Passive Surveillance Method for Tick Species and Associated Tick-borne

Pathogens in Wisconsin Darby Murphy, DVM

University of Wisconsin-MadisonMaster of Public Health candidate

Outline Introduction/Background

Goals

Methods/Results

Discussion/Conclusions

Acknowledgments

Background Ehrlichiae and Anaplasma phagocytophilum are obligate

intracellular gram-negative bacteria

Transmission- infected tick vector

Human A. phagocytophilum- Ixodes scapularis- blacklegged tick

Ehrlichia chaffeensis and E. ewingii- Amblyomma americanum– “lone star tick”

Ehrlichia muris-like (EML)- vector not yet established

Veterinary- E. canis, A. platys- Rhipicephalus sanguineusE. ewingii, A. phagocytophilum, E. muris-like (?)

Background In humans:

A. phagocytophilum- human granulocytic anaplasmosis (HGA) Ehrlichia chaffeensis- human monocytic ehrlichiosis (HME) E. ewingii ehrlichiosis E. muris-like ehrlichiosis

Signs/Symptoms: fever, chills, headache, muscle pain, malaise, nausea/vomiting, +/- rash

Clinical Laboratory Findings: anemia, thrombocytopenia, leukopenia, elevated liver enzymes

Laboratory detection: PCR, peripheral blood smear, indirect immunofluorescence assay (IFA)

Treatment: doxycycline

WI incidence= 6.1 cases/100,0000

E. muris-like confirmed cases

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

Reported cases of novel E. muris-like (EML) infections in Wisconsin residents, 2009-2012 (n= 22)

Burnett

Kenosha

This map is based on the county of residence of cases and not by the county of exposure 

n=7

n=3

n=1n=3

n=2n=4

n=1

n=1

E. muris confirmed case in out of state resident(exposure likely occurred here)

Active vs. Passive Tick Surveillance

Active surveillance- involves collection of host-seeking ticks, capturing sentinel host animals/wildlife reservoirs, testing collected ticks and specimens from host animals, etc.

Passive surveillance- involves submission of ticks collected from humans, domestic animals or wildlife species from participating medical clinics, veterinary clinics or wildlife rehabilitation centers to detect presence of tick species in a location

Project Goals

Surveillance for geographic extent of Ehrlichia muris-like (EML) in Wisconsin

Surveillance for potential E. chaffeensis in ticks in Wisconsin

Surveillance in southeastern Wisconsin for A. phagocytophilum (survey for establishment of pathogen)

Surveillance for invasive (non-native) tick species not known to currently occur in Wisconsin e.g. Amblyomma americanum.

Surveillance for invasion of eastern Wisconsin by Ixodes scapularis

Project Methods Veterinary clinics, humane shelters and wildlife

rehabilitation centers contacted and provided with tick collection materials for passive tick collection

13 Veterinary Clinics, 17 humane shelters and 6 wildlife rehabilitation centers

Identification of specimens- species, life stage, sex, host species, other information (e.g. recent travel history)

DNA extraction and real-time PCR testing for pathogen identification on I. scapularis submitted ticks

Sequencing unusual amplicons from PCR

Post‐amplification displaying melting curve analyses using a real‐time PCR assay. From left to right: groEL PCR melting temperature peaks of E. ewingii, Ehrlichia sp. Wisconsin (EML) (red arrow), E. chaffeensis, and A. phagocytophilum. (Pritt et al. 2011)

Results Current total of 1778 tick

submissions identified

10 tick species – Ixodes scapularis Dermacentor variabilis Amblyomma americanum Rhipicephalus sanguineus D. albipictus Haemaphysalis

leporispalustris I. cookei I. dentatus I. sculptus I. texanus

From 19 species of mammals and 1 avian species

Still awaiting Fall 2012 submissions from some participating locations…

Veterinary Clinic(s)

Humane Society

Wildlife Rehab Center

No active participants

Douglas

Bayfield

Ashland

SawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

CrawfordRichland

Grant

LaFayette Green

DaneIowa

Columbia

Dodge

JeffersonWaukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

Wisconsin Tick Surveillance Participants 2011-2012

Burnett

Veterinary Clinic & Humane Society

All three

OtherKenosha

Humane Society & Wildlife Rehab

I. scapularis submissions

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

JeffersonWaukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

Ixodes scapularis on all species by county 2011-2012

Burnett

Kenosha

N= 647  

Awaiting submissions

Current total I. scapularis Tested

Total I. scapularis collected from animals tested= 225

Total tick specimens collected from dogs tested= 38

Total specimens collected from cats tested= 7

Total specimens collected from humans tested= 4

A. phagocytophilum PCR positiveI. scapularis ticks

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

Anaplasma phagocytophilum real-time PCR positive Ixodes scapularis 2011-2012

Burnett

Kenosha

0/6

2/6

7/448/43

0/4

0/4

0/3

0/2

Total:21/225

2/70

Would like to focus remaining testing on southeastern and eastern regions

2/42

0/1

E. ewingii/E. canis/? PCR positiveI. scapularis ticks

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

E. ewingii/E. canis /(??) real-time PCR positive Ixodes scapularis 2011-2012

Burnett

Kenosha

1/4

4/43

Total:16/225

4/44

0/6

0/4

0/60/2

0/3

6/700/1

1/42

Major Conclusions I. scapularis detected in eastern Wisconsin

Amblyomma americanum in southern Wisconsin-established?

Rhipicephalus sanguineus rare (via this method of surveillance)

Unexpected rates of E. ewingii/E. canis/? based on known abundance and distribution of recognized vectors for these pathogens

No Ehrlichia chaffeensis or E. muris-like detected via this method of surveillance thus far

Acknowledgements Susan Paskewitz, PhD- professor, UW-Madison Dept. of

Entomology

Diep Hoang-Johnson- vector-borne epidemiologist Wisconsin Division of Public Health

Christopher Olsen, DVM, PhD- Vice Provost for Teaching and Learning UW-Madison

Xia Lee, MS - UW-Madison Doctoral candidate

Gretchen Gantz- UW-Madison

Acknowlegements

Aves Alliance Wildlife Rehab Bay Beach Wildlife Sanctuary Bay Area Humane Society Cadott Animal Hospital Care Animal Clinic Chippewa Co. Humane Association Coulee Region Humane Society Crivitz Veterinary Clinic Dane County Humane Society Dunn Co. Humane Society Four Lakes Wildlife Center Green County Humane Society Humane Animal Welfare Society Humane Society of Barron Co. Humane Society of Burnett Co. Humane Society of Marathon Co. Humane Society of Jefferson Co. Lake Wissota Animal Hospital Lancaster Veterinary Clinic/Grant Co. Pound

Langlade County Humane Society Northwoods Wildlife Center Northwoods Veterinary Clinic Peesh Wildlife Rehab Oakwood Hills Animal Hospital Oconto Veterinary Center Rock County Humane Society Rusk County Animal Shelter Shorewood Animal Hospital Spooner Veterinary Clinic St. Croix Medical Center Town & Country Veterinary Clinic Vernon Co. Humane Society Washburn Co. Humane Society Westside Family Pet Clinic Wildlife In Need Center Wildwood Animal Hospital

Participating veterinary clinics, humane societies and wildlife rehabilitation clinics

Questions

R. sanguineus submissions

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

R. sanguineus on all species by county 2011-2012

Burnett

Kenosha

N= 1

A. americanum submissions

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood Portage

Juneau

Adams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

A. americanum on all species by county 2011-2012

Burnett

Kenosha

N= 1

Approximate distribution of Amblyomma americanum

Dark green= historical rangeLight green= areas of recent expansion and establishment

M.J. Yabsley / Veterinary Parasitology 167 (2010) 136–148

A. americanum adult(s)

A. americanum adult(s) and nymph(s)

Douglas

Bayfield

AshlandSawyerWashburn

Polk Barron Rusk

Price

Iron

Vilas

Oneida

TaylorChippewaSt. Croix

Pierce

Dunn

Pepin

Eau Claire

Buffalo

TrempealeauJackson

Monroe

Clark

Wood

Portage

JuneauAdams

Sauk

La Crosse

Vernon

Crawford

Richland

Grant

LaFayette Green

DaneIowa

Columbia Dodge

Jefferson Waukesha

Racine

Ozaukee

SheboyganFond du LacGreen Lake

Marquette

Waushara Winnebago

Calumet

Manitowoc??????

Kewaunee

Brown

OutagamieWaupaca

Shawano

MenomineeOconto

Langlade

Forest Marinette

Florence

Door

Milwaukee

Washington

Lincoln

Marathon

Wisconsin River Rock

Walworth

Wisconsin Amblyomma americanum records 2006-2012

Burnett

Kenosha

A. americanum submission from Tick Surveillance study

Amblyomma sp. nymph or larvae

A. americanum & Amblyomma sp.

n=1

n=1n=1

n=1

n=2

n=1

n=1

n=9n=2 n=2

n=2

n=1

n=1

A. americanum nymph(s)