A Practical Approach to Aeroallergen Identification

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Workshop 1: A Practical Approach to Aeroallergen Identification

Estelle Levetin, PhD

How to Set Up a Sampling Station

Learning Objectives and Disclosure Information Upon completion of this workshop, participants

should be able to: Set up a sampling station to collect airborne pollen and fungal

spore

Recognize the most common types of pollen found in the atmosphere

Recognize the most common types of fungal spores found in the atmosphere

No conflicts to disclose

Aerobiological Sampling

Sampling plan or objective

Choosing samplers: Rotorod, Burkard Spore Trap, Lanzoni, Allergenco

One day head or 7-day head for Burkard or Lanzoni

Location

Preparing the samples

Slide Analysis and Identification

Data Analysis

ACAAI Annual MeetingNov. 7 - 11 2013, Baltimore

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Sampling Objective

Pollen only or both pollen and spores

Sampling frequency 7 days a week

5 days a week

3 days a week

Time commitment

Rotorod Samplers

Rotorod Samplers

Models most often used by allergists have retracting rods for intermittent operation

Standard is 10% sampling time

Head rotates at 2400 rpm

Leading edge of rod coated with grease

Pollen and spores impacted on greased surface

Efficient for pollen and spores >10 m


3

Rotorod Samplers

Older Model Rotorod Sampler

Rotorod Analysis

Collector rods placed in a special adapter for microscopic examination

Rods stained with Calberla’s pollen stain

Entire surface of each rod counted unless pollen/spore load very high (then a subset of the surface is analyzed)

Atmospheric concentrations determined


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Rotorod Calculations

C = N / VC is concentration, N is the total number of pollen or spores counted on both rods, V is the volume of air sampled by the rods

V = Rod area (m2) x D x x RPM x tRod area = width of rod (1.52 mm = 0.00152m) x length of the rod (23 mm = 0.023m) x 2 (both rods), D is the diameter of the Rotorod head (8.5 cm = 0.085m), RPM is 2400, t is minutes sampled per day

With a 5% sampling time (72 min) V = 3.226 m3

Concentration = N/3.226 m3

With a 10% sampling time (144 min) V = 6.452 m3

Concentration = N/6.452 m3

Hirst Spore Trap

Burkard Spore Trap

Lanzoni Spore Trap

Allergenco

Burkard and Lanzoni Spore Traps


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Allergenco – Samplair MK-3

Advantages of Burkard Spore Trap

High efficiency down to less than 5 m Allows for greater

accuracy for small fungal spores

Time discrimination Permits analysis for

diurnal rhythms

Permanent slides for future reference

Location

Roof of a building - ideal 3 to 6 stories above ground (30 to 60 ft)

Not close to overhanging vegetation

Air flow not obstructed by nearby buildings or other structural features


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Parapet around roof requires platform to elevate the orifice above the wall

Telescoping mast elevates sampler above local vegetation.

Burkard 7-day sampler head

Standard is the 7-day sampling head

Sampler drum mounted on 7-day clock

Drum moves by orifice at 2 mm per hr

Melenex tape mounted on drum and greased (Lubriseal, High Vacuum Grease, other)

Air is brought in at 10 l/min and impacts on greased Melenex tape

Drum changed each week

Seven Day Sampling Head


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Processing the 7-day drum

Melenex tape removed from drum Tape cut into seven 24 hour segments each 48 mm

long Segments mounted on microscope slides in 10%

gelvatol (polyvinyl alcohol) and dried Glycerin-jelly mounting medium added and a 50 mm

cover slip Mounting medium contains pollen stain - either basic

fuchsin or phenosafarin

Melenex tape on cutting board

One-day sampling head

Alternate head is the 24 hour head Standard glass microscope slide is greased

and placed on the head Alternatively Melenex tape can be fixed on the

slide and greased

Slide is changed daily, carrier realigned Mounting medium with stain and coverslip

are added


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24 hour sampling head

Outdoor air sample from Tulsa

Analysis

Microscopy - 400X for pollen; 1000X for fungal spores

Different methods of microscopic analysis are used to obtain Average daily concentration - Single longitudinal

traverse

Hourly or bihourly concentrations which can then be averaged to obtain a daily average - 12 transverse traverses


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The Single Longitudinal Traverse Method

The Twelve Transverse Traverse Method

Burkard Counting Methods

Comparison of methods

Single Longitudinal Traverse Quicker

Produces average daily concentration

Good for routine monitoring

3 or 4 longitudinal traverses can increase accuracy

12 Transverse Traverses Takes longer

Can determine diurnal rhythm of airborne allergens

All traverses can be averaged to determine average daily concentration

Conversion to Concentrations

Microscope counts are entered into a database such as Excel

Formulas added to convert counts into concentrations

Information needed Field diameter of objective lens - Variable Flow rate (10 liters/minute) and exposure time (normally 24

hrs) for a total volume of air sampled of 14.4 m3


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Calculating Concentrations for Single Longitudinal Traverse

C = Concentration - pollen grains/m3

N = number of pollen counted on traverse W = Width of entire sample - 14 mm F = field diameter of objective lens - 0.48 mm V = total volume of air sampled- 14.4 m3

C = N x W/F x 1/VC = N x 14mm/0.48mm x 1/14.4m3

C = N x 2.025

Cushing Daily Pollen concentrationsDate Cupress Ulmus Ambrosia Artemisia Cheno/AmCompositaCyperaceaPoaceae

1-Oct-01 0 0 181 4 4 34 0 132-Oct-01 0 0 170 8 2 42 0 173-Oct-01 0 2 284 13 6 48 0 274-Oct-01 0 0 269 2 6 21 0 365-Oct-01 6 6 231 48 8 19 0 86-Oct-01 0 0 19 0 0 19 0 27-Oct-01 0 0 57 4 2 4 0 138-Oct-01 0 0 164 0 8 27 0 179-Oct-01 0 0 189 0 0 6 2 8

10-Oct-01 2 0 80 8 6 2 0 811-Oct-01 2 0 27 2 2 2 0 612-Oct-01 4 0 50 4 0 17 0 213-Oct-01 19 0 29 2 6 21 0 1314-Oct-01 2 0 36 2 2 6 0 415-Oct-01 95 0 63 6 4 15 0 4

Example of an Excel Spreadsheet with 15 Days of Pollen Data

Identification

AAAAI and ACAAI Aeroallergen courses

Other aerobiology courses

Reference slides NAB/AAAAI Pollen Slide Library

Reference slides from local specimens

Consult a botanist at a local university

Identification Manuals


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Identification Manuals

Grant Smith. 2000. Sampling and Identifying Allergenic Pollens and Molds, AAAAI, Milwaukee

R.O. Kapp, How to Know Pollen and Spores - originally published in 1950s - new edition

Richard Weber. 1998. Pollen Identification Ann Allergy Asthma Immunol 80:141–7.

Lewis WH, Vinay P, Zenger VE. 1983. Airborne and Allergenic Pollen of North America. Johns Hopkins University Press, Baltimore, MD.

Aeroallergen Photo Library, Steve Kagan, http://allernet.net/ Lacey M and West J. 2006. The Air Spora: A manual for

catching and identifying airborne biological particles. Springer.

Essential Reference

Grant Smith’s Sampling and Identifying Allergenic Pollen and Molds

Sample Pages


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How the data can be used

Average daily concentrations can be graphed to look at the seasonal and yearly pollen levels

Develop regional pollen calendar

Data can be compared with patient symptoms, peak flow readings, office visits, emergency room visits

Prepare for peak seasons - staffing, etc


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Average Daily Pollen Concentration in the Tulsa Atmosphere - 2011

0

500

1000

1500

2000

2500

3000

3500

4000

J F M A M J J A S O N D

Po

llen

gra

ins/

cub

ic m

eter

of

air

J F M A M J J A S O N D

Airborne Ambrosia pollen in Tulsa Fall 1999

0

100

200

300

400

500

600

700

8/15 8/29 9/12 9/26 10/10 10/24

Polle

n g

rain

s/m

3

Multiple Years of Data

Data from several years can be averaged to produce a graph of the pollen season

Smoothing techniques such as 5 day running mean can be used to generate a smoother curve and better estimate of the typical peak period


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Airborne Ambrosia pollen in Tulsa: 20 year mean

0

100

200

300

400

500

600

15-A

ug

22-A

ug

29-A

ug

5-Sep

12-S

ep

19-S

ep

26-S

ep

3-Oct

10-O

ct

17-O

ct

24-O

ct

31-O

ct

Po

llen

gra

ins/

m3

Five day running mean of airborne Ambrosiapollen in Tulsa: 20 year mean

Peak on or about Sept 10

0

50

100

150

200

250

300

350

400

450

500

17-A

ug

24-A

ug

31-A

ug

7-Sep

14-S

ep

21-S

ep

28-S

ep

5-Oct

12-O

ct

19-O

ct

26-O

ct

Conclusion

Air sampling allows the allergist to get a first hand understanding of the local aeroallergens, their concentration, and season occurrence

Several years of sampling will allow for the development of a pollen calendar which can benefit the physician and his or her patients


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Introduction to the Fungi


should be able to: Set up a sampling station to collect airborne pollen and

fungal spore





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Fungi are eukaryotic organisms that are neither plant nor animal

Fungi include molds

Mushrooms


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Puffballs

Bracket Fungi

Fungi can be unicellular such as yeast


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Fungi usually have a thread like body made up of hyphae

Hyphae make up the mycelium

Hyphae also make up the structure of fruiting bodies such as mushrooms


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Fungal Life Styles

Fungi are absorptive heterotrophs

As absorptive heterotrophs they exist as Pathogens

Mutualistic symbionts

Saprobes

Common Human Pathogen


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Apple-Cedar Rust A Destructive Plant Pathogen

Lichens are symbiotic organisms composed of an alga and a fungus

The Majority of Fungi Are Saprobes


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Most of the common airborne fungi are saprobes naturally occurring on leaf surfaces, decaying plant material, or in soil

Fungi reproduce by spores

Spores can result from sexualor asexual reproduction

Mycelium

Sexual Spores

Mycelium

Asexual Spores


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Spore Release Mechanisms

PASSIVE: Frequently related to wind speed and turbulence – include members of the “Dry Air Spora” which peak in the afternoon

ACTIVE: Generally require moisture – common mechanism for ascospores and basidiospores Basidiospores most abundant in predawn hours

Ascospores most abundant during or following rain; however, a number of ascospores only require high humidity and are abundant in predawn hours

Sporangium Conidia on hyphae

Sporangiospore or just “spore”

Conidium

Sporangiophore Conidiophore

Types of Asexual Spores

Rhizopus Sporangium


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Chains of Conidia

Fungal Classification

Mycologist recognize 8 phyla in the Kingdom Fungi based phylogenetic analysis of DNA

Three of these phyla contain important allergens Zygomycota

Ascomycota

Basidiomycota

ZYGOMYCOTA -- Zygospores

ASCOMYCOTA -- Ascospores

BASIDIOMYCOTA -- Basidiospores

ASEXUAL (ANAMORPHIC) STAGE – Conidia


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Asexual /Anamorphic Fungi

Most are members of the Ascomycota with a small percent members of the Basidiomycota

Formerly called Deuteromycetes Imperfect Fungi (Fungi Imperfecti)

Also called Mitosporic Fungi Mold Spores


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Members of the Zygomycota produce asexual spores in a sporangium

The zygospore (zygosporangium) is the characteristic sexual structure of the Zygomycota

Characteristic sexual structure of the Ascomycota is the ascus containing 8 ascospores


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Ascomycota

Many members of the Ascomycota develop asci within a fruiting body

Asexual spores are called conidia

Fruiting bodies often called ascocarps

Ascocarps can be Flask-shaped

Cup-shaped

Other

Cup-shaped fruiting body

Morels are also cup-shaped fruiting body


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Asci with ascospores from a morel

Abundant Airborne Ascospores Following Rain

Airborne Ascospores Still in Group of 8


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Majority of Asexual Fungi Are Ascomycota

Basidiomycota The most conspicuous fungi in

the environment

Basidiospores are typically produced in a large fruiting body such as Mushrooms

Bracket Fungi

Puffballs

Characteristic spores are basidiospores and four basidiospores are produced externally on basidia


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Basidia line the pores of bracket fungi and the gills of mushrooms

Single basidium with 4 basidiospores

Basidiospores are small and single-celled often with an asymmetric attachment peg


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Rusts and Smuts

Basidiomycota also includes two groups of fungi that lack fruiting bodies

Rust fungi and smut fungi

Important pathogens on both native and cultivated plants

Stem Rust of Wheat

Loose Smut of Wheat


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Common Fungal Spores

Estelle Levetin, PhD


should be able to: Set up a sampling station to collect airborne pollen and fungal

spore





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Fungal Spore Characteristics

Spore size

Spore shape

Number of cells

Attachment Scars

Wall characteristics

Spore color

Spore Size, Shape, and Septation

SIZE: 2m to 100 mm SHAPE: Globose, elliptical, fusiform,

asymmetric, lemon-shaped, barrel-shaped, curved

SEPTATION: Non-septate (one cell), single septum, transverse septa, transverse and longitudinal septa, random septa, pseudoseptate

Other Characteristics

ATTACHMENTS: Attachment scars, attachment pegs

APPENDAGES WALL CHARACTERISTICS: Smooth,

granular, reticulate, spines, warts, wall thickness

COLOR: Hyaline (colorless) to deeply pigmented


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Globose Barrel-shaped Non-septate Random septa

Lemon-shaped Club-shaped Transverse septa Attachment scars

Elliptical Curved Transverse and Longitudinal septa

Ornaments: spines

Asymmetric and germ pore

Cylindrical Pseudoseptate Appendages

Spore color

Asexual Spores in the Ascomycota

Also known as Deuteromycetes, Fungi Imperfecti (imperfect fungi),

or Mitospores


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Asexual Spores

Typically the most abundant spores in the atmosphere

Asexual stage of ascomycetes

Conidia often formed on specialized hyphae called conidiophores

Look for attachment scars where the spores were attached to the conidiophore

Cladosporium

Note the septum

Cladosporium


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Several species of Cladosporium are common in the atmosphere

Note the prominent attachment scars on Cladosporium conidia

Alternaria


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Alternaria

Curvularia

Curvularia

Nigrospora

Drechslera


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Drechslera-type spores

Several genera of fungi have similar cylindrical spores Drechslera Bipolaris Exserohilum Helminthosporium

Drechslera-type spores

Pithomyces


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Note the colorless attachment at the base of Pithomyces spores

Epicoccum

Penicillium species

Produce distinctive conidiophores (spore bearing structures)

Spores are usually spherical to oval and form in chains


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Aspergillus species

Produce distinctive conidiophores (spore bearing structures)

Spores are usually spherical to oval and form in chains

Penicillium-Aspergillus type spores

Fusarium


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Botrytis

Oidium

Nigrospora

Nigrospora Culture Air Sample


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Periconia

Cercospora

Polythrincium Peronospora


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Stemphylium

Torula

Tetraploa


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Spegazzinia

Stachybotrys

Phylum Ascomycota: Sexual Stage


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Ascospores are produced in an ascus. Eight ascospores are found in each ascus without any attachment scars

Ascospores are sometimes found in groups of eight in air samples

Chaetomium ascospores


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Leptosphaeria ascospores

Pleosporaascospores

Diatrypella ascospores


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Sporomiella ascospores

Venturia ascospores

Many ascospores on a rainy day


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Phylum Basidiomycota

Basidiospore

Basidium

Ganoderma basidiospores


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Agrocybe - type

Coprinus

Other basidiospores that are easy to recognize

Coprinus and Ganoderma basidiospores

Psathyrella Psathyrella velutina

Russula

Stropharia


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Lycoperdon Calvatia

Pisolithus Scleroderma

Mixed Basidiospores

Other Spores in the Basidiomycota

Rusts and Smuts


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Rust spores: Puccinia

Puccinia uredospores Puccinia teliospores

Smut Spores

Smut Spores


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Other Fungal-like Spores

Myxomycetes – Slime Molds

Myxomycete (slime mold) spores

Other slime mold spores


Documents

A Practical Approach to Aeroallergen Identification