Microsatellite genotyping of environmental Aspergillus fumigatus isolates Nicole

Microsatellite genotyping of environmental Aspergillus fumigatus isolates Nicole Abel The University of Tulsa

Introduction • J&M FARMS – Miami, OK (Ottawa Co. ) – Large outdoor compost facility – Commercial mushroom farm – Complaints about smell and perceived health effects

Concerning Statistics • 13. 4% of Ottawa County residents have asthma • Average asthma rate for rest of OK is 7. 2%

Compost & Bioaerosols • Several microorganisms break down the organic matter in compost to create a usable product • During composting process, compost is churned mechanically • Mechanical agitation cause these biological agents to become aerosolized – “bioaerosol” • Increasing concern about the potential impact of composting on human health

Aspergillus fumigatus • Fungus of the genus Aspergillus • Dominant bioaerosol from compost • Most common species in this genus to cause disease in individuals with a compromised immune system

Aspergillus fumigatus

Aspergillus fumigatus • Bioaerosol health concerns are focused on Aspergillus fumigatus for several reasons: – Grows abundantly in compost – Can survive at peak compost temperatures – Can survive temperature of the human body – Conidia are easily airborne – When inhaled, small enough to reach lung – Several well-known health effects

Health effects of AF • AF is known to cause a wide span of invasive diseases collectively called “Aspergillosis” • Traditionally viewed as weak pathogen, causing problems with asthma, allergies, and other diseases: – “Farmer’s lung” – Aspergilloma – Fungal sinusitis

Health effects of AF – “Farmer’s lung” • Inflammation of the lungs due to repeated exposure to the spores • Symptoms of acute farmer’s lung: – Chills, Cough, Fever, Shortness of breath • Symptoms of chronic farmers lung: – Breathlessness, Cough, Loss of appetite, weight loss • Often reverseable by avoiding additional exposure • Chronic form may lead to scarring of lung tissue

Health effects of AF – Aspergilloma • an overgrowth of the fungus on the surface of preexisting cavities in the lungs • This often occurs in patients treated successfully for tuberculosis • Often no symptoms, but most common symptom is coughing up blood (hemoptysis) • Most cases do not require treatment • With severe hemoptysis, surgery may be required to remove the aspergilloma and stop the bleeding

Health effects of AF – Fungal sinusitis • Inflammation of sinuses • Patients often have allergies or asthma • Thick fungal debris and sticky mucus that must be surgically removed in order to keep the inflammatory condition under control • At times this massive nasal polyposis and fungal debris can expand erode towards the eyes or brain.

Health effects of AF • The situation has become much more serious in recent years – increase in the number of immunosuppressed patients – degree of severity of modern immunosuppressive therapies, • Now most common airborne pathogenic fungus • Causes wide span of severe and usually fatal invasive infections in the immunocompromised

Aspergillosis • Extrinsic asthma- most common, occurs in allergic individuals, causes cough, wheezing, chills, aches, and pains • Extrinsic allergic alveolitis- can occur in those without allergies, but with repeated exposure to AF spores and many of the spores reach lung tissue, causes coughing, difficulty in breathing, and fever • Allergic bronchiopulminary aspergillosis- more serious form of “Farmer’s lung”, may follow entrinsic asthma, but symptoms are chronic and more severe, treatment is required or this disease can be fatal

Aspergillosis • Invasive Aspergillosis – – – Most dangerous and rare Occurs in the immunocompromised AF grows from the lung into other organs Often fatal Causes 30% of fungal infections in cancer patients Occurs in 10 -25% of patients being treated for leukemia. Even treated, the mortality rate for these patients is 8090% – Major cause of death at transplant and leukemia centers

Peoria Tribe/University of Tulsa Air Quality Studies in Miami, OK

Phase 1 • Previous study recorded elevated airborne A. fumigatus within 400 m of compost facility • A. fumigatus spores were significantly higher downwind of compost facility than control sites • Problem: Air samples not done in populated areas so not pertinant to public health

Present Study 2009 -2010 • Objectives: – Determine AF conc. in facility’s compost – Determine airborne AF conc. in Ottawa Co. vs. Tulsa Co. – Determine genetic relation between airborne AF in Ottawa Co. , AF in the facilty’s compost, and if distinct from Tulsa AF controls

Hypothesis • AF concentrations in facility’s compost will be high • Airborne AF concentration will be greater in Ottawa Co. vs. Tulsa Co. • AF from compost & Ottawa Co. air will be genetically identical • AF from Tulsa air will be genetically different from AF from Ottawa Co. air and compost isolates

Compost Windrows Miami, OK

Compost • Purchased 6 truckloads of spent compost from facility • 1 gallon bag from each truckload was brought to our lab at TU • 10 random samples obtained from each bag of compost • Samples dilution plated onto MEA containing Streptomycin in dilutions of 10 -1, 10 -2, and 10 -3 • Plated AF concentrations in the compost ranged from 0 -100, 000 CFU/g

Compost 1 Sample 2 10 -1 10 -2 840 CFU/g 10 -3

Compost 1 Sample 1 10 -2 100, 000 CFU/g 10 -3

Air Samples • Ottawa Co. - samples collected from 5 locations within 5 miles of facility (selected by the EPA) • Tulsa Co. - samples collected from 5 random locations in Tulsa (144 km upwind from compost site) to serve as control • Verified control sites do not use compost from this facility • Sampling done once a week with Anderson Single Stage Samplers onto MEA plus streptomycin plates for a total of 39 weeks

Ottawa Co. Air Sample Locations Commerce Picher North Miami Quapaw Miami Compost Facility/ Mushroom farm

Tulsa Air Sample Locations Kendall. Whittier Mohawk Park Woodward Park University of Tulsa campus Lakewood Gardens

Average AF for Each Sample Location -------Ottawa Co. -------Tulsa Co. -------

Average Airborne AF each Month Ottawa Co. vs. Tulsa Co. Number of times AF collected from air in 39 weeks: Ottawa Co. - 17 Tulsa Co. -8

Overall Average Airborne AF CFU/m 3 Ottawa Co. vs. Tulsa Co. P=0. 0238 (Statistically Significant)

Mushroom Side Project • Several samples of the compost had extremely high levels of AF • Commercial mushrooms are grown in the compost • Could their mushrooms have elevated AF on their surface when sold in grocery store?

Mushrooms • Purchased 7 brands of mushrooms (including J&M) • Compost from each brand was removed from surface of mushrooms • Samples diluted to 10 -1, 10 -2, and 10 -3 onto MEA plates with Streptomycin and incubated • AF colonies were identified and counted

J&M Grocery Mushrooms

Mushroom Side project #2 • J&M mushrooms have excessive AF on their surface • AF is hydrophobic and becomes airborne easily • If these mushrooms are washed in a sink, will they become airborne in the kitchen?

Mushroom Washing • J&M mushrooms were purchased and washed in a sink to mimic home use • Air samples collected next to the sink using an Anderson single stage sampler with MEA plates before, during, and after washing • This process was repeated with separate packages of mushrooms for a total of three trials

Airborne AF During Mushroom Washing

Subcultures • All plates from air samples, compost, and mushrooms were incubated at 45° C for 48 hours • All AF isolates were sub-cultured & grown in malt extract broth

Microsatellite • A microsatellite is a specific sequence of DNA bases which are repeated a certain # of times. For example: – GTGTGT---’GT’ X 6 – CTGCTG---’CTG’ X 4 – ACTCACTC---’ACTC’ X 4 • Microsastellites can differ in # of repeats & are hereditary, so # of repeats can determine relatedness • AKA – simple sequence repeats (SSR) – short tandem repeats (STR) – variable number tandem repeats (VNTR)

Microsatellite Applications • Forensics – Link a suspect with a sample of blood, semen or hair taken from a crime – Link a sample found on a suspect's clothing with a victim – Investigating paternity in order to establish rape or incest – Linking DNA samples with relatives of a missing person

Microsatellite Applications – Diagnosis & identification of human diseases • Change in length early in development of some cancers • Early cancer detection • Polymorphic, so useful in locating genes responsible for various genetic disorders

Microsatellite Applications – Population studies • Variation of microsatellites in populations can help to make inferences about pop. – Structures – Differences – Genetic drift – Genetic bottlenecks – Date of a last common ancestor

Microsatellite Applications • Conservation biology • Detect sudden change in population, • Effects of population fragmentation • Interaction of different populations • Identification of new and incipient populations.

DNA extraction • The DNA was extracted from all isolates using EZNA kit • DNA concentrations extracted from each isolate were documented using the Nanodrop 8000

Identification and PCR • Isolates positively identified as AF using: – Microscopic analysis – DNA analysis of ITS gene • DNA extraction • PCR • Sequencing

Genotyping • 9 microsatellites were used to discriminate between the isolates of AF using the method of de Valk et al. (2005) – Genotyped using complete panel of 9 microsatellites (ABI 3130 xl Capillary Sequencer) – Genotypes analyzed – Strains and relatedness determined (Gene. Mapper v 4. 0)

AF STRAINS

Conclusions Compost • Aspergillus fumigatus concentration in J&M’s compost was extremely variable (0100, 000 CFU/g) • Bags #1 & #2 contains extremely high amounts of AF • Some J&M compost contains very high levels of AF and is mechanically agitated, it is possible this could cause elevated airborne AF in the surrounding area.

Conclusions Air Samples • Total airborne spore concentration significantly higher in Ottawa Co. vs. Tulsa Co. • P=0. 0238 • The sampling location with highest airborne conc. Is most often downwind from J&M

Conclusions Genetic Analysis • Genetic variation exists amongst the isolates; however: – 3 of 3 isolates from mushrooms match compost – 2 of 2 Isolates from air sampling during mushroom washing match compost – 12 of 19 isolates from Ottawa Co. air match compost – 30 of 48 isolates from compost match Ottawa Co. air samples – Tulsa isolates distinct from all J&M isolates

Acknowledgments • • • Dr. Estelle Levetin Dr. Mark Buchheim Dr. Ron Bonett Jacob Crowley Justin Downs and Brandon Bartley Funding for this study was provided by the Peoria Tribe of Indians through an • EPA grant

Thank you! Questions?