Microsatellite genotyping of Microsatellite genotyping of environmental environmental Aspergillus Aspergillus

fumigatusfumigatus isolates isolates

Nicole AbelNicole Abel

The University of The University of TulsaTulsa

IntroductionIntroduction

• 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 can 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 fumigatusAspergillus fumigatus

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 and 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 80-90%

– 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 StudyPresent Study2009-20102009-2010

• 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

Compost Windrows

Miami, OK

CompostCompost• 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 2Compost 1 Sample 2

10-1 10-2 10-3

840 CFU/g

Compost 1 Sample 1Compost 1 Sample 1

10-1 10-2 10-3

100,000 CFU/g

Average AF Concentrations in Facility's Compost

13904

11904

154

1938

1632

660

0 5000 10000 15000

1

2

3

4

5

6

Co

mp

os

t B

ag

#

AF CFU/g

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 withAnderson Single Stage Samplers onto MEA plus streptomycin plates for a total of 39 weeks

Ottawa Co. Air Sample Locations

North MiamiNorth Miami

MiamiMiami

CommerceCommerce PicherPicher

QuapawQuapaw

Compost Compost Facility/ Facility/

Mushroom farmMushroom farm

Tulsa Air Sample LocationsTulsa Air Sample Locations

Mohawk Park

University of Tulsa campus

Lakewood Gardens

Kendall-Whittier

Woodward Park

-------Ottawa Co.------- -------Tulsa Co.-------

0

5

10

15

20

25A

vera

ge A

F C

FU/m

3

Sample Location

Average AF for Each Sample Location

Number of times AF collected from air in 39 weeks:Ottawa Co.- 17 Tulsa Co.-8

0

5

10

15

20

25

30

35

40

Avera

ge C

FU

/m3

Apr May Jun Jul Aug Sep Oct Nov Dec Jan

Month

Ottawa

Tulsa

Average Airborne AF each Month

Ottawa Co. vs. Tulsa Co.

0

1

2

3

4

5

6

7

8

Avera

ge C

FU

/m3

Ottawa Tulsa

P=0.0238

(Statistically Significant)

Overall Average Airborne AF CFU/m3 Ottawa Co. vs. Tulsa Co.

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

AF on Surface of 7 Brands of Grocery Store Mushrooms

16933

613 20

2400

0 40

8000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

AF

CFU

/g

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

0 0 0

35 35

70

0 0

35

0

10

20

30

40

50

60

70

AF

(C

FU

/m3 )

before during after

trial 1

trial 2

trial 3

SubculturesSubcultures• 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:– GTGTGTGTGTGT---’GT’ X 6– CTGCTGCTGCTG---’CTG’ X 4– ACTCACTCACTCACTC---’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 extractionDNA 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 3130xl Capillary Sequencer)

– Genotypes analyzed

– Strains and relatedness determined (GeneMapper v 4.0)

AF STRAINS

ConclusionsCompost

• Aspergillus fumigatus concentration in J&M’s compost was extremely variable (0-100,000 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.

ConclusionsAir 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

ConclusionsGenetic 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 BuchheimDr. Mark Buchheim• Dr. Ron Bonett Dr. Ron Bonett • Jacob Crowley Jacob Crowley • Justin Downs and Brandon Bartley Justin Downs and Brandon Bartley • Funding for this study was provided by the Funding for this study was provided by the

Peoria Tribe of Indians through an Peoria Tribe of Indians through an • EPA grantEPA grant

Thank you!Questions?