had high concentrations of C. gattii ranging from 1,850 to 60,300 CFU/g wet weight soil. When temperature was increased to 65°C after the 21 day incubation in 55°C, C. gattii cells continued to persist in many samples for weeks. In contrast, in the compost heat survival experiment, laboratory grown C. gattii cells were eliminated with a 24-hour incubation period at 62°C in screened compost. The difference observed in C. gattii survival in similar temperatures may be due to differences in substrate during heat treatment (endemic region soil versus compost) as well the use of laboratory grown C. gattii cells versus naturally grown cells. Based on the results of the pilot experiments and the feasibility of conducting the entire composting experiment with one consistent and standardized culture medium, the 1/16th strength MEB was chosen as the growth medium for subsequent composting experiments. SEB would have been the ideal medium for the composting study because the medium was derived from soil endemic for C. gattii on Vancouver Island and was very likely to have similar nutrient concentrations compared to those available to C. gattii cells growing in the wild. However, because a large amount of media was needed for the composting experiment, it was important to have one consistent medium for culturing C. gattii cells for all experimental trials. Because SEB could only be made in small batches, it was not possible to eliminate slight variations between batches. Therefore, the next best candidate, 1/16th strength MEB, was selected for its consistency and reliability. Although C. gattii cell growth and cell sizes in 1/16th MEB were faster and larger than those observed in SEB, respectively, MEB was commercially available in a standardized recipe. C. gattii growth rates observed in 1/16th strength MEB were very similar to those observed in more diluted 1/32nd and 1/64th dilutions, while the cell concentrations achieved in 41  1/16th MEB were higher than the other media tested. A higher final cell concentration in culture allowed smaller inoculation volumes to be used when conducting the composting experiments, which made the transport and handling of the inoculation cultures safer and more convenient.  42  3  Methods  3.1  C. gattii concentrations in composting feedstock and compost  product from the Comox Valley Biosolids Composting Facility 3.1.1 Study site: Comox Valley Biosolids Composting Facility The Comox Valley Biosolids Composting Facility in Cumberland, BC is a composting facility that composts biosolids from Comox Valley Regional District (CVRD). The composting facility produces approximately 80 tonnes of compost product per year. An in-vessel composting system is used by the composting facility to compost biosolids from CVRD. Biosolids received are mixed in a 1:1 weight ratio with chipped wood waste to achieve a final C/N ratio of 12 in the final composting feedstock. The wood waste used is a mix of wood waste from a nearby landfill and waste wood from land-clearing activities in the region. The composting feedstock mix goes first into one of four in-vessel composters with automated aeration control for 4 weeks. After the in-vessel phase, the composting material is moved to a covered area and arranged in piles. A positive aeration system aerates the static piles during this phase, which lasts for approximately 16 weeks. Finally, the composting material is moved outside into static piles for curing for another 4-12 weeks without turning. Once the compost is mature, it is screened to produce the final compost product. This composting facility was selected for sampling because it incorporates wood waste from regions with C. gattii colonization. Therefore, C. gattii may enter the composting system with contaminated wood waste.  3.1.1.1Testing of C. gattii in composting feedstock and product Approximately 40 grams of compost feedstock and screened compost product were collected weekly into Ziploc bags by staff members at the Comox Valley Biosolids Composting Facility. Sampling began in August 2008 and concluded in July 2009. One compost feedstock sample and one compost product sample were collected every week and stored in a cardboard box left in a cool, dark area. Every five weeks, the collected samples were sent via courier to the laboratory in 43  the School of Environmental Health at UBC. All samples were stored in a 4°C walk-in cooler (Foster, Huston, NY) in the UBC laboratory prior to analysis. The concentrations of C. gattii were determined using methods already described in Section 2.2.1.5.  3.2  Air sampling for C. gattii during wood waste chipping in Parksville,  BC Occupational exposure to C. gattii among city workers in Parksville, BC were measured between October 15th and 21st, 2008 when the city Wood Chipping Program was taking place. The City of Parksville offered free wood chipping for residents in spring and fall. Small trees and branches discarded by city residents were processed through a wood chipper by city workers and the chipped wood waste was either composted or spread on park trails in Parksville. Sampling was performed over four 8-hour workdays of wood chipping. Two city workers operated the chipper and truck together. A total of six different city workers were involved over the four-day sampling period. Area air samples were taken using a Biotest Hycon Air Sampler RCS Plus (Biotest, Dreieich, Germany) fitted with Staib agar media strips (Biotest, Dreieich, Germany). For each sample, 200 litres of air were sampled. Staib agar was prepared as previously described in Section 2.2.1.5. The air samples were taken at roughly 1.5 metres off the ground to approximate the workers’ breathing zones around the chipper and truck when the chipper was in operation. Additional air samples were collected when the truck was emptied at the end of each shift. A total of 63 air samples were taken, representing wood chipping exposures at more than 140 residential locations where chipped wood was processed. The Staib media strips were kept cool in a cooler with ice-packs until they reached the laboratory where they were incubated at 30°C (Johns Scientific, Toronto, ON) for up to 7 days. Brown yeast colonies matching the characteristics of C. gattii colony were identified and counted. Ten bulk wood chip samples were also taken at the end of each workday. All 40 bulk wood chip samples were brought back to the laboratory and tested for the presence of C. gattii with methods described in Section 2.2.1.5.  44  3.3  C. gattii composting simulation  All composting simulation experiments were housed inside a 10 feet by 10 feet polyester canopy with walls on all four sides. The canopy was set up outdoors at the Parksville Public Works Yard. All composting simulation experiments were inoculated with C. gattii culture described below. C. gattii genotype VGIIa isolate #152 (Laboratory Storage Identification Code: 94) was used to prepared experimental inocula for the composting experiments. The isolate was collected in 2002 at Rathtrevor Park in Parksville, BC. Genotyping of the isolate was previously determined by URA5-RFLP (restriction fragment length polymorphism) technique by Sara Kidd (7). In URA5RFLP, the URA5 genes in C. gattii isolates were amplified using polymerase chain reaction (PCR) and amplified DNA segments were cut using restriction enzymes. Differences in the DNA sequences, represented by different sizes of URA5 gene restriction enzyme digestion products, were visualized and identified using agarose gel electrophoresis. The inoculation culture was grown in 300-700 ml batches in reduced (1/16th) strength MEB for at least 48 hours in a continuous shaker at room temperature (20 ± 1°C). Reduced strength media was used to culture cells for the experiment because pilot experiments showed that the rate and characteristics of cell growth in reduced strength media closely resemble cell growth in soil. For details of the pilot experiments, refer to Section 2.3.1.1-2.3.1.2. Cell concentrations in the final culture were determined by cell counts under a light microscope (Nikon, Tokyo, Japan) with a hemocytometer (Hausser Scientific, Horsham, PA). All liquid cultures were kept cold inside a cooler with ice-packs prior to inoculation in composting experiments.  3.3.1 In-vessel composting simulation Yard waste (chipped wood, shrubs, grass, etc.) feedstock from the Delta Vancouver City Landfill in Delta, BC was used for the experiment. Composting feedstock from the Vancouver Landfill mostly originate from garden waste collected in the City of Vancouver. Feedstock used in the experiment was collected and prepared according to standard protocol at the landfill. By the time of collection for use in the composting experiment, the feedstock material was already chipped, 45  mixed and incorporated into windrows for at least four days at the Vancouver Landfill to allow for initial thermophilic heating of the composting material. For each composting trial, a 10 kg batch of composting feedstock was inoculated with liquid culture of C. gattii. A total of six trials were conducted between September 4th and November 9th 2009. The starting concentrations of C. gattii varied from 50,000 colony forming units (CFU) per gram (wet weight) of composting feedstock to 1,000,000 CFU/g. A cement mixer was used to evenly mix the liquid culture with the feedstock material. Appropriate amounts of water (4-6 litres) were added to the mixture to ensure proper moisture content in the starting composting material. After the feedstock was thoroughly mixed, the loaded cement mixer was placed inside an insulated wooden enclosure fitted with two 250-watt heat lamps as heating elements (Picture of the in-vessel composting shown in Figure 10).  Figure 10. Cement mixer and enclosure setup for in-vessel composting experiment.  46  The heat lamps, controlled by preset temperature switches, operated during the first three days of each trial to heat up the feedstock to ≥55°C and ≤84°C to simulate the thermophilic phase in the composting process. 55°C was chosen as the lower temperature limit to satisfy regulatory temperature requirements for in-vessel composting in BC (see Section 1.4.3 for details). 84°C was the upper temperature limit for the temperature switches and temperature sensing devices used in the experiment. The cement mixer was attached to a timer which was set to turn the feedstock for two minutes every two hours to allow even heating and aeration. After the first three days of heating, the heat was turned off and the feedstock remained in the cement mixer inside the enclosure for seven days with the mixer still turning for two minutes every two hours. After a total of ten days inside the enclosure, the compost material was removed and allowed to cure for an additional 50 days in covered plastic bins. Ten temperature sensing dataloggers (ACR SmartButton, ACR Systems) were placed inside the cement mixer in every trial to monitor feedstock temperature throughout the process. One temperature sensor was placed outside of the enclosure to monitor ambient temperature, and one was placed beside the preset temperature switches to ensure its proper functioning. All temperature dataloggers were placed inside sealed polyvinyl chloride (PVC) plastic piping filled with desiccant for protection against moisture and mechanical mixing (Figure 11).  47  Figure 11. ACR Smart Button temperature datalogger (right) and protective PVC piping.  3.3.1.1 C. gattii analysis To measure the concentration of C. gattii throughout the composting process, five bulk feedstock samples were taken at each of these time points: 1) before feedstock inoculation, 2) immediately after inoculation and mixing, 3) after the first three days, 4) when the feedstock was removed from the enclosure, and 5) every 10 days during curing. After collection into Ziploc bags, the bulk samples were kept cool in a cooler with ice packs until analysis in the laboratory. Samples were generally analysed within two weeks of collection. Measurement of C. gattii concentrations in compost simulation samples was analysed with identical procedures used to analyse compost samples in Section 2.2.1.5.  3.3.1.2Compost pathogen analysis The concentrations of coliform bacteria, thermo-tolerant coliform bacteria and Escherichia coli in compost feedstock during different stages of the composting simulation experiment were 48  measured using methods based on compost pathogen testing procedures published in the Test Methods for the Examination of Composting and Compost (TMECC) by the US Composting Council (62). Compost pathogen testing were performed in samples taken 1) immediately after feedstock inoculation and mixing, 2) after the first three days of heating, and 3) when the feedstock was removed from the enclosure. The most probable number (MPN) method and plate count method were used to determine bacterial concentrations in compost samples. For both the MPN and plate count methods, two samples from every sampling time point were used in compost pathogen testing. In 250 ml polypropylene jars (Nalgene, Penfield, NY), 20 g of the collected bulk sample was mixed with 180 ml of buffered peptone water, prepared by adding 10 g of special peptone (Oxoid, Hampshire, UK) with 1000 ml of phosphate buffered saline (Fisher Scientific, Hampton, NH). The mixture was homogenized with a blender (Oster, Rye, NY) on high setting for 2 minutes to produce a sample homogenate with 10 -1 dilution. Five additional dilutions were made by performing serial dilutions in 9 ml of sterilized 0.85% saline water for the 10-1 sample, producing 10 -2, 10-3, 10-4, 10-5, and 10-6 dilutions of sample homogenates used in both the MPN and plate count compost pathogen analysis methods described in upcoming sections. Serial dilutions and all subsequent 9 ml media tubes were prepared in 16 mm x 150 mm glass test tubes with plastic caps (Fisher Scientific, Hampton, NH).  3.3.1.1.1 MPN method For total coliform concentration measurement using the MPN method, 9-ml lauryl-trypose (LT) (Oxoid, Hampshire, UK) media tubes were prepared in glass test tubes with inverted gas tubes (Fisher Scientific, Hampton, NH). 1 ml of the 10 -1, 10-2, and 10-3 sample homogenate dilutions were aseptically transferred into the 9-ml LT tubes in triplicate. The completed LT samples were capped and incubated for 24 hours at 37ºC (NuAire, Plymouth MN). Positive samples were indicated by turbidity of the media and the presence of gas in the inverted glass tube. For thermo-tolerant coliform concentration measurements using the MPN method, 20 µl of positive LT broth from the previous step was aseptically transferred into 4-methylumbelliferyl-pD-glucuronide (EC-MUG) tubes (Oxoid, Hampshire, UK) with inverted gas tubes. Inoculated 49  EC-MUG tubes were capped and incubated for 24 hours at 45ºC (Precision Scientific, Winchester, VA). Positive samples were indicated by turbidity of the media and the presence of gas in the inverted glass tube. For the presence of E. coli in positive EC-MUG samples, positive EC-MUG samples from the previous step were examined under ultraviolet (UV) light (365 nm wavelength) (Fisher Scientific, Hampton, NH). Positive samples were indicated by fluorescence under the UV light. Concentrations of compost pathogen were computed using the 3-tube MPN online calculator by TMECC (62).  3.3.1.1.2 Plate count method For the plate count method, sample homogenates were plated directly on MacConkey agar (BD Diagnostics, Franklin Lakes, NJ) culture plates (Fisher Scientific, Hampton, NH). Three different dilutions of the sample homogenates were plated on six MacConkey plates using standard spread plating technique: 100 µl of liquid homogenate was placed onto the surface of the agar and spread evenly with a sterilized bent glass rod. For feedstock samples collected prior to heating, the 10-4, 10-5, and 10-6 sample homogenate dilutions were selected for plating. For samples collected after the heating phase, the 10-1, 10-2, and 10-3 sample homogenate dilutions were selected for plating. Three of the six plates were incubated at 37ºC for 24 hours. The other three were incubated at 45ºC for 24 hours. Bright pink or red colonies, which indicate growth of lactose positive cells, were counted after incubation. The total coliform concentrations were obtained from colony counts in plates incubated at 37ºC. Thermo-tolerant coliform concentrations were obtained from colony counts in plates incubated at 45ºC.  3.3.1.1.3 Moisture content and pH Additional composting parameters including moisture content and pH were tested using methods published in TMECC (62).  50  To measure moisture content, approximately 10 g of bulk sample was placed inside pre-weighed 10 ml ceramic cups (CoorsTek, Golden, CO) , weighed (Sartorius, Goettingen, Germany), and placed inside an 80ºC oven (Thermolyne, Waltham, MA) for 48 hours. After drying in the oven, the cup with its content was weighed once more and the moisture content of the sample was calculated based on the equation below (62): Moisture content (%) = 1 – [net dry weight/net wet weight] x 100 Moisture contents were measured at the following times in each trial: 1) immediately after feedstock inoculation and mixing, 2) after the first three days, 3) at the end of the curing phase after 60 days, and 4) any sampling time point with positive samples for C. gattii. pH analysis was performed for each of the following time points in each composting trial: 1) immediately after feedstock inoculation and mixing and 2) at the end of the curing phase after 60 days. pH analysis was performed by adding 10 ml of distilled water to 2 g of collected bulk sample inside a 50 ml polypropylene sterile plastic test tube (Fisher Scientific, Hampton, NH). The mixture was vortexed (Scientific Industries, Bohemia, NY) on high setting (#9) for 30 seconds and allowed to settle for 10 minutes. pH of the settled samples was measured by a calibrated electrometric pH meter (Fisher Scientific, Hampton, NH).  3.3.1.1.4 Statistical analysis Results collected from the in-vessel composting simulation trials were analysed with Microsoft Excel (Version 2010).  3.3.2 Yard composting experiment In addition to the in-vessel composing simulations, two passive composting batches were set up to simulate backyard composting in which management of the compost batches are minimal. Two 30 kg batches of feedstock from Vancouver Landfill were inoculated with 200,000 CFU/g starting concentration of C. gattii, mixed thoroughly with the cement mixer, and placed in clean plastic 55-gallon barrels that were cut in half. Ventilation holes (1 inch diameter, 12 holes per 51  half-barrel) were drilled in the barrel wall to allow better aeration. The top openings of the halfbarrels were covered loosely with polyethylene sheets to retain moisture. The concentrations of C. gattii were measured at the following times: before inoculation of the feedstock, immediately after inoculation and mixing, and every 10 days for a total of 60 days. Five samples were taken during each sampling time point and the presence of C. gattii was analysed with procedures described in Section 2.2.1.5. The concentrations of coliform bacteria, thermo-tolerant coliform bacteria and E. coli, as well as composting material pH and moisture content, were also measured using methods described in Section 3.3.1.2. Compost pathogen testing was performed in samples taken immediately after feedstock inoculation and mixing and on every 10 th day of the experiment. Moisture content and pH measurements were made once immediately after feedstock inoculation and once after 60 days of passive composting. All compost material samples collected were kept cold in a cooler with ice-packs until they returned to the laboratory, where all the analyses were carried out.  3.3.2.1Statistical analysis Results collected from the yard composting simulation trials were analysed with Microsoft Excel (Version 2010).  3.4  Air sampling during the composting of C. gattii contaminated  material In order to determine whether aerosolized C. gattii cells were present in air during composting of contaminated waste material, air samples were collected at various times in the composting experiment: 1) the inoculation of composting feedstock with C. gattii, 2) the mixing of inoculated composting feedstock with the cement mixer, 3) the transfer of composting material from the cement mixer to curing plastic bins, and 4) daily visits to the experiment canopy during the three-day heating phase in each in-vessel composting simulation trial. All area air samples were taken using a Biotest Hycon Air Sampler RCS Plus (Biotest, Germany) fitted with Staib agar media strips. Air sampling volume for each sample was 200 L. All air samples were taken from approximately 1.5 m off the ground to approximate a worker’s breathing zone. The Staib 52  media strips were kept cold in the field inside a cooler with ice-packs. Upon return to the laboratory, the samples were incubated at 30°C for up to 7 days and brown yeast colonies matching the characteristics of C. gattii colony were identified and counted.  53  4  Results  4.1  C. gattii concentrations in composting feedstock and compost  product from the Comox Valley Biosolids Composting Facility Concentrations of C. gattii were below the limit of detection (10 CFU/g; wet weight) for all feedstock and screened compost samples from the Comox Valley Biosolids Composting Facility. Table 11 shows the dates for which compost and feedstock samples were collected.  54  Table 11. C. gattii concentrations in Comox Valley Biosolids Composting Facility feedstock and compost samples. Sampling date August 11, 2008 August 18, 2008 August 25, 2008 September 1, 2008 September 8, 2008 September 15, 2008 September 22, 2008 September 29, 2008 October 6, 2008 October 13, 2008 December 1, 2008 December 8, 2008 December 15, 2008 December 22, 2008 December 29, 2008 January 5, 2009 January 12, 2009 January 19, 2009 January 26, 2009 February 2, 2009 February 9, 2009 February 16, 2009 April 13, 2009 April 20, 2009 April 27, 2009 May 4, 2009 May 11, 2009 May 18, 2009 May 25, 2009 June 1, 2009 June 8, 2009 June 15, 2009 June 22, 2009 June 29, 2009 July 6, 2009 July 13, 2009 July 20, 2009 CFU = colony forming unit  Sample C. gattii concentration (CFU/g) Feedstock Screened Compost < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10  55  4.2  Air sampling for C. gattii during wood waste chipping in Parksville,  BC All air samples had C. gattii concentrations that were below the limit of detection (5 CFU/m3). Sampling was performed at 128 different residential addresses in Parksville, BC. Most of the samples were collected from residential neighbourhoods in Western Parksville. Figure 12 is a map of Parksville with sampling locations highlighted. The sampling addresses and number of samples taken are listed in Table 12. One air sample typically included chipping work performed at two residential addresses. Weather was sunny and mild on days which sampling took place; ambient temperatures were between 8-10oC. All 40 bulk woodchip samples collected also had C. gattii concentrations that were below the limit of detection (10 CFU/g; wet weight).  Figure 12. Garden waste chipping sampling locations (n=128) in Parksville, BC.  56  Table 12. Airborne C. gattii concentrations in locations where garden waste chipping took place.  Sampling address in Parksville, BC 400 - 700 Block Temple St 400 - 600 Block Pym St 700 Block Stanhope Rd 500 Block Belson St 500 - 700 Block Soriel Rd 600 - 700 Block Sanderson Rd Coldwater Rd Park 600 Block Wedgewood Cre 300 - 600 Willow St 500 Block Panorama Pl Alberni Hwy at Hirst Ave 400 Block Todd Pl 600 - 700 Block Doehle Ave 700 Block Bradbury Ave 400 Block Heather Pl Forester Park (unloading) 300 and 500 Block Bay Ave 300 Block Dogwood St 200 Block Roscow St 300 Block Wisteria St 300 Block Rheumer Cre 700 Block Daffodil Dr 600 - 700 Block Forsyth Ave 700 Block Camas Way 700 Block Lilac Dr 700 Block Camellia Dr 200 Block Violet Pl 700 Block Nanoose Ave 700 Block Newcastle Ave 700 Ermineskin Ave 600 Block Blenkin Ave 0 and 20 - 30 Block Gerald Pl 10 and 40 - 50 Block Jenkins Pl 700 Block Ironwood Ave  Number of  C. gattii concentration  samples  (CFU/m3)  5 5 1 3 3 3 2 2 9 5 1 1 2 1 1 4 2 2 2 1 1 1 5 1 2 1 3 3 2 2 1 1 1 2  <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5  CFU = colony forming unit  57  4.3  C. gattii composting simulation  4.3.1 In-vessel composting simulation A total of 6 trials were conducted for the in-vessel composting experiment. 4.3.1.1Temperature Heating time and average temperatures during the heating period for all 6 in-vessel composting simulation trials are shown in Table 13. Figure 13 shows the average temperature inside the composter during the heating period and ambient temperature for one of the six composting simulation trials (Trial 2). Temperature graphs for all other trials are included in Appendix A. The heating period was defined as starting when the average temperature detected by the ten temperature dataloggers inside the cement mixer reached 55 oC and ending when the average temperature dropped below 55oC after 3 days when the heating elements were switched off. With the exception of Trial 1, all composting trials achieved an average temperature beyond 55 oC for approximately 3 days. Table 13. Heating time and average temperature during the heating phase of in-vessel simulation.  In-vessel  Heating time1 (hour)  Average temperature  composting  during heating time  simulation  [range] (oC)  Trial 1  17.5  52.3 [46.0 - 70.5]  Trial 2  72  70.8 [44.0 - 85.0]  Trial 3  68  66.5 [55.0 - 85.0]  Trial 4  69.5  67.1 [51.0 - 85.0]  Trial 5  67.5* (53.5 + 14)  62.6 [50.5 - 84.5]  Trial 6  70  68.0 [45.0 - 85.0]  1. Heating time is time in which the composting material is held at an average temperature of 55 oC or above during the heating phase  *Temperature in Trial 5 dropped slightly below 55oC to 53-54oC for two hours.  58  Figure 13. Average temperature (ºC ) in composter and ambient temperature during heating period for Trial 2.  59  4.3.1.2 pH pH values for samples taken from all 6 in-vessel composting simulation trials are shown in Table 14. pH values were measured for samples taken at the beginning (Day 0) and end (Day 60) of the trials. Generally, only small changes in pH values were observed. Table 14. pH values for composting material at the beginning (Day 0) and end (Day 60) of the in-vessel composting simulation.  In-vessel  pH  composting  Day 0  Day 60  simulation Trial 1  8.28  7.77  Trial 2  8.44  8.75  Trial 3  8.05  8.58  Trial 4  8.17  8.59  Trial 5  7.74  8.21  Trial 6  7.57  7.22  4.3.1.3 Moisture content Moisture contents for samples collected at different time points for all 6 in-vessel composting simulation trials are shown in Table 15. Moisture content was measured for samples taken on Day 0 (immediately after C. gattii mixing), Day 3 (after heating to 55oC), Day 10 (as composting material exited the mixer and into curing bins) and Day 60 (end of experiment). Day 60 moisture contents for Trial 1, 2 and 4 were not available because the curing bins were exposed to the rain in the curing period. In general, the composting materials in all 6 trials became drier as the composting experiment progressed.  60  Table 15. Moisture content values for in-vessel simulation composting material at different sampling time.  In-vessel  Moisture content (%)  composting  Day 0  Day 3  Day 10  Day 60  simulation Trial 1  62  60  56  n/a  Trial 2  70  56  56  n/a  Trial 3  67  59  59  62  Trial 4  66  47  46  n/a  Trial 5  80  67  63  60  Trial 6  73  62  60  57  4.3.1.4 Bacterial pathogen concentrations in in-vessel composting simulation experiment Concentrations of coliform and thermo-tolerant coliform bacteria were measured for composting material in the in-vessel simulations using two methods (MPN and plate count) at three different times. Day 0 samples were collected immediately after C. gattii inoculation, Day 3 samples were collected immediately after heating to 55oC, and Day 10 samples were collected as the material exited the cement mixer and into curing bins. 4.3.1.4.1 Coliform bacteria Coliform bacteria concentrations in samples collected from all 6 in-vessel composting trials are shown in Table 16. In general, coliform concentrations found with the MPN method are consistent with those found using the plate count method. Overall, coliform concentrations in Day 3 post-heating samples were much lower than those found in Day 0. Concentrations found using the plate count method indicate that heating resulted in coliform bacteria reductions ranging from 3 to 6 orders of magnitude from the starting sample. A slight increase in coliform concentrations was observed in most trials after the heat was turned off. In 4 out of 6 trials, Day 10 samples had coliform concentrations that were higher than those found in corresponding Day 3 samples. 61  Table 16. Coliform concentrations for composting material in in-vessel simulation measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 37ºC.  Coliform concentration (CFU/g; dw)  In-vessel composting simulation  Day 0 MPN  Plate  Trial 1  Day 3 MPN  Plate  >910  Count 1.7E+08  Trial 2  >710  Trial 3  Day 10 MPN  Plate  >530  Count 1.3E+05  >1100  Count 1.4E+06  3.8E+07  340  6900  >1100  950  >790  1.1E+08  >540  8000  320  <42  Trial 4  >810  3.6E+08  >660  1100  20  5.2E+04  Trial 5  >490  2.6E+06  20  <33  >660  5.3E+05  Trial 6  >650  3.7E+07  2  60  20  130  4.3.1.4.2 Thermo-tolerant coliform bacteria Thermo-tolerant coliform bacteria concentrations in samples collected from all 6 in-vessel composting trials are shown in Table 17. Due to some samples being inadvertently destroyed during analysis, thermo-tolerant coliform concentrations were not available for the first three trials. Thermo-tolerant coliform concentrations found by the MPN method and plate count method were not always consistent with each other. Plate count thermo-tolerant coliform concentrations showed decreases ranging from 3 to 5 orders of magnitude after 3 days of heating for three trials where thermo-tolerant coliform bacteria information were available.  62  Table 17. Thermo-tolerant coliform concentrations for composting material in in-vessel simulation measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 45ºC.  Thermo-tolerant coliform concentration (CFU/g; dw)  In-vessel composting simulation  Day 0 MPN  Plate  Trial 1  Day 3 MPN  Plate  n/a  Count n/a  Trial 2  n/a  Trial 3  Day 10 MPN  Plate  n/a  Count n/a  n/a  Count n/a  n/a  n/a  n/a  n/a  n/a  n/a  n/a  n/a  n/a  n/a  n/a  Trial 4  >810  >2.0E+06  >150  790  1  <57  Trial 5  670  1.2E+07  20  <33  50  2500  Trial 6  >120  4.3E+05  <1  <38  <1  <38  4.3.1.5C. gattii concentrations in in-vessel composting simulation experiment Average concentrations of C. gattii for all sampling time points for all 6 trials are shown in Table 18. All control samples taken before the inoculation of compost feedstock had C. gattii concentrations that were below the limit of detection (<2.0 CFU/g dw, assuming a moisture content of 60% for sample material). In 5 out of 6 in-vessel composting simulation trials performed, detectable levels of C. gattii were recovered from composting feedstock immediately after inoculation with culture media (Day 0), indicating that C. gattii inoculation of composting material was successful. Trial 1 had no detectable concentrations of C. gattii immediately after liquid culture inoculation (Day 0), but detectable levels of C. gattii were later detected in collected samples. C. gattii concentrations for all 6 trials were below the limit of detection in samples taken immediately after heating (Day 3). During the curing phase, (Day 10 – 60), detectable levels of C. gattii were found in 4 out of 6 trials. On Day 60 of the experiment, C. gattii was still present in composting material for 4 of the 6 trials conducted. The highest average C. gattii concentration measured during the curing phase was 870 CFU/g (dw), which occurred on Day 40 in Trial 3. 63  Table 18. Average C. gattii concentrations in in-vessel composting simulation samples at different times.  C. gattii concentration (CFU/g dw) Trial  Inoculation  Day  Day  Day  Day  Day  Day  Day  Day  concentration  0  3  10  20  30  40  50  60  1  5.0E+4  <LoD  <LoD  60  30  <LoD  10  20  2.0  2  1.0E+6  1.1E+4 <LoD  <LoD  <LoD  <LoD  <LoD  <LoD  <LoD  3  1.0E+6  1810  <LoD  2.0  150  560  870  260  8.0  4  1.0E+6  850  <LoD  <LoD  4.0  <LoD  2.0  <LoD  2.0  5  5.24E+5  1.7E+4 <LoD  <LoD  <LoD  200  <LoD  20  20  6  4.33E+5  1.1E+4 <LoD  <LoD  <LoD  <LoD  <LoD  <LoD  <LoD  LoD = 2.0 CFU/g dw assuming moisture content of material is 60%  4.3.2 Yard composting experiment A total of 2 trials were conducted for the yard composting experiment. 4.3.2.1Temperature Average temperatures measured in yard composting trials are shown in Table 19. No significant heating was observed for the composting material in the yard composters and temperatures within the composters were nearly identical to ambient temperatures measured. Table 19. Average yard composter temperatures and ambient temperatures.  Yard composting  Average temperature [range] (oC)  Trial 1  13.4 [3.0 – 30.5]  Trial 2  14.5 [3.5 – 33.0]  Ambient*  15.3 [-1 – 33.5]  *Data from October 18-20 were not available.  64  4.3.2.2pH pH values for samples collected from the 2 yard composting trials are shown in Table 20. pH was measured for samples collected at the beginning (Day 0) and end (Day 60) of each trial. Composting material in both composters became more acidic as the trial progressed. Table 20. pH values of composting feedstock and finished compost for yard composters.  Yard  pH  composting  Day 0  Day 60  Trial 1  7.92  7.03  Trial 2  7.88  6.82  4.3.2.3Moisture Content Moisture contents of samples collected from the 2 yard composting trials at different times are shown in Table 21. Moisture content was measured for samples collected at the beginning (Day 0) and end (Day 60) of each trial. Composting material in both yard composting bins were drier at the end of the experiment than at the beginning of the experiment. Table 21. Moisture content of composting feedstock and finished compost for yard composters.  Yard  Moisture content (%)  composting  Day 0  Day 60  Trial 1  72  66  Trial 2  74  70  4.3.2.4Bacterial pathogen concentrations in yard composting experiment Concentrations of coliform and thermo-tolerant coliform bacteria were measured for composting material in the yard composting bins using two methods (MPN and plate count) at two different times. Day 0 samples were collected immediately after C. gattii inoculation and Day 60 samples were collected at the end of each trial.  65  4.3.2.4.1 Coliform bacteria Coliform bacteria concentrations in the yard composting bins at different times are shown in Table 22. For both trials, coliform bacteria concentrations measured by the MPN method were similar between samples collected at the beginning and end of each trial. Samples collected at the end of both trials had coliform concentrations that were approximately 2 orders of magnitude lower by plate count than those collected in the beginning. Table 22. Coliform concentrations for composting material in yard composters measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 37ºC.  Yard  Coliform concentration (CFU/g; dw)  composting  Day 0  Day 60  MPN  Plate Count  MPN  Plate Count  Trial 1  >680  1.61E+08  >800  2.11E+06  Trial 2  >630  8.99E+07  >720  4.08E+05  4.3.2.4.2 Thermo-tolerant coliform bacteria Thermo-tolerant coliform bacteria concentrations found in samples collected from the yard composting trials are shown in Table 23. Day 0 MPN concentrations were not available for both trials due to samples being inadvertently destroyed during analysis. Thermo-tolerant coliform plate count concentrations were found to be lower by approximately 2 orders of magnitude for Day 60 samples compared to Day 0 samples for both trials conducted. Table 23. Thermo-tolerant coliform concentrations for composting material in yard composters measured by the Most Probable Number (MPN) method and plate count method on MacConkey agar incubated at 45ºC.  Yard  Thermo-tolerant coliform concentration (CFU/g; dw)  composting  Day 0  Day 60  MPN  Plate Count  MPN  Plate Count  Trial 1  n/a  1.93E+07  370  2.24E+05  Trial 2  n/a  1.92E+07  500  8.22E+04  66  4.3.2.5C. gattii concentrations in yard composting experiment All 80 bulk composting material samples except one collected from the yard composting simulation experiment had C. gattii concentrations that were below the limit of detection (2 CFU/g; wet weight, assuming 60% moisture content). Levels of C. gattii were below the limit of detection in samples collected immediately after inoculation and mixing of the compost feedstock (Day 0) for both trials. The positive C. gattii sample was collected on Day 60 in Trial 1. C. gattii concentration was 2,900 CFU/g (dry weight) for the lone positive sample.  4.4  Air sampling during the composting of C. gattii contaminated  material A total of 27 RSC air samples were taken during the composting simulation experiment. All samples collected had C. gattii concentrations that were below the level of detection (5 CFU/m3). Table 24 shows the dates and activities during which the air samples were collected.  67  Table 24. Air C. gattii concentrations on different dates and during different activities in simulation composting trials. Sampling date  Activity  C. gattii concentration  September 4, 2009  Mixing C. gattii inoculum with feedstock  (CFU/m3) <5  September 5, 2009  In-vessel simulation trial visit  <5  September 6, 2009  In-vessel simulation trial visit  <5  September 7, 2009  Collecting sample from in-vessel simulation  <5  September 14, 2009  Mixing C. gattii inoculum with feedstock  <5  September 14, 2009  Mixing C. gattii inoculum with feedstock  <5  September 14, 2009  Mixing C. gattii inoculum with feedstock  <5  September 16, 2009  In-vessel simulation trial visit  <5  September 17, 2009  Collecting sample from in-vessel simulation  <5  September 24, 2009  Mixing C. gattii inoculum with feedstock  <5  September 25, 2009  In-vessel simulation trial visit  <5  September 26, 2009  In-vessel simulation trial visit  <5  September 27, 2009  Collecting sample from in-vessel simulation  <5  October 4, 2009  Mixing C. gattii inoculum with feedstock  <5  October 5, 2009  In-vessel simulation trial visit  <5  October 6, 2009  In-vessel simulation trial visit  <5  October 14, 2009  Mixing C. gattii inoculum with feedstock  <5  October 15, 2009  In-vessel simulation trial visit  <5  October 16, 2009  In-vessel simulation trial visit  <5  October 17, 2009  Collecting sample from in-vessel simulation  <5  October 24, 2009  Mixing C. gattii inoculum with feedstock  <5  October 25, 2009  In-vessel simulation trial visit  <5  October 26, 2009  In-vessel simulation trial visit  <5  October 27, 2009  Collecting sample from in-vessel simulation  <5  November 6, 2009  Mixing C. gattii inoculum with feedstock  <5  November 7, 2009  In-vessel simulation trial visit  <5  November 8, 2009  Collecting sample from in-vessel simulation  <5  CFU = colony forming unit  68  5  Discussion  5.1  Was C. gattii present in compost feedstock and product in Comox  Valley Biosolids Composting Centre between August 2008 and July 2009? The presence of C. gattii in composting material is the prerequisite for any potential occupational and environmental exposure. In this study’s year-long survey, no compost feedstock and product samples collected at the composting facility had detectable levels of C. gattii. The Composting Centre was a biosolids composting facility which used plant waste as bulking material. The facility was located in Courtenay, BC, which was a region with confirmed C. gattii colonization (43). However, compared to other C. gattii colonization hotspots such as Parksville and Duncan, environmental sampling found fewer permanently colonized locations in Courtenay and nearby Comox (8). Based on the Cryptococcus Environmental Sampling Database, a Microsoft Access database for environmental samples collected by the UBC Laboratory from 2001 to 2010, 125 of 772 (16.2%) environmental samples taken in Courtenay and Comox were positive for C. gattii. Sampling performed in Parksville, in contrast, found 1175 positive C. gattii samples out of 2275 (51.6%). Geographically, Courtenay and Comox are near the northern boundary of the C. gattii colonization region on Vancouver Island. It has already been hypothesized that C. gattii may establish colonization more easily in areas with higher average temperature and lower precipitation (8). It is possible that the extent of C. gattii colonization in Courtenay is low, leading to the absence of positive compost feedstock and compost product samples from the composting facility monitored. Due to the fact that the site was a biosolids composting facility and plant waste was used only as bulking material, the composting process may not be optimized for the breakdown of plant waste in compost piles. Plant waste not completely degraded was removed from the finished compost by screening and this material, known as “overs,” would be reincorporated into new batches of composting material for further degradation. As a result of the recycling of plant waste overs as bulking material, the actual amount of plant waste processed by the composting facility may be limited. In addition, there was no way of verifying that the plant waste processed in the plant 69  originated from nearby regions. Wood waste may have come from other non-colonized areas on Vancouver Island during the time of sampling. There were some sampling gaps in which samples were not collected (in November 2008 and March 2009). Sampling was performed according to schedule for the rest of the monitoring period. Most importantly, sampling was performed according to plan during the summer months, in which C. gattii environmental concentrations, if present, were expected to be high. It is unlikely that any positive samples were missed due to the sampling gaps. Conclusion: C. gattii was not present in sampled compost feedstock and product in Comox Valley Biosolids Composting Centre between August 2008 and July 2009.  5.2  If present in the compost feedstock, does C. gattii persist through  the composting process of a composting system meeting BC provincial regulatory composting requirements in Canada? Significant log-reduction and removal of pathogens from feedstock waste material is one of the most widely advertised benefits of composting. Current composting regulations in Canada require in-vessel composting material to be held at above 55ºC for at least three days for the pathogen inactivation (50). The regulatory requirement, however, was developed mainly to protect the general public from exposure to pathogens such as E. coli and Salmonella sp during the handling and use of compost (63). Survival of C. gattii in compost may lead to inhalation exposure in compost facility workers and compost product users. The first in-vessel composting simulation trail was a pilot trial for optimizing the composter temperature and other experiment parameters such as C. gattii inoculation concentration and moisture content for the composting material. Therefore, compared with subsequent trials, the first trial had lower temperature and lower inoculation concentration for C. gattii. With the exception of the pilot trial, average temperatures for the heating period were over 60ºC, with maximum temperatures reaching 85ºC for all in-vessel composting simulation trials. However, out of the six in-vessel composting simulation trials, only one trial achieved the 70  regulatory standard and maintained composting material temperature at > 55ºC for 72 hours. Three other trials succeeded in maintaining the temperature of the composting material over 55ºC for at least 68 hours, which was very close to the condition set out by the regulations. Failure to meet the strict time-temperature criteria is not uncommon in full-scale, as well as experimental small-scale composting due to the heterogeneity of composting material (63,64). Completely uniform composting material consistency, heating, moisture content, aeration and mixing are extremely difficult to achieve in composting systems. Temperature performance for the composter used in the current study was similar to other pilot scale and full scale in-vessel composters (64,65). Vinnerås and colleagues conducted pilot scale experiments with a 90 L insulated composter composting thermo-tolerant and food waste for 35 days (64). Temperature in the centre of the composter was above 55ºC for 120 hours but temperature near the wall of the composter was above 55ºC for only 48 hours (64). Temperatures during the thermophilic phase in two industrial-scale in-vessel composters in Sweden and Finland were investigated by Christensen and colleagues (65). The facility in Sweden had mean temperatures ranging from 45.3-73.9 ºC and maximum temperatures ranging from 71.1-80.1ºC, depending on the sampling position within the composting vessel (65). The facility in Finland had mean temperatures ranging from 43.1-56.6ºC and maximum temperatures ranging from 57.9-74.6ºC (65). Coliform bacteria and thermo-tolerant coliform bacteria concentrations measured in raw feedstock were within concentration ranges reported by other published studies (66). Overall, coliform and thermo-tolerant coliform concentrations in the composting material in the in-vessel composting experiment reduced by at least 1000-fold immediately after the 3-day heating phase compared to those measured in the beginning of the experiments. Regrowth of coliform and/or thermo-tolerant coliform after the 3-day heating phase was detected in trial number 4 and 5. The magnitudes of indicator organism reduction for the composting simulation were similar to those reported by other composting studies (63,66), suggesting that, in terms of pathogen reduction, the current in-vessel composting simulation is comparable to other full-scale industrial composting systems.  71  No other studies investigated the survival of C. gattii in composting systems. Similarly, temperature inactivation of C. gattii was not specifically investigated by any study. However, one heat growth and survival study conducted by Martinez and colleagues on 19 different clinically isolated strains of C. neoformans in different serotypes (A and D) reported that no growth was observed in any strain at 44ºC (67). The study also reported that, when grown in the laboratory and suspended in warm distilled water, most of the 19 strains tested were susceptible to heat killing at 47ºC for 60 minutes (67). Temperatures achieved during the heating phase in the in-vessel composter used in this study and the exposure time to these temperatures were well beyond the experimental time-temperature inactivation criteria reported by Martinez and colleagues (69). Even for the pilot trial, which ran the coolest and for the shortest time, composting material was heated continuously to at least 46ºC for 17.5 hours. In subsequent in-vessel simulation trials, composting material was heated continuously to at least 44-55ºC for 53.5-72 hours. According to the heat inactivation results reported by the aforementioned study by Martinez and colleagues, these temperatures achieved and exposure times should be sufficient in eliminating Cryptococcus cells added to the composting material (67). Contrary to heat inactivation results observed by Martinez and colleagues, C. gattii was able to persist in most in-vessel composting trials in this study. In all 6 in-vessel composting simulation trials, C. gattii concentrations initially decreased to below the limit of detection after the three day heating phase. This initial decrease in C. gattii concentrations suggests that heating was effective at killing C. gattii introduced into the composting material. However, C. gattii became detectable again as early as seven days after the heating phase in two trials, and eventually detectable levels of the fungus was found in four out of six trials. The apparent regrowth of C. gattii may be due to incomplete killing during the heating phase. Cooler pockets may exist in the composting material which allowed some C. gattii cells to persist through the heating phase and re-establish colonization once the composting batches were cooled and mixed. Compared to the study conducted by Martinez and colleagues, this study used a Cryptococcus strain that was originally isolated from the environment rather than clinically isolated Cryptococcus cells that have been maintained in the laboratory for a number of generations. 72  After infecting mammalian hosts and lengthened in vitro culture, laboratory strains may change significantly from wild type cells (68) and may become less tolerant to environmental stressor such as heat and desiccation. Cells survival in heated composting material may also differ from survival in water or other laboratory media. For instance, growing in a more natural substrate such as composting material may result in the melanization of Cryptococcus cells, which may increase cellular resistance to heat stress. Cells used in this experiment were also enriched inside the laboratory using laboratory growth medium. A dilute growth medium was used to culture the cells in the laboratory in order to approximate low-nutrient growth conditions in the wild, but other growth conditions, such as moisture, temperature, pH and the presence of other microorganisms cannot be reproduced in the laboratory. Therefore, compared to cells in the wild, cells used in this experiment may also have adjusted to growing inside the laboratory and became less adapted to surviving in natural substrates such as compost. The fact that our laboratory cultured environmental isolate, when added to compost feedstock at real-world concentrations, was able to survive the composting process in the in-vessel composting simulator suggests that the more robust wild C. gattii cells are likely to persist through the composting process with greater success. Because the experiment was carried out in an endemic region for C. gattii, it was possible for the heat treated composting material to be contaminated and colonized with environmental C. gattii. However, because both the in-vessel composter and the curing bins were well sealed and the experiment took place within a closed canopy tent, it was unlikely that the composting material was contaminated with environmental C. gattii. A number of air samples were taken within the experiment tent at various times to assess the concentration of C. gattii in air. No positive air samples for C. gattii were found in or around the tent which housed the experiment. Compared to Day 0 concentrations measured immediately after C. gattii inoculation, C. gattii concentrations measured post heating phase were generally low and well below 100 CFU/g dw. Higher concentrations were detected in one trial (Trial 3), in which up to 870 CFU/g dry weight of C. gattii was found. The regrowth of C. gattii after the heating phase may depend on a number of environmental factors such as temperature, pH, moisture content and nutrient content of the composting feedstock. Because the in-vessel simulations were carried out in the fall (early 73  September to mid November), average ambient temperatures were generally below 15ºC when the composting batches were curing. It has been well established that C. gattii prefers warmer temperatures and concentrations in the environment generally increase in the summer (8). Low ambient temperatures during the curing phase may limit the regrowth of C. gattii in the composting material after the heating phase. During warmer months, C. gattii regrowth in compost may reach higher concentrations than those observed in this study. In the yard composting simulation experiment, temperatures measured inside the composting bins were basically identical to ambient temperatures. The volume of composting material may be too small for the generation of significant self-heating. As a result of the low temperature, both coliform bacteria and thermo-tolerant coliform bacteria concentrations were only reduced by approximately 100-fold in both trials. Although both yard composting trials were inoculated with 200,000 CFU/g of C. gattii on a wet weight basis, no C. gattii were recovered in composting material samples taken immediately after inoculation. This may be due to the high degree of heterogeneity in the composting material, resulting in uneven mixing and uneven distribution of added C. gattii cells. The sudden introduction of C. gattii cells grown in laboratory media into composting material may also be a significant stressor on the cells, affecting their survival. Only one sample (Trial 1 Day 60) in the yard composting experiment tested positive for C. gattii (2,900 CFU/g dw). The fact that C. gattii was not detected in multiple samples taken during the first 50 days of the experiment in both composting bins suggest that overall C. gattii concentrations were probably very low. The detection of C. gattii in one sample suggests that there may be “pockets” of composting material where C. gattii persisted. Due to the lack of heating, the moisture content of the yard composting material remained high compared to material which was composted with the in-vessel simulator. The low temperature and moist environment inside the yard composters may limit C. gattii survival and colonization, as the pathogen was reported to favour drier and warmer environments in the wild (8). Conclusion: If present in the original feedstock, C. gattii is likely to persist through composting processes, even when provincial regulatory composting requirements are met. 74  5.3  If present in the composting feedstock, does C. gattii aerosolize and  become detectable in air during the composting of contaminated feedstock, leading to potential occupational exposure? Microorganisms rarely grow without attaching to a surface or substrate. For C. gattii in composting material, it is reasonable to assume that aerosolization would first require the attachment of C. gattii onto a soil or composting material particle, then the particle would somehow become air-borne. When composting C. gattii-contaminated waste, the fungus may aerosolize during various activities such as preparation of waste material for composting (i.e. chipping and mixing), and processing and handling of the composting material (i.e. pile turning and moving). In this study, no positive air samples were observed during the composting simulations with C. gattii inoculated composting material. While extensive personal sampling during the composting experiments was not performed, grab air samples were collected during activities when C. gattii was most like to become aerosolized, such as mixing and transferring of contaminated composting material. Some factors in the composting experiment may affect aerosolization of C. gattii. Because C. gattii inoculation was performed via the introduction of a solution containing the organism, any composting material that came in contact with added pathogen had to be moistened by the inocula. High moisture content in the contaminated composting material may decrease the amount of aerosolized particles and dust produced during the composting process. The in-vessel composter used was very well insulated for heat retention during the heating phase of the experiment. The sealed composter may have acted as an enclosure during the experiment and prevented the release C. gattii into ambient air. However, air samples were also taken during site visits when the composter was opened and inspected; no positive samples were found amongst the site inspection samples. Finally, C. gattii cells used in the inocula were cultured in the laboratory and, based on the results of the pilot study, were likely larger in size compared to cells growing in the environment. Larger cell sizes may also make aerosolizaion less likely to occur.  75  Conclusion: C. gattii is unlikely to aerosolize from contaminated composting feedstock or material given the material is sufficiently moist (>50% moisture content).  5.4  Was C. gattii present in air when city workers in Parksville, BC  were chipping garden waste from residential homes in fall 2008? Extensive C. gattii environmental sampling conducted on Vancouver Island consistently reported Parksville and surrounding areas to be “hotspots” for colonization (8). C. gattii has been found in air, tree-swab and soil samples in many locations in Parksville, particularly in coastal forested areas in southeastern parts of the city (8). The widespread colonization of C. gattii in the city is clearly a factor that would influence work exposure to the pathogen, particularly in arborists and other outdoor workers working with plants and soil. This study was the first to measure worker C. gattii exposure while treating residential yard waste. Occupational exposure to C. gattii for arborists and other outdoor city workers was investigated by only one other study (44). C. gattii was found in 36 out of 283 (12.7%) occupational monitoring samples for arborists performing limbing, chain sawing, and chipping tasks in parks and other public green areas in Parksville (44). However, the previous study did not specifically investigate exposure during the processing of residential green waste. C. gattii was not found in any air nor bulk wood chip samples collected while city workers were chipping residential yard waste in Parksville in October 2008. Because air sampling was conducted for every residential address where yard waste was processed and the duration of sampling covered the entire wood waste chipping period, missing truly positive C. gattii samples was unlikely. A number of factors may be associated with the absence of C. gattii in residential waste samples. Compared to natural forests and parks, home gardens provide a very different environment for C. gattii colonization. The amount of sun exposure, moisture, organic matter content and pH in soil in gardens may be very different compared to those found in soil in nearby natural areas. It has been suggested that C. gattii prefers soil that is more dry and with less organic matter content. Because gardens are usually watered and have soil which is richer in organic matter, it may be more difficult for C. gattii to establish colonization in these environments. 76  Even within a well recognized hotspot for C. gattii colonization such as the Parksville, the presence and concentrations of environmental C. gattii display a high degree of spatial and seasonal variability. In the Cryptococcus Environmental Sampling Database mentioned in Section 5.1, 51.6% of all environmental samples collected from Parksville from 2001 to 2010 were positive for C. gattii. However, environmental sampling was not performed using a randomized design method. Rather, sampling was often performed by either targeting previously known positive sites or focusing on areas around the residences of human and animal cases. Therefore, the true proportion of positive sites versus negative ones may be different than the proportion of positive versus negative samples reported by the database. The residential addresses where the yard waste originated were also located in areas of Parksville where few positive environmental C. gattii samples were found. Figure 14 shows locations where positive environmental samples had been found in Parksville, BC since 2000 (S. Mak, 2011, personal communication). The positive sites showed very little overlap when compared to the locations where air samples were taken during yard waste chipping for this study. An overlap of two maps is shown in Figure 15. Concentrations of C. gattii in the environment may be low in the areas sampled. Previous environmental sampling found that environmental C. gattii concentrations were usually higher in the summer and lower in other seasons. As the residential wood chipping occurred in the fall during cool, cloudy days, seasonal and meteorological conditions may have also affected the concentrations of C. gattii during the study period. Conclusion: C. gattii was not detected in air during the chipping of residential garden waste in Parksville BC in fall of 2008. Workers performing residential waste chipping in the sampled area in the fall are unlikely to be exposed to significant concentrations of C. gattii.  77  Figure 14. Locations where positive C. gattii had been found in Parksville BC from 2000 – 2011 2000 (S. Mak, 2011, personal communication).  78  Figure 15. Garden waste chipping locations overlaid above positive historical sampling locations in Parksville, BC.  79  5.5  Strengths and limitations  5.5.1 Strengths This thesis project was the first study to investigate C. gattii survival in composting systems. A custom-designed in-vessel composting simulator was used to study the survival of the organism during the composting process. In-vessel composting is regarded as the most efficient composting method for pathogen reduction and elimination. Based on the results of the composting indicator pathogen concentrations before and after the heating period, the composting simulator used in the current study was as efficient as full-scale industrial composting processes in terms of pathogen elimination. The custom composter also allowed live C. gattii cells to be used in the inoculums without contaminating any composting facilities nor the environment. Further, an experimental approach was employed to study C. gattii survival during the composting simulation. This study design allowed precise control over a number of composting factors such as composting material type, batch size, moisture content, C. gattii inoculation concentration, material mixing, and temperature and duration of the heating phase. Consistent composting parameters allowed for repeated trials to be conducted for the experiment to increase study power. This study is also unique in that environmental isolates of C. gattii were used in all pilot and core experiments. Due to either convenience or the lack of access to environmental isolates, many studies involving Cryptococcus species used strains isolated from clinical infections. Clinically isolated strains may have phenotypic properties that are different than wild type strains in the environment because the cells had to adapt to surviving in human or animal hosts. Differences between wild and clinical strains may become even more significant when clinical strains are cultured in the laboratory for multiple generations, which causes the organism to further change and adapt to the laboratory growth environment. Since this study aimed to investigate C. gattii survival in real-world settings, the use of wild environmental isolates increased the validity of the results.  80  5.5.2 Limitations For most in-vessel composting simulation trials, the length of the heating phase lasted less than the intended 72 hours due to variations in ambient temperature. Because later trials were conducted in the fall, ambient temperatures were significantly colder than when the first few trials were conducted. This had lead to difficulties in maintaining high composter temperature for the required length of time. Heating was originally intended to last exactly 72 hours to meet the minimum regulatory requirement for top grade compost. In future experiments, heating phase target length should be set at around 75 hours to ensure that the heating time meets the minimum regulatory requirement. Some samples from the composting experiments were inadvertently destroyed during analysis, resulting in the loss of thermo-tolerant coliform bacteria concentration results for the first three trials of the in-vessel composting experiment. There were also gaps in composting feedstock and product sampling performed in the composting facility in Comox, BC. The two gaps in November and March were 6 and 8 weeks long, respectively. In future studies, more communications should be made to the on-site staff collecting samples in order to remind them of the sampling schedule.  81  6  Conclusions and recommendations  Cryptococcus gattii has demonstrated the ability to survive through the in-vessel composting process and re-establish colonization in the composting material after the thermophilic phase. Due to sampling locations and seasonality of sampling period, air sampling during composting did not detect any positive samples. However, occupational exposure to C. gattii during the composting of contaminated waste may still occur in other locations and warmer weather. C. gattii that persist through the composting process may lead to additional occupational and general public exposure during the transport, handling and application of the compost product. Furthermore, C. gattii may migrate with compost products and spread to un-colonized areas. An important finding was that although C. gattii was susceptible to heat generated during the composting process, current provincial composting regulatory time-temperature requirements are unlikely to be adequate in completely eliminating C. gattii from contaminated composting material. Further studies may investigate more precisely the time-temperature conditions needed for complete inactivation of C. gattii inside contaminated composting material. Once the timetemperature criteria have been scientifically established and composting facilities are capable of meeting the inactivation criteria, composting may be used as an effective and efficient method of decontaminating plant waste with C. gattii. Future studies may also monitor other composting facilities on Vancouver Island for C. gattii in plant waste. Plant waste collected from areas with more positive environmental C. gattii samples, such as Parksville and Duncan, may be more likely to be contaminated with C. gattii and incorporated into municipal and commercial composting processes. Although no occupational exposure to C. gattii occurred during the chipping of residential yard waste, potential exposure to wood dust may have occurred. No quantitative measures were made for the concentration of wood dust or general particles in air, but air samples for C. gattii were heavily loaded with wood dust and other plant debris. 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Cryptococcus neoformans var. neoformans (serotype D) strains are more susceptible to heat than C. neoformans var. grubii (serotype A) strains. J Clin Microbiol 2001 Sep;39(9):3365-3367. (68) Franzot SP, Mukherjee J, Cherniak R, Chen LC, Hamdan JS, Casadevall A. Microevolution of a standard strain of Cryptococcus neoformans resulting in differences in virulence and other phenotypes. Infect Immun 1998 Jan;66(1):89-97.  88  Appendix A: Supplemental Figures  Figure 16. Concentrations of VGIIa cells in 1/16th strength MEB.  89  Figure 17. Concentrations of VGIIa cells in 1/32nd strengh MEB.  90  Figure 18. Concentrations of VGIIa cells in 1/64th strengh MEB.  91  Figure 19. Concentrations of VGIIa cells in SEB  92  Figure 20. Concentrations of VGIIb cells in 1/16th strengh MEB.  93  Figure 21. Concentrations of VGIIb cells in 1/32nd strength MEB.  94  Figure 22. Concentrations of VGIIb cells in 1/64th strength MEB.  95  Figure 23. Concentrations of VGIIb cells in SEB.  96  Figure 24. Concentrations of VGI cells in 1/16th strength MEB.  97  Figure 25. Concentrations of VGI cells in 1/32nd strengh MEB.  98  Figure 26. Concentrations of VGI cells in 1/64th MEB.  99  Figure 27. Concentrations of VGI cells in SEB.  100  Figure 28. Average temperature in composter and ambient temperature during heating period for Trial 1.  101  Figure 29. Average temperature in composter and ambient temperature during heating period for Trial 3.  102  Figure 30. Average temperature in composter and ambient temperature during heating period for Trial 4.  103  Figure 31. Average temperature in composter and ambient temperature during heating period for Trial 5.  104  Figure 32. Average temperature in composter and ambient temperature during heating period for Trial 6.  105  Figure 33. Average temperature in yard composting bin and ambient temperature for home composting Trial 1.  106  Figure 34. Average temperature in yard composting bin and ambient temperature for home composting Trial 2.  107  

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