Identifying Amphibian Pathogens Using Quantitative PCRAlexis Gramera*, Jessica Krempp, and Gregory Ruthig, Ph. D.Department of Biology, North Central College, Naperville, IL

AbstractWater molds are a major source of mortality for amphibians. It is essential to understand the diseases that contribute to these amphibian declines and to develop methodology for the identification of amphibian pathogens. We designed techniques to identify the presence and abundance of pathogens in the environment. Pathogens were isolated from frog egg samples (either Lithobates catesbeianus or L. clamitans). The abundance of a pathogen was determined using real-time quantitative PCR analysis. The formation of this technique provides future researchers a less costly and more time efficient method for water mold identification.

Introduction• Amphibian populations have

declined drastically since 1989 (Wilkinson 2003).

• Water molds are amphibian pathogens that can live on many different host species (Ruthig and Provost-Javier 2012).

• It is difficult to identify pathogenic water mold species using morphology.

• A critical component of the study of water molds includes the detection of the pathogen on infected amphibians, as well as in the environment (Ruthig and DeRiddler 2012).

Methods• We collected frog egg masses from bodies of

water in Naperville, IL (A-B).• We isolated water molds from infected eggs

onto cornmeal agar (C-D).• We extracted and amplified the ITS regions of

the genomes of collected pathogens. • PCR products were purified and sequenced at

the University of Chicago.• Using MEGA6.06, we aligned our sequences

with known sequences from the database, Genbank to determine the taxonomic identity of our strains.

• We designed a qPCR probe that is species specific, that does not bind to other strains.

• We are now testing the ability of our qPCR probe to determine its efficacy. We want to determine if:1. The probe works on the strain for which it

was designed. 2. The probe works on other water mold

strains. A B

DC

Results

E

Acknowledgements• Thank you to Dr. Gregory Ruthig for his assistance and guidance throughout this project.• Thank you to North Central for making this project possible.• Thank you to Chris Boffa and Jacquelyn Pfaff for their laboratory assistance.• Thank you to Andrew DuBois, Joel DiBernardo, and Katy Reese for their contribution to the

Ruthig Lab.• Thank you to Dr. Jonathan Visick, Dr. Stephen Johnston, and Dr. Jennifer Sallee for their

guidance.

Leptolegnia sp. EM32A

Leptolegnia sp. NT

Leptolegnia sp. SP

Achlya papillosa

Achlya oligacantha

Achlya racemosa

Achlya colorata

Leptolegnia sp. SAP248

Achlya aquatica

Achlya primoachlya

Achlya americana

Achlya intricata

Achlya ambisexualis

Achlya heterosexualis

2014 Gramera RvWk Mating B ITS 1 (Leptolegnia sp.)

Saprolegnia semihypogyna

Achlya sp. O3EG1

Achlya sp. DG

Leptolegnia sp. CBS

Saprolegnia diclina

2014 Gramera RvWk Mating A ITS 1 (Saprolegnia sp.)

Saprolegnia salmonis

Saprolegnia hypogyna

Saprolegnia parasitica

2014 Gramera Field Saprolegnia ferax ITS 1

Saprolegnia oliviae

Saprolegnia ferax

Saprolegnia sp. AESB

Saprolegnia bulbosa

Saprolegnia anomalies

Saprolegnia longicaulis

Aphanomyces sp. NVA

Aspergillus tubingensis

Pythium sp. OAK

Pythium erinaceum

Pythium parvum

Pythium takayamanum

2014 Gramera RvWk Mating C ITS 1 (Pythium sp.)

2014 Gramera Field Phytophthora sp. ITS 1

Phytophthora brassicae

Phytophthora botryosa

Phytophthora tropicalis

Phytophthora riparia

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65

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27

40

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9

18

12

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55

7893

36

50

0.1

1. We collected three egg masses.2. We obtained DNA sequences from the

ITS region of fourteen water mold isolates (E).

3. We classified these isolates using Maximum Likelihood Phylogenetic analysis (F).

4. We designed a locked nucleic acid (LNA) primer/probe set for qPCR using Primer Express 3.0 for strain 2014 Gramera RvWk Mating A, a likely member of the genus Saprolegnia.

5. We are currently testing the LNA probe’s efficacy, with the goal that the probe will only amplify our desired strain (G).

FForward Primer

[TTGCTTGTGCTTCGGTACGA]Reverse Primer

[ATTTCGGCGAGGCTGTTG]LNA Probe

[TGGACATATATTGCTTTTTG](Bold letters indicate LNAs)

G

B

Arrow indicates samples

collected

Positive Amplifications

• Quantitative PCR (qPCR) is a molecular technique that allows researchers to identify the presence and abundance of a particular pathogen strain.