Biosurveillance Utilizing Engorged Ticks As Phlebotomists for Xenodiagnosis Charles Elliott Lewis Iowa State University

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2018 Biosurveillance utilizing engorged ticks as phlebotomists for xenodiagnosis Charles Elliott Lewis Iowa State University

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Biosurveillance utilizing engorged ticks as phlebotomists for xenodiagnosis

Charles Elliott Lewis

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Microbiology

Program of Study Committee: Julie Blanchong, Major Professor Lyric Bartholomay Bradley Blitvich

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure the thesis is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2018

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iv ABSTRACT ...... v CHAPTER 1. GENERAL INTRODUCTION ...... 1 Thesis Organization ...... 2 CHAPTER 2. LITERATURE REVIEW ...... 3 Introduction ...... 3 Antibodies: a Basic Review of the Vertebrate Immune Response ...... 3 Current Approaches to Sample Collection: Restrictions and Risks ...... 4 Ticks: Considerations as Phlebotomists for Xenodiagnosis ...... 7 cELISA: Simple Diagnostics for Vertebrate Antibody Detection ...... 9 LFA: a Reliable Pen-side and Field Immunoassay ...... 11 Conclusions and Objectives ...... 13 Figures...... 14 References ...... 16 CHAPTER 3. MAINTENANCE OF THE ARGASID TICK ORNITHODOROS TARTAKOVSKYI USING A MODIFIED ARTIFICIAL MEMBRANE FEEDING SYSTEM ... 22 Abstract ...... 22 Brief Communication ...... 23 Acknowledgments...... 27 Figures...... 29 Tables ...... 31 References ...... 32 CHAPTER 4. DETECTION OF ANTIBODIES IN ARGASID TICKS UTILIZING COMMERCIALLY AVAILABLE ENZYME-LINKED IMMUNOSORBENT ASSAYS ...... 34 Abstract ...... 34 Introduction ...... 35 Materials and Methods ...... 37 Results ...... 40 Discussion ...... 42 Acknowledgements ...... 44 iii

Tables ...... 46 Figures...... 51 References ...... 57 CHAPTER 5. GENERAL CONCLUSIONS AND AREAS OF FUTURE RESEARCH ...... 60 References ...... 63 APPENDIX: THEILERIA EQUI COMPETITIVE ELISA KIT WORKSHEET ...... 64

ACKNOWLEDGMENTS

I would like to thank my committee chair, Dr. Julie Blanchong, and my committee

members, Dr. Brad Blitvich and Dr. Lyric Bartholomay, for their guidance and support

throughout this research.

In addition, I would like to thank the United States Department of Agriculture National

Veterinary Services Laboratories for the Extended Graduate Training Program, which provided

financial support for my Master program work. I would also like to thank my friends and

colleagues at the USDA, including Dr. Kim Dodd for motivating me to get this project

completed, Josh Thompson for assistance with the laboratory work, and Dr. Monica Reising for

assistance with the statistical analysis and graphics, and the faculty and staff in the

Interdepartmental Microbiology program for making my time at Iowa State University a wonderful experience. Finally, I would also like to thank my family and friends for their encouragement throughout the program.

ABSTRACT

Emerging infectious diseases pose a global threat to both human and animal health. The majority of novel zoonotic pathogens occur along the interface between humans, domestic animals, and wildlife creating complex ecological interactions that can be intricate and difficult to decipher. The availability of a diverse array of methods and techniques of sampling and diagnosing these diseases expands the effectiveness of the surveillance programs needed to enact countermeasures to counteract their effects. The purpose of the research reported here is to investigate the use of ticks as biological samplers for the purpose of antibody-based xenosurveillance. We show that antibody diagnostic techniques can be applied to tick blood meal samples and discuss the implications of this technology to understanding host immune status. In order to complete this work, we t also evaluated and expanded the use of membrane feeding in laboratory-derived tick colonies, and describe the use of a novel feeding apparatus for Argasid

tick species which will expand the techniques available for use in arthropod laboratory research.

When combined with other techniques, determining host antibody presence in tick blood meals

could prove useful as part of a passive surveillance toolkit. This study confirms that ticks can

function as bioaccumulators of host animal antibodies and provides a proof of concept that this

information can be translated into a component of a serosurveillance program.

CHAPTER 1. GENERAL INTRODUCTION

The diverse ecology of emerging infectious diseases warrants novel methods for searching for clues to elucidate pathogen-host dynamics, pathogen-environment dynamics, species spillover, and drivers of emergence and re-emergence of pathogens. These investigations often are limited by the expense, difficulty and associated dangers of trapping and/or sampling from animals, like bats, birds, and small rodents, that can be difficult to impossible to obtain.

Remote or non-contact sampling methods are an opportunity to overcome some of these hurdles.

The use of hematophagous arthropods as efficient samplers of vertebrate blood offers an elegant alternative to traditional blood collection techniques.

The purpose of the research reported here is to investigate the use of ticks as a xenodiagnostic tool to take a biological sample from a host for the purpose of antibody-based surveillance. This thesis includes the following:

(i) A literature review conducted to describe the historical use of arthropods as

biological samplers and the laboratory methods utilized to evaluate the samples

they provide.

(ii) A description of the establishment of a tick colony and refinement of methods for

membrane feeding of laboratory-derived tick colonies including the description of

a novel feeding apparatus for Argasid tick species.

(iii) The application of commercially available antibody diagnostic techniques (lateral

flow and competitive enzyme-linked immunosorbent assays) to tick blood meal

samples and the information this provides in the context of both pathogen

surveillance and host immune status. 2

Thesis Organization

This thesis is organized into five chapters. Chapter one provides a general introduction to the rationale guiding this research. The literature review following the general introduction,

Chapter Two, describes the challenges associated with sampling animals for biosurveillance and details the accomplishments from other researchers’ use of hematophagous insects as the phlebotomist for xenodiagnosis. Chapter Three details the development of a novel membrane feeding apparatus for maintenance of argasid tick research colonies. Chapter Four details a proof-of-concept study applying two immunoassay platforms to test for the presence of blood meal antibodies in engorged ticks fed using the method described in chapter three. Chapter Five contains general conclusions of this research and suggestions for future research.

CHAPTER 2. LITERATURE REVIEW

Introduction

The threat posed by emerging infectious diseases to human and animal health is a global concern and the ecological interactions that facilitate pathogen spread and persistence are

exacerbated by the fact that the majority of these diseases have both wildlife and domestic

animal involvement, some species of which can be daunting to sample, and these factors are

impacting the ability to predict, prevent, and control these diseases. (Daszak et al., 2000;

Haydon et al., 2002; Joneset al. 2008). Studies focused on the ecology of infectious diseases rely

on robust surveillance, routinely based on serology (Gilbert et al., 2013). The benefit to this

approach is the ability to infer infection history based on detection of specific antibodies. The

measurement of antibody presence in a population has become an increasingly important tool in

disease surveillance and control programs, as well as disease ecology studies, as antibodies are

typically easier to detect and usually persist longer than the causative agent (Gilbert et al., 2013).

The information achieved through pathogen surveillance has greatly improved the understanding

of both human and veterinary pathogen ecology (Gilbert et al, 2013). The availability of effective

diagnostic assays has greatly expanded with advances in technology while methods for obtaining

the samples needed for the assays has stayed relatively stagnant. The many benefits of obtaining

surveillance data, however, are dependent on the safe and effective collection of the specimen on

which the diagnostic assay will be performed.

Antibodies: a Basic Review of the Vertebrate Immune Response

The vertebrate immune system has to be highly dynamic and diverse to meet its task of

protecting the individual from environmental agents that are foreign to the body (Batista and 4

Harwood, 2009; Kennedy, 2010). This system is divided into two major functional areas: the

innate and the adaptive (or acquired) immune responses (Kennedy, 2010). The innate immune response involves those defenses that an individual is born with and involves components that can be called upon at very short notice (Linde et al., 2008). The adaptive response, which the rest of this section will focus on, is present only in vertebrates and is acquired as the individual is exposed to foreign agents. Adaptive responses take days to weeks to initially develop and generation of pathogen-specific lymphocytes and memory cells is the hallmark of the response

(Kennedy, 2010; Murphy and Weaver, 2016).

Antibodies, also known as immunoglobulins (Ig), are glycoproteins produced by activated B cells that have differentiated into plasma cells as a component of the humoral response of acquired immunity in response to infection (Batista and Harwood, 2009; Kennedy,

2010; Murphy and Weaver, 2016). These are produced to bind and neutralize a pathogen and are highly specific to a molecule that is unique to that invader, an antigen. The antibody binds to the matched antigen and then signals other components of the immune system to respond.

Antibodies are produced in several varieties, isotypes, of which IgG is the most relevant to the topics discussed here. IgG provides the majority of antibody-bound immunity for microbes and plays a key role in the diagnostic assays to be described (Rajewsky, 1996; Murphy and Weaver,

2016). The immune response in ticks should not conflict with immunoassays designed to detect and measure vertebrate antibodies, including the enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA) used in Chapter 4 of this thesis.

Current Approaches to Sample Collection: Restrictions and Risks

Regardless of the diagnostic assay, test results and the usefulness of the data are directly related to the quality of the sample tested. One factor that affects sample quality is the technique 5 utilized to obtain the sample. Sampling campaigns can be complicated through unique target species biology and ecology, the scarcity of the target species, and the influence of human activity on that species (Witmer, 2005). Capture and sampling of fragile, den-dwelling, flying and nesting species can be difficult to impossible to perform. Not only is capture taxing and highly dependent on the experience and skill of the technician, it can be stressful and dangerous for the animal, including the risk of mortality (Stadler et al., 2011). Bleeding these animals by conventional methods is also not without risk and can result in hematomas, other trauma, or even the death of the sampled animal (Baer and McLean, 1972; Helversen et al., 1986; Smith et al.,

2010; Stadler et al., 2011).

Directly handling animals in field settings is also a risky endeavor for those conducting the study. There are both biotic risks, including bites and contact with pathogens, as well as abiotic risks like environmental exposure. Individual target species, like bats and birds, may require extensive specialized handling experience (Sikes et al., 2011). Another consideration for field capture and handling of animals can be the requirement of obtaining permits for associated work, which can be time consuming and require extensive negotiations. Logistical difficulties

(for example, small blood vessels) may also prevent investigators from obtaining an adequate sample (Voigt et al., 2004). Blood collection in unsedated, fractious animals may lead to decreased sample quality, for example suboptimal volume collection and hemolyzed red blood cells, especially in the presence of unconditioned, physical restraint. The presence of hemoglobin from hemolysis could lead to inconclusive serology results, depending on the assay utilized

(Setia et al., 2014; Hodgkinson et al., 2017). Regardless of the approach used, the potential for pain, distress, and suffering of animals being utilized for research purposes must be accounted for. This is required by Federal regulations in the United States, as well as professional 6 organizations like the American Society of Mammologists (Sikes et al., 2011). Therefore, it is imperative to evaluate less invasive methods that produce minimal and transient distress.

An Alternative Sample Collection Approach: Less Invasive Methods Utilizing

Hematophagous Insects

Hematophagous insects, those who feed on blood, have been used to mitigate the risks associated with traditional collection methods from animals both in the wild and in captive settings (Stadler et al., 2011). Numerous hurdles can be overcome by using hematophagous insects as biological samplers and use of these insects has been shown to be a promising method of minimally invasive blood sampling and a variety of insects have been employed as samplers of small animals (Helversen et al., 1986; Voigt et al., 2003; Voigt et al., 2004; Voigt et al., 2005;

Thomsen and Voigt, 2006; Voigt et al., 2006; Voigt et al., 2006; Arnold et al., 2008; Vos et al.,

2010; Bauch et al., 2013). These methods have expanded in scope since the first report from von

Helversen et al. (1986) wherein triatomine bugs were used to collect blood from bats for doubly- labelled water experiments and lymphocyte culture. Other reports have included the use of triatomine bugs for a variety of study objectives in numerous species, including serologic and endocrinologic studies in rabbits, mice, bats, and birds, as well as larger animals, including tapirs and elephants (Stadler et al., 2011).

In species where conventional blood-drawing is difficult, insects have also been applied to trapped individuals in lieu of needles in an effort to avoid hematomas (Voigt et al., 2004).

Mosquitos, ticks, sandflies, and blow flies have all been utilized for the purpose of xenosurveillance for a variety of pathogens including influenza virus, myxoma virus, and Lyme disease, as well as broad viral surveillance (Barbazan et al., 2008; Meiser and Schaub 2011; Ng 7 et al., 2011; Marques et al., 2014; Millán et al., 2014; Brugman et al., 2015; Walter et al., 2016;

Bitome-Essono et al., 2017; Fauver et al., 2018). Stadler et al (2011) published a review of the use of triatomine bugs, specifically Rhodinus, Triatoma, and Dipetalogastor species, as “living syringes” (Stadler et al., 2011). The “bug method” described by Arnold et al (2008) utilized

Dipetalogastor maximus in false eggs to sample nesting birds with no evidence of behavior change or nest abandonment. These researchers collected samples from common sterns (Sterna hirundo) using traiatomine bugs contained in false eggs with small windows to allow the bug to feed to compare corticosterone concentrations between this method and the same birds using conventional methods (trapping the individual and directly drawing blood). They found no significant differences between the corticosterone levels in sterns sampled via the two methods suggesting that the bug method is a viable alternative for sampling (Arnold et al., 2008). Based on these studies, it can be presumed that the use of hematophagous insects reduces the pain and distress associated with capture, handling, and traditional venipuncture collection of blood.

Ticks: Considerations as Phlebotomists for Xenodiagnosis

Utilizing a hematophagous feeding strategy, ticks are obligate blood-feeding ectoparasites with natural feeding behavior that makes them ideal candidate phlebotomists for indirect sampling purposes as they have evolved strategies that allow them to feed while causing minimal disruption to the animal supplying the blood meal (Nava et al., 2009; Mans 2011). Some tick species are promiscuous while most species have a preference for feeding on certain groups of wild animals, including some ticks that are extremely host-specific (Jongejan and Uilenberg

2004; Sonenshine and Roe, 2013). These preferences may be able to be exploited when using ticks for xenodiagnostics or xenosurveillance. As a case in point, Ixodes scapularis nymphs and 8

adults have proven utility in sampling hosts for infection with the causative agent of Lyme disease, Borrelia burgdorferi (Walter et al., 2016).

Along with mites, ticks encompass the arthropod subclass Acari, order Metastigmata. The order is subdivided into three families; Ixodidae encompassing the hard ticks, Argasidae encompassing the soft ticks, and Nuttallellidae with a lone tick species. The diversity of this order is expansive and includes eighteen genera with over 900 species (Black and Piesman,

1994; Crampton et al., 1996; Klompen et al., 1996; Barker and Murrell, 2004; Nava et al., 2009;

Burger et al., 2014). Ticks are widely distributed throughout the world, predominately in warm, humid climates. They are present on every continent, except Antarctica and there are at least 93

species identified in the continental United States (Personal Communication – United States

Department of Agriculture, National Veterinary Services Laboratories, National Tick

Surveillance Program). Of the multiple arthropod vector groups, ticks transmit the greatest

variety of pathogenic microbes, including viruses, protozoa, rickettsiae, and spirochetes. For this

reason, they are generally thought of as the most important vectors of disease affecting humans

and animals (Jongejan and Uilenberg, 2004).

The two main groups of ticks, the argasid and ixodid families, have life styles reflecting

their feeding strategies. The Argasidae live in close proximity to their preferred hosts, for

example in nests and dens, and those life stages that actively feed typically do so for short

periods of time (minutes to hours) before detaching to digest the meal (Jongejan and Uilenberg,

2004; Sonenshine and Roe, 2013). They attach quickly, feed quickly, and then detach from the

host to begin processing the blood meal. In comparison, Ixodidae encompasses tick species with

one-, two-, or three-host life stages reflecting the number of animals they attach to during their

life cycle (Jongejan and Uilenberg, 2004; Sonenshine and Roe, 2013). These species take blood 9 meals for extended periods of time when compared to soft tick species and engorgement can take days to weeks to feed to repletion (Sonenshine and Roe, 2013). The host species-specificity of some species and the promiscuous, multi-species feeding of others could be of great value in sampling schema for targeted collection of host blood and sera.

The use of ticks as samplers can have numerous benefits. If the target vertebrates must be handled, engorged ticks could be removed from the animal in place of conventional venipuncture blood collection. Collection of engorged ticks from dens, nests, or below roosts could reduce the need to directly handle target animals and, therefore, eliminate the stress of sample collection for both the handler and the target animal species.

cELISA: Simple Diagnostics for Vertebrate Antibody Detection

In Chapter 4 of this thesis, two immunoassays will be utilized to evaluate the detection of host antibodies in ticks: competitive enzyme-linked immunosorbent assay (cELISA) and lateral flow assay (LFA). There are 205 veterinary diagnostic kits licensed by the United States

Department of Agriculture’s Center for Veterinary Biologics with approximately half of those representing ELISAs (Personal Communication – United States Department of Agriculture,

Center for Veterinary Biologics). This type of assay was first described by two research teams in

1971 (Engvall and Perlmann, 1971; Van Weemen and Schuurs, 1971). The general principal behind the ELISA is an antigen-antibody reaction demonstrated through a color change through the use of an enzyme-linked conjugate and enzyme substrate. This color change not only identifies the presence of the molecule of interest, it can also indicate the relative concentration

(Hornbeck, 1991; Engvall, 2010; Aydin, 2015). ELISAs can be formatted to detect antigen using antibody or to detect antibody using antigen, but all forms of ELISA require at least one antibody 10

with specificity for a particular antigen. There are multiple detection strategies that can be

utilized in the immunoassay (Gan and Patel, 2013; Aydin, 2015). The chromogenic assay

detection method will be briefly discussed here due to its relevance to the competitive ELISA

(cELISA) that is utilized in Chapter four.

The chromogenic ELISA assay results in a color reaction that absorbs light in the visible

range. The complex formed by the binding of antigen and antibody is attached to a solid surface,

for instance a polystyrene microtiter plate, and antibody labeled with an enzyme is added

followed by the addition of a substrate that utilizes that enzyme to produce a color change. The

absorbance of the color change is measured as Optical Density (OD) and is directly proportional

to the amount of analyte being measured based on the Lambert-Beer Law (Wild, 2013). A

common enzyme utilized to label antibodies for ELISA is horseradish peroxidase (HRP) (Gan

and Patel, 2013).

The cELISA format involves the simultaneous addition of competing antibodies. The

central event of this assay is the competitive binding process between the sample antibody and

the antibody added as part of the assay (Gan and Patel, 2013; Aydin, 2015). The cELISA kit used

in Chapter 4 to detect antibodies to the intraerthyrocytic protozoan Theileria equi is a

competitive assay. In this test, the bottom of each well of a 96 well plate is coated with T. equi

antigen. The serum being tested is then added to the appropriate well and allowed to adsorb for a specified amount of time. The test wells are washed to remove debris and then a monoclonal antibody is added. If the serum sample being tested is negative, the primary monoclonal antibody

binds to the antigen coated to the bottom of the well. If the serum sample being tested has T. equi

antibodies, it will inhibit binding of the primary monoclonal antibody, which will then be washed

away. A secondary antibody that is labeled with HRP is added to bind to the primary monoclonal 11

antibody followed by adding the enzyme substrate. The combination of the HRP and its substrate

produces a subsequent color change (Figure 1). The OD of this change is measured by

spectrophotometry and then test wells are compared to the controls provided in the kit to

determine which meet the criteria to be deemed positive or negative. In competitive assays,

strong color development indicates little or no inhibitory effect on the binding of primary monoclonal antibody which, therefore, indicates the absence of antibody in the serum being tested. Weak color development indicates the inhibition of binding of the primary antibody to the antigen on the coated well, which indicates the presence of antibody in the test serum (Veterinary

Medical Research and Development, Catalog #274-2 kit insert, Pullman, WA, USA). The protocol produced to collect data during this study is provided in Appendix A. This form provides step-by-step details for this assay and was used to standardize each test performed for this project.

LFA: a Reliable Pen-side and Field Immunoassay

The lateral flow assay (LFA), when used as an immunoassay, is a paper-based format for the rapid detection (5-30 minutes) of antibody or antigen in complex mixtures. LFAs have been utilized to detect targets in a wide variety of samples, including whole blood, serum, saliva, feces, and urine (Pacifici et al., 2001; Magambo et al., 2014; Carrio et al., 2015; de Lourdes

Moreno et al., 2015; Schramm et al., 2015; Koczula and Gallotta, 2016). This format is regularly accepted by regulatory authorities and end users and is reported to be cost effective and easy to produce, as well as easy to use (Koczula and Gallotta, 2016). The compact size and stability, as well as not requiring advanced laboratory equipment, make this format ready to use patient-side at the point-of-care. These same features also make it a preferred field diagnostic. 12

The basic principle for a LFA-based immunoassay is based on the absorbent power of the materials utilized, predominately paper. As these assays are typically in a linear format, absorbent paper is placed at the terminal end of the test strip. A sample is added to the opposite end and is pulled through reaction stages by capillary action powered by the wicking nature of the paper. This movement pulls the sample through multiple reactions. The sample typically mixes with a conjugate and then a test line where the analyte of interest is captured (Figure 3)

(Koczula and Gallotta, 2016).

The Idexx Feline Immunodeficiency Virus (FIV)/Feline Leukemia Virus (FeLV) SNAP® assay was utilized in the study reported in Chapter 4 (Idexx, Part Number 92-0000657-00). This

LFA is a simultaneous test for the presence of antibodies specific to FIV and antigen specific to

FeLV in feline anticoagulated whole blood, serum, or plasma with results in ten minutes. The

SNAP® format uses bidirectional flow through a wash step to increase the opportunity for target analyte binding. Per the test kit insert, a positive result for FeLV is diagnostic for infection based on the presence of FeLV p27 antigen. Presence of the specific antibodies required for a positive result for FIV indicates that the animal has been exposed to FIV and may have active FIV infection. This assay is reported by the manufacturer to have a relative sensitivity of 93.5% (95%

CI) and a relative specificity of 100% (95% CI) for detection of FIV antibody. The relative sensitivity and specificity for FeLV antigen detection is 98.6% (95% CI) and 98.2% (95% CI), respectively (Idexx Laboratories, Inc., Product #FTIV1 kit insert, Westbrook, ME, USA).

The FIV/FeLV SNAP® assay is performed by adding three drops of sample to a tube followed by four drops of anti-FeLV/FIV antigen conjugated to horseradish peroxidase provided in the kit. After the sample is mixed, it is poured into the sample well on the device. The sample moves by capillary action from the well to the activation circle in 30-60 seconds at which time 13

the activator is snapped to release the test reagents. The test results are read in the device window ten minutes after activation (Figure 3a). The results are validated by the presence of a

chromogenic color change in the positive control spot location and the lack of a color change in

the negative control spot location. A positive sample is seen as a color change (blue dot) at the

FIV antibody and/or FeLV antigen sample spot locations (Figure 3b and 3c).

Conclusions and Objectives

Deciphering the intimate relationship between biotic and abiotic components involved in

the ecology of emerging disease can be a daunting task. The availability of a variety of tools at

the hands of researchers allows the complex interactions to be pieced together. The use of techniques, like that described in the forthcoming, can be used to gain insight into these complex systems. For the work reported in this thesis, soft ticks (Acari: Argasidae) were chosen because of their feeding strategy and lifestyle, as they feed quickly and adapt readily to membrane

feeding. Due to tick containment concerns, a modified transmembrane feeding apparatus was

developed and reported in Chapter 3 of this thesis. This feeding apparatus was utilized in a proof-of-concept to evaluate the detection of host antibodies by two immunoassay platforms,

LFA and cELISA, and is reported in Chapter 4.

Figures

Figure 1: The competitive Theileria equi enzyme-linked immunosorbent assay demonstrated the color change that results from the combination of horseradish peroxidase and substrate in the last step of the assay. In the competitive format, a stronger color change represents a more negative result. The green box represents a negative result and the red box represents a positive result.

Figure 2: The lateral flow assay is typically configured in a linear format. The sample is added to one end of the assay strip and is pulled by capillary action of the absorbent pad at the terminal end of the test. During the samples movement, it is pulled through several reactions, including interaction with a conjugate on the conjugate release pad and to the test line containing antigen to capture antibody in the sample (or antibody to capture sample antigen). This figure was modified from Koczula, 2016.

Figure 3: The Idexx Feline Immunodeficiency Virus and Feline Leukemia Virus SNAP® lateral flow assay. (a) Components of the assay and (b) interpretation of the results (per the product insert), and (c) an example of a positive FIV antibody result from Chapter 4.

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CHAPTER 3. MAINTENANCE OF THE ARGASID TICK ORNITHODOROS TARTAKOVSKYI USING A MODIFIED ARTIFICIAL MEMBRANE FEEDING SYSTEM

Modified from a short communication submitted to the Journal of Medical and Veterinary

Entomology

C. E. Lewis1, 2, L. C. Bartholomay3, and J. A. Blanchong2

1National Veterinary Services Laboratories, Plum Island Animal Disease Center, United States Department of Agriculture,

Greenport, NY, U.S.A., 2Department of Natural Resource Ecology and Management, Iowa State University, Ames, IA, U.S.A,

3Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, U.S.A,

Abstract

Development and maintenance of laboratory tick colonies provides reliable access to a

variety of tick species at multiple life stages. Advances in techniques for membrane feeding ticks

reduces the number of laboratory animals needed for colony maintenance. In this study,

modifications to the existing protocol for in vitro feeding of an Argasid species, Ornithodoros

tartakovskyi, were made. Adult O. tartakovskyi ticks, of both genders, were allowed to feed to

engorgement using a novel, transmembrane apparatus in a six-well laboratory plate format with

well-inserts of laboratory-grade, wax sealing film. Of the 193 ticks placed on the membrane,

89% (n=172) fed until engorgement and subsequently detached. The modified feeding method

described herein will aid in future laboratory tick-based research as it allows for increased

containment, ease of sorting, successful in vitro feeding, easy replacement of blood meals, and

reduction in the total volume of blood meal required.

Key words. Ornithodoros tartakovskyi, Argasid ticks, in vitro, artificial feeding, arthropod

biocontainment 23

Brief Communication

Techniques for laboratory propagation of hematophagous arthropods have been

established for more than a century (Hindle and Merriman, 1912). Since that time, these methods

have been utilized to study variables that would have been difficult, if not impossible, to elucidate without this work, including such areas as host-vector-pathogen interactions, vector

competence, and arthropod biology (Levin and Schumacher, 2016). These techniques have also

been utilized to develop tick tissue-derived cell lines and to improve testing of acaricides and

vaccines (Kröber and Guerin, 2007). Despite the benefit provided by laboratory tick colonies, the

long term establishment and maintenance of colonies is time-consuming, expensive, and

complicated by the use of laboratory animals as a blood meal source for the ticks (Costa-da-Silva

et al., 2014; Levin and Schumacher, 2016). The use of laboratory animals has come under

increased public scrutiny and alternatives must be investigated for reduction, replacement, and refinement of their use (Costa-da-Silva et al., 2014).

Artificial feeding techniques have been applied to both Ixodid and Argasid ticks, though they have been most successful in species of Argasidae, whose feeding requirements more readily adapt to a laboratory environment (Levin and Schumacher, 2016). A variety of in vitro methods have been utilized, including the use of capillary tubes and numerous membrane-based systems. Membrane materials used have included artificial skin, harvested mouse skin, and a variety of laboratory-grade film membranes (Schwan et al., 1991; Abbassy et al., 1994; Kröber and Guerin, 2007; Levin and Schumacher, 2016; Kim et al., 2017). Though these feeding systems are an improvement on the traditional direct-feeding animal methods, there is room for advancement, including the use standard materials found in most microbiology laboratories, improved reproducibility using the standard material, and ease of use in a biocontainment 24

environment. The objective of this study was to develop improved methodology to artificially

feed adult soft ticks to repletion across membranes.

We developed our method utilizing Ornithodoros tartakovskyi soft ticks (Ixodida:

Argasidae), a species geographically distributed across southwest Asia and the Middle East.

These ticks are indiscriminate feeders, known to parasitize reptiles, birds, and mammals, and

typically quest in burrows or nests (Piazak et al., 2000; Bondarenko, 2015). This tick species, a

known vector of Acanthocheilonema viteae, is routinely used as a model for human filariasis,

and is hypothesized to be associated with the ecology of numerous diseases, including

mammalian tick-borne flaviviruses, Yersinia pestis, and Borrelia species (Londoño, 1976;

Bilialov et al., 1988; Piazak et al., 2000; Turell, 2015).

Adult male and female O. tartakovskyi were acquired from a colony at the University of

Wisconsin – Oshkosh National Institutes of Health Filariasis Research Reagent Resource (FR3)

Center through the National Institutes of Health’s Biodefense and Emerging Infections Research

Resources Repository (www.beiresources.org; adult females, NR-48929; adult males, NR-

48930). This study utilized 193 adult ticks with 112 females (58%) and 81 males (42%). Ticks

were housed in a dark humidified insect incubator at 27oC ±2oC and 80% ±2% humidity.

For this study we expanded on modifications to a blood-feeding apparatus developed by

Abbassy et al (1994), Costa-de-Silva et al (2014), Kim et al (2017), Kröber and Guerin (2007),

and Schwan et al (1991). Our design utilizes six-well laboratory-grade, tissue culture-treated,

polysterene plates with 24 mm diameter inserts with 3.0 µm pore size, polycarbonate membranes

(Transwell®, product number 3414, Corning Inc., Kennebunk, ME, U.S.A.) that were modified by removing the manufactured membrane using a scalpel (figure 1, A). A piece of Parafilm-M® 25

(Bemis, Co., Inc., Oshkosh, WI, U.S.A.) was pulled thin and stretched over the insert, replacing

the pre-existing membrane and creating a “cup” to hold the ticks (figure 1, B).

Once prepared, the plate was placed in a glass dish with double-sided tape as a form of secondary containment around the edge of the dish. A small weight was placed on top of a plate lid, to prevent escape of the ticks. The dish was then placed on a heated laboratory rocker

(Product 4637, Thermo Fisher Scientific Co., Fair Lawn, NJ, U.S.A.) to maintain the temperature at 37.0oC and to ensure movement of the blood meal. A reusable temperature monitoring device

was fabricated by placing a digital thermometer probe through the lid of a 35 mm x 10 mm petri

dish (Product: FB0875711YZ, Thermo Fisher Scientific Co., Fair Lawn, NJ, U.S.A) filled with

water as a substitute for blood, and sealing the dish with glue to prevent evaporation (figure 1,

C). Rocker-specific settings were determined to maintain a blood meal temperature of 37oC using the temperature monitoring device and set to ensure mixing and movement of the blood during feeding.

Five to ten adult ticks were placed into the cup created by the modified insert and membrane. This cup was then placed into a plate well containing blood (figure 1, D). Two

milliliters of blood per well was determined to adequately cover the membrane while giving

enough volume to support adequate movement and mixing of the blood meal when on the

laboratory rocker.

Ticks were fed during their second or third adult feeding on either rabbit-derived or

horse-derived, mechanically defibrinated whole blood acquired through HemoStat Laboratories

(product DRB030 and DHB250 respectively, Dixson, California, U.S.A.). Adult female ticks

were fed separately from adult males. Ticks were allowed to feed until self-detachment. Once detached, ticks were sorted into containers for housing and further use. Individual tick feeding 26

times ranged from ten minutes to one hour to complete. Ticks were visually monitored to prevent

them from feeding on each other. The feeding inserts were disinfected with 0.1 N sodium

hydroxide for repeat use. The six-well plates and the Parafilm-M® membranes were not reused.

A total of 193 adult O. tartakovskyi ticks were fed on the modified feeding apparatus.

Eighty-nine percent (n=172) of those attached to the membrane and successfully fed (Table 1).

Eleven percent (n=21) did not engorge. A total of 112 adult females were allowed to feed. One hundred three attached to the membrane and successfully fed (92% of females), nine did not (8% of females). A total of 81 adult males were allowed to feed. Sixty-nine (85% of males) attached to the membrane and successfully fed, 12 (15% of males) did not.

In vitro feeding systems, such as the one described here, provide numerous advantages when compared to the feeding of ticks on live animals. As there are currently no alternatives to the use of blood, these systems should at least reduce the number of animals used to maintain tick colonies. Our method further limits the volume of blood meal required to feed ticks, as well as incorporating a method that allows for the conservation and reuse of the material through exchange of inserts and membranes. Future work investigating non-animal derived synthetic blood meals would further the initiative to replace animals in this area of scientific research. The inserts and membrane allow for attachment of ticks to be visualized and this, we propose, should allow for easier evaluation of methods to prevent tick attachment by direct visualization of the structures utilized to access the host skin and the lesions they create for blood uptake (figure 2, D and E). This feeding system could also be applied in vector competence studies. By control of the blood meal, the infection rates of ticks should prove more efficient and, therefore, this efficacy should be extended to the rate of infection of animals the ticks are used to inoculate. 27

Effective in vitro systems and feeding techniques allow the study of vector-pathogen

interactions under controlled circumstances (Butler et al., 1984; Krull et al., 2017). Methods to

easily manipulate blood meals, as reported here, will facilitate the ability to formulate meals by

spiking with virus, manipulating serum components, or adding infected erythrocytes. This

system utilizes a small volume of blood, two milliliters, compared to four to five milliliters

required in previous feeding methods for O. tartakovskyi (Oskkosh, 2014). Experiments to

evaluate the probability of successful feeding of nymph and larva stages or of other Argasid

species, remain to be performed. Further evaluation utilizing Ixodid ticks and additional

experimental parameters described by Kröber and Guerin (2007) and Krull et al. (2017) should

be incorporated, such as incorporation of glucose or tick pheromones as components of the blood

meal, controlled exposure to CO2, and incorporation of animal hair on the membrane

Effective and efficient use of vertebrate animals in research is paramount, as is the

continued availability of laboratory-derived ticks for research. We report on a method of feeding that adds to the repertoire of available techniques to limit the use of animals in the maintenance of tick colonies, as well as propose its use as a means to further future tick-based research.

Acknowledgments

This project was supported by the U. S. Department of Agriculture’s National Veterinary

Services Laboratories (NVSL) Extended Graduate Training Program. The authors would like to

acknowledge Steven Schaar at the University of Wisconsin, Madison for his assistance and consultation in establishing the tick colony, Dr. Monica Reising at the USDA Center for

Veterinary Biologics for her review of data and statistics, and Dr. Michael Puckette with

Department of Homeland Security for his review of the manuscript. We would also like to 28

extend our thanks to Joshua Thompson at the NVSL for his technical assistance throughout the project. The following reagents were provided by the NIH/NIAID Filariasis Research Reagent

Resource Center for distribution by BEI Resources, NIAID, NIH: Uninfected Adult Female

Ornithodoros tartakovskyi (Live), NR-48929 and Uninfected Adult Male Ornithodoros

tartakovskyi (live), NR-48930.

Figures

A B C

Figure 1: Tick feeding apparatus. (A) Plate insert with the original membrane attached. (B) Original membrane removed and replaced by Parafilm M®. (C) The temperature monitoring deviceD used to estimate the temperature of the blood while on the heated rocker. (D) Complete feeding apparatus (lid and weight removed) with feeding Ornithodoros tartakovskyi ticks showing differing levels of engorgement.

Figure 2: Images of Ornithodoros tartakovskyi ticks feeding on the membrane feeding apparatus. (A) adult females displaying varying degrees of engorgement, (B) unfed adult females, (C) adult females with varying degrees of engorgement, (D and E) reverse (blood meal) side of the membrane with an adult female tick attached.

Tables (Max 2) B C

D E 31

Tables

Table 1: Cross classification of Ornithodoros tartakovskyi ticks by sex and feeding status. Percentages are presented relative to the total number of ticks fed.

Feeding Status Fed Unfed Total Female 103 (53%) 9 (5%) 112 (58%) Male 69 (36%) 12 (6%) 81 (42%) Total 172 (89%) 21 (11%) 193

References

Abbassy, M. M., K. J. Stein and M. Osman (1994). New artificial feeding technique for experimental infection of Argas ticks (Acari: Argasidae). Journal of Medical Entomology 31(2): 202-205.

Bondarenko, D. (2015). Relations of Central Asian Tortoise (Agrionemys horsfieldii) parasites in nature. Zoologichesky Zhurnal 94(7): 801-815.

Butler, J., W. Hess, R. Endris and K. Holscher (1984). In vitro feeding of Ornithodoros ticks for rearing and assessment of disease transmission. Acarology VI/editors, DA Griffiths and CE Bowman.

Hindle, E. and G. Merriman (1912). The sensory perceptions of Argas persicus (Oken). Parasitology 5(3): 203-216.

Kröber, T. and P. M. Guerin (2007). An in vitro feeding assay to test acaricides for control of hard ticks. Pest Management Science 63(1): 17-22.

Krull, C., B. Böhme, P.-H. Clausen and A. M. Nijhof (2017). Optimization of an artificial tick feeding assay for Dermacentor reticulatus. Parasites and Vectors 10(1): 60.

Levin, M. L. and L. B. Schumacher (2016). Manual for maintenance of multi-host ixodid ticks in the laboratory. Experimental and Applied Acarology 70(3): 343-367.

Oskkosh, U. o. W. (2014). Tick Rearing Techniques Using Commercially Obtained Blood as Food Source. Filariasis Research Reagent Resource Center. Standard Operating Procedure.

CHAPTER 4. DETECTION OF ANTIBODIES IN ARGASID TICKS UTILIZING COMMERCIALLY AVAILABLE ENZYME-LINKED IMMUNOSORBENT ASSAYS

Modification from an original article to be submitted for publication.

C. E. Lewis1,2,, L. C. Bartholomay3, B. J. Blitvich4, and J. A. Blanchong2

1National Veterinary Services Laboratories, Plum Island Animal Disease Center, United States Department of Agriculture,

Greenport, NY, U.S.A., 2Department of Natural Resource Ecology and Management, Iowa State University, Ames, IA, U.S.A,

3Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, U.S.A,

4Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University,

Ames, IA, U.S.A

Abstract

Serological surveillance is commonly used to decipher the ecology and epidemiology of

emerging infectious diseases, and to evaluate the efficacy of programs designed to manage and

control diseases in animals. Previous studies have shown that hematophagous insects can be used

as biological samplers for the detection of pathogens and pathogen-specific antibodies. In this

study, the ability to detect antibodies to two distinct pathogens in engorged ticks was

demonstrated by lateral flow enzyme-linked immunosorbent assay (LFA) and competitive

enzyme-linked immunosorbent assay (cELISA). Adult Argasid ticks (Ornithodoros tartakovskyi;

Acari:Argasidae) were allowed to feed on blood containing antibodies to feline

immunodeficiency virus (FIV) or Theileria equi using a transmembrane method. Ticks were

homogenized and assayed for antibodies to FIV and T. equi using commercially available LFA

and cELISA kits, respectively. A total of 115 adult ticks were tested, 54by LFA and 65 by

cELISA. Of the 20 females fed FIV antibody-positive blood meals, 19 (95%) tested positive and

one (5%) tested negative. Of the six female ticks FIV antibody-negative blood meals, all six 35

(100%) tested negative. Adult male ticks fed FIV antibody-positive blood meals tested negative

when tested individually (two individuals) or in pairs (one pair). Adult male ticks fed FIV antibody-positive blood meal tested positive when pooled into groups of five (four groups of five were tested). Of the four male ticks fed FIV antibody-negative blood, all (100%) tested negative.

Of the 42 females fed T. equi antibody–positive blood meals, 32 tested positive (76.2%) and 10

tested negative (23.8%). Of the 15 females that were fed T. equi-negative blood meals, all 15

tested negative (100%). Engorged female and male ticks yielded 20-36 µl and no more than 4 µl

of sample, respectively for the serological analysis. The false-negative results for both males and females by LFA and cELISA is hypothesized to be due to low sample volume. The results of this study suggest that ticks are a viable option as biological samplers for indirect, non-invasive sampling of animals during serologic studies.

Introduction

The threat posed by emerging infectious diseases to human and animal health is a global concern and the ecological interactions that facilitate pathogen spread and persistence are exacerbated by the fact that most have both wildlife and domestic animal involvement (Daszak et al., 2000; Haydon et al., 2002; Jones et al., 2008). The detection of pathogen-specific antibodies in target species has become an increasingly important tool in disease surveillance and control programs, as well as disease ecology studies, because antibodies are often easier to detect and persist longer than the causative agent (Gilbert et al., 2013). Traditional collection methods often

involve the capture, direct handling, and bleeding of target animals. This approach has

limitations that can result from the sensitivity of the target species to disturbance and stress,

animal size, capture-shyness, and the requirement of technical experience for blood collections. 36

Additionally, many species of animals are small, fragile, or endangered. Bleeding animals by conventional methods is not without risk because it can result in hematomas and other trauma, or even death of the sampled animal (Baer and McLean, 1972; Helversen et al., 1986; Smith et al.,

2010; Stadler et al., 2011). Therefore, it is important that less invasive methods for serologic surveillance are evaluated.

The use of hematophagous insects has been shown to be a promising method of minimally invasive blood sampling and has employed a variety of insect species as samplers of blood from small animals for the purpose of xenodiagnostics (Helversen et al., 1986; Voigt et al.,

2003; Voigt et al., 2004; Voigt et al., 2005; Thomsen and Voigt, 2006; Voigt et al., 2006; Voigt et al., 2006; Arnold et al., 2008; Vos et al., 2010; Bauch et al., 2013). These methods have expanded in scope since the first report from von Helversen et al. (1986) where triatomine insects were used to collect blood from bats for doubly-labelled water experiments and lymphocyte culture. Other reports have included the use of triatomine bugs for serologic and endocrinologic studies in rabbits, mice, bats, and birds, as well as larger animals, including tapirs and elephants (Stadler et al., 2011). A method described by Arnold et al. (2008) utilized artificial bird eggs containing Dipetalogastor maximus were placed in bird clutches and successfully used to collect blood from nesting birds without any evidence of behavior change or nest abandonment. Rhodinus, Triatoma, and Dipetalogastor spp. have also been used to acquire blood from vertebrate animals (Stadler et al., 2011). Ticks have been utilized for human xenodiagnostics and this technique should be evaluated for expansion to animal pathogen surveillance.

Ticks (Ixodida) are obligate blood-feeding ectoparasites composed of two large families, the Ixodidae (hard ticks) and Argasidae (soft ticks), in addition to a third family, 37

Nuttalleliellidae, which consists of a single species (Nava et al., 2009). The natural feeding

behavior of ticks makes them ideal for xenosurveillance. Ticks have evolved strategies that allow

them to feed while causing minimal disruption to its vertebrate host which is ideal for indirect sampling purposes (Mans, 2011). The present study was designed as a proof-of-concept to examine the use of ticks as samplers for serologic purposes. A laboratory colony of the argasid tick, Ornithodoros tartakovskyi, was used as a model for other tick species and two immunoassay platforms, competitive and lateral flow assays, were used to test ticks for pathogen-specific antibodies that had been added to artificial blood meals.

Materials and Methods

Ticks and their maintenance. Adult O. tartakovskyi were acquired from an established colony at the University of Wisconsin – Oshkosh National Institutes of Health Filariasis Research Reagent

Resource (FR3) Center through the National Institutes of Health’s Biodefense and Emerging

Infections Research Resources Repository (www.beiresources.org; adult females, NR-48929; adult males, NR-48930). Ticks were housed in a dark humidified insect incubator at 27oC ±2oC and 80% ±2% humidity. O. tartakovskyi ticks were selected for these experiments due to their availability and ease of feeding on membranes.

FIV serum. Feline immunodeficiency virus (FIV) antibody positive serum was chosen due to the availability of serum and a reliable commercial diagnostic kit in a LFA format. The serum was acquired from the Cornell University Veterinary College Animal Health Diagnostic Center

(Ithaca, New York, USA). The serum was originally collected from a three year old, domestic shorthair cat that was naturally infected with FIV. 38

T. equi serum. Theileria equi antibody positive serum was chosen due to the availability of serum and a reliable commercial diagnostic kit in a cELISA format. The serum was obtained from an experimentally infected horse at the United States Department of Agriculture’s National

Veterinary Services Laboratories at the National Centers for Animal Health campus in Ames,

Iowa, USA.

Source of blood meal. For the LFA negative blood meal, rabbit-derived defibrinated whole blood was acquired through a commercial source. A manual packed cell volume (PCV) was calculated to be 37%. For the cELISA negative blood meal, horse-derived defibrinated whole blood was acquired through a commercial source. A manual packed cell volume (PCV) was calculated to be

40%. The FIV antibody positive blood meal was prepared by centrifuging 4.0 ml of the negative blood meal for 10 minutes at 2,000 x g. One ml of serum was removed from the centrifuged product and replaced with 0.5 ml of FIV positive serum. The tube was then gently mixed to resuspend the blood without rupturing the erythrocytes. The PCV of the final product was 34%.

The T. equi positive blood meal was prepared by centrifuging 4.0 ml of the negative blood meal for 10 minutes at 2,000 xg. One ml of serum was removed from the centrifuged product and replaced with 0.5 ml of T. equi positive serum. The tube was then gently mixed to resuspend the blood without rupturing the erythrocytes. The PCV of the final product was 32%.

Membrane feeding. Membrane feeding was conducted as previously described (Chapter 3).

Adult female ticks were fed separately from adult males and ticks were allowed to feed until they 39

detached on their own. Detached ticks were placed inside individual tubes and stored at -20oC until tested. Only fully engorged ticks were used for subsequent experiments.

Tick homogenizations. Ticks were homogenized individually without the addition of diluent using a tube and pestle system. For those samples to be tested by cELISA, homogenates were pulse centrifuged to pellet residual debris. Homogenate sample volumes were measured using a calibrated, single channel, manual pipette.

Lateral flow assay. Serum, blood and homogenized ticks were assayed for antibodies to FIV

(Idexx, Westbrook, Maine, USA). This assay has been developed for the detection of antibodies to FIV in addition to feline leukemia virus (FeLV) antigen. Serum and blood meals were tested following the manufacturer’s instructions. Homogenized were also tested per kit instructions with one exception: the four drops of conjugate were added directly to homogenates and not the sample tubes provided with the kit. Results were interpreted as described in Figure 1.

Competitive enzyme-linked immunosorbent assay. Serum, blood and homogenized ticks were assayed for antibodies to T. equi by competitive ELISA (Veterinary Medical Research and

Development, Pullman, Washington, USA). Serum and blood meals were tested according to the manufacturer’s instructions. Tick samples were tested per kit instructions with one exception; the serum dilution buffer was added directly to the homogenates instead of being added to a dilution plate and transferred to the antigen-coated plate provided in the kit. A sample was considered positive if its optical density (OD) was greater than or equal to the OD of the positive control. 40

For the assay to be valid, the OD of the positive control was required to be greater than or equal

to 1.5 times the OD of the negative control.

Results

In this study, a total of 115 ticks were utilized during their second or third adult feeding.

Fifty ticks were utilized for the LFA (26 adult females, 24 adult males) and 65 ticks were utilized for the competitive ELISA (cELISA; 57 adult females, 8 adult males).

Detection of antibodies to FIV by LFA

Prior to use, the FIV serum utilized in making the FIV-positive blood meal was confirmed FIV positive and FeLV negative using the LFA. The negative blood meal was confirmed negative by the LFA. After the positive blood meal was created using the serum and negative rabbit blood, it was tested and confirmed positive (Table 1; Figure 2). A total of 26 adult female ticks were tested for antibodies to FIV by LFA (Figure 3). Twenty ticks had engorged on blood spiked with antibodies to FIV and six ticks had fed on non-spiked (negative control) blood. Of the 20 adult female ticks that fed on FIV antibody-positive blood, 19 (95%) tested positive (95%) and one tested negative (5%). The one positive-fed tick that tested negative contained the smallest (19µl) sample volume. The ticks that tested positive yielded an average sample volume of 27.6 µl (range 23-36 µl) (Table 2, Table 3, Figure 4). The six adult female ticks that had fed on non-spiked blood were negative for antibodies to FIV.

A total of 28 adult male ticks were tested individually or pools of two to five for antibodies to FIV (Table 4, Figure 5). Twenty-four ticks had engorged on blood spiked with antibodies to FIV and four ticks had fed on non-spiked (negative control) blood. The two male 41 ticks that fed on spiked blood were tested individually, and both tested negative. Two males that fed on spiked blood were pooled into a single sample and also tested negative (Figure 6). Four pooled samples of five ticks each were tested and all tested positive. The four male ticks that were negative blood meals tested negative (100%). As expected, all ticks were negative for

FeLV antigen.

Detection of antibodies to T. equi by cELISA

Prior to use, the T. equi positive serum utilized in making the T. equi-positive blood meal was confirmed positive using the cELISA. The negative blood meal was confirmed negative by the cELISA. After the positive blood meal was created using the serum and negative equine blood, it was tested and confirmed positive using the same assay. (Table 5). A total of 65 adult ticks were tested by cELISA, including 57 females. The 15 adult female ticks that were fed on the negative blood meal tested negative. Of the 42 adult female ticks that were fed on the T. equi- positive blood meals, 32 tested positive (76.2%) and ten tested negative (23.8%; Table 6). Of the positive-fed adult females that were tested, 28 had their sample volumes measured. The positive- fed female ticks that tested positive had an average sample volume of 24.7 µl (range 15-34 µl).

The positive-fed females that tested negative had an average sample volume of 8.9 µl (range 2-

19 µl; Table 7).

Accounting for sample volume, positive-fed adult females with a volume ranging from

20 µl to 34 µl tested positive by cELISA (n=14, Figure 7). Positive-fed adult females with sample volumes ranging from 2 µl to 13 µl tested negative (n=7). Positive-fed adult females with sample volumes greater than 13 µl, but less than 20 µl, had a mix of positive and negative results

(n=7). Of those ticks in the mixed range, negative results were obtained from samples with 42

volumes of 14 µl (n=1), 17 µl (n=1), and 19 µl (n=1). Positive results were obtained from

samples with volumes of 15 µl (n=2) and 18 µl (n=2).

Due to the results provided by male ticks tested by LFA, only a total of 8 adult males

were tested on the T. equi cELISA. The adult male ticks showed little variation in volume (all ≤4

µl). The male ticks that were fed on the T. equi positive blood meal all tested negative (Table 7).

Male ticks were not feed on negative blood meals and tested by cELISA.

Discussion

The successful collection of blood samples for large scale serosurveillance programs can

be a daunting task involving numerous challenges and risks. Here an alternative, less invasive

technique of blood collection was evaluated for use in the serologic survey of animals utilizing

ticks as biological samplers. In this proof-of-concept study, ticks were evaluated for their ability

to acquire antibodies to FIV and Theileria equi in spike blood meals. Results demonstrate that it

is possible to detect vertebrate host antibody presence in engorged ticks.

Previous studies have reported the successful uptake of intact antibodies and large

proteins during tick feeding, including across membranes (Ackerman et al., 1981; Minoura et al.,

1985; Ben-Yakir et al., 1986; Ben-Yakir et al., 1987; Chinzei and Minoura, 1987; Ben-Yakir,

1989; Rutti and Brossard, 1992; Wang and Nuttall, 1994; Jasinskas et al., 2000; Kim et al.,

2017). Studies focused on the feasibility of vaccination of cattle to internal tick antigens

performed by Jasinskas et al (2000) demonstrated that immunoglobulin-G remained intact

through the midgut and passage into the tick hemolymph with capillary feeding of Amblyomma americanum, an ixodid tick (Jasinskas et al., 2000). The same study showed that 70-80% of biological activity, measured as immunoglobulin G binding in an ELISA, was retained (Jasinskas 43 et al., 2000). Thus, the present study confirms previous findings that intact antibodies are transferred during feeding and revealed that these antibodies can be successfully detected by commercial LFA and cELISA.

One factor that we demonstrated affected the ability to detect antibodies was the size of the engorged tick due to its impact on the volume of the sample available for testing. Male O. tartakovskyi ticks are smaller than females (Figures 8 and 9) and single males did not provide a sufficient sample volume for reliable antibody detection when tested individually. This effect was confirmed by successful antibody detection once male ticks were pooled into a single, batched sample (Table 6). Female O. tartakovskyi ticks were usually large enough to be tested individually. Review of the sample volumes suggests a minimum volume of approximately 20

µL is necessary for reliable results (Table 7 and Figure 7). The one female tick that fed on FIV antibody-positive blood did not test positive by LFA. This tick yielded a smaller sample volume than the other females indicating that the volume was below this threshold, as were all the individual male ticks that were tested. Alterations to the commercial kit protocols were minimal and only involved reducing the transfer of sample between tubes to conserve the available sample volume. In this case, the whole tick was added directly to the kit sample dilution buffer and then homogenized with a tube pestle. This modification proved successful at minimizing the sample volume loss.

The study reported here was a proof-of-concept and was not designed to determine the accuracy, precision, or robustness of utilizing ticks as samplers. One limitation of this study was the small number of ticks utilized, therefore, there are numerous factors that will require further evaluation to accurately assess the usefulness of this technique One limitation of the study is that all ticks were processed within hours of self-detachment from the feeding membranes. 44

The processing of ticks several days or weeks after engorgement needs to be evaluated to

determine the effect of the digestive processes, as antibody could be prone to chemical

degeneration and enzymatic digestion via heme processing (Voigt et al., 2004; Vos et al., 2010).

This could lead to a decrease in antibody titers. The blood meal concentration through the water

secretion process utilized by numerous tick species could also lead to changes and may result in

inflated titers (Balashov, 1967). Sample volumes obtained for individual Ixodid ticks are likely

to be greater for both males and females overcoming one of the main challenges in this study

(Balashov, 1967; Coons et al., 1986). To further evaluate the applicability of this technique, the

same approach should be applied to other Argasid ticks, as well as expanded to include Ixodid

species. Ticks that have feed on live animals, instead of transmembrane feeding, should be

included in the approach, specifically vertebrate species likely to be included in surveillance

studies. This would verify that the results reported here are not biased towards artificial feeding.

Our results suggest that using ticks as biological samplers in a xenodiagnostic context

should be further validated as a viable alternative to directly collecting samples from vertebrates

in situations where animals are difficult to trap or their capture introduces undue stress. The

method described here warrants further investigation as it has the benefit of being minimally

invasive and not requiring direct contact with the target vertebrate species of interest.

Acknowledgements

This project was supported by the U. S. Department of Agriculture’s National Veterinary

Services Laboratories (NVSL) Extended Graduate Training Program. The authors would like to

acknowledge Steven Schaar at the University of Wisconsin, Madison for his assistance and consultation in establishing the tick colony and Dr. Monica Reising at the USDA Center for 45

Veterinary Biologics for her review of data and statistics. We would also like to extend out thanks to Joshua Thompson at the NVSL for his technical assistance throughout the project. The

following reagents were provided by the NIH/NIAID Filariasis Research Reagent Resource

Center for distribution by BEI Resources, NIAID, NIH: Uninfected Adult Female Ornithodoros tartakovskyi (Live), NR-48929 and Uninfected Adult Male Ornithodoros tartakovskyi (live),

NR-48930.

Tables

Table 1: Cross classification of serum status (positive or negative for antibodies) and lateral flow assay Feline Immunodeficiency Virus antibody test results of female Ornithodoros tartakovskyi ticks. A total of 26 female ticks were tested.

Serum Status Test Result Negative Positive Total Negative 6 1 7 Positive 0 19 19 Total 6 20 26

Table 2: Lateral flow assay results for adult female Ornithodoros tartakovskyi ticks (n=26).

Total Tested Total Tested Negative Negative 6 6 False Positive 0

Total Tested Total Tested Positive Positive 20 19 False Negative 1

Table 3: Approximate sample volume and test result for adult female Ornithodoros tartakovskyi ticks fed on Feline Immunodeficiency Virus positive blood meal and tested by lateral flow assay.

Approximate Sample Tick ID Volume (µl) Test Result 156 30 Positive 157 25 Positive 158 32 Positive 159 28 Positive 160 23 Positive 161 29 Positive 162 25 Positive 163 32 Positive 164 19 Negative 165 26 Positive 172 25 Positive 173 28 Positive 174 24 Positive 175 27 Positive 176 25 Positive 177 30 Positive 178 23 Positive 179 29 Positive 180 27 Positive 181 36 Positive

Table 4: Number of male Ornithodoros tartakovskyi tick lateral flow assay results classified by number of ticks pooled for the test (ticks in test) and serum status (positive or negative). A total of 24 male ticks were tested.

Ticks in Test Serum Status Test Result Count Individual Negative Negative 4 Positive Negative 2 Pool of 2 Positive Negative 1 Pool of 5 Positive Positive 4

Table 5: Results for the components utilized to fabricate the artificial blood meal fed to the Ornithodoros tartakovskyi ticks for competitive enzyme-linked immunosorbent assay evaluation.

Sample Test Result by cELISA T. equi Serum Positive Equine Blood Negative Spiked-blood meal Positive

Table 6: Cross classification of serum status (positive or negative for Theileria equi antibodies) and competitive enzyme-linked immunosorbent assay test results of adult Ornithodoros tartakovskyi ticks, including both male and female ticks.

Serum Status Test Result Negative Positive Total Negative 16 17 33 Positive 0 32 32 Total 16 49 65

Table 7: Tick number, serum status, test result, sex, and size (male vs female Ornithodoros tartakovskyi ticks) and sample volume for each tick tested for antibodies to Theileria equi by the competitive enzyme-linked immunosorbent assay. Sorted by sex and sample volume. Samples with conflicting results are highlighted in blue (ND, not determined). Sample Tick Serum Status Test Result Sex Volume (µl) 34 - - Female <2 40 + - Female <2 42 + - Female <2 60 + - Female <2 39 + - Female 5 58 + - Female 12 41 + - Female 13 62 + - Female 14 43 + + Female 15 51 + + Female 15 44 + - Female 17 49 + + Female 18 61 + + Female 18 52 + - Female 19 33 - - Female 20 48 + + Female 20 54 + + Female 20 56 + + Female 21 53 + + Female 24 65 + + Female 26 50 + + Female 27 46 + + Female 28 47 + + Female 28 55 + + Female 28 38 + - Female 3 36 - - Female 30 45 + + Female 30 64 + + Female 30 35 - - Female 31 57 + + Female 31 59 + + Female 31 50

Table 7 Continued Sample Tick Serum Status Test Result Sex Volume (µl) 37 - - Female 34 63 + + Female 34 1 - - Female ND 2 - - Female ND 3 - - Female ND 5 + + Female ND 6 + + Female ND 8 - - Female ND 11 - - Female ND 12 + + Female ND 13 + + Female ND 14 + + Female ND 15 + + Female ND 16 + + Female ND 17 + + Female ND 19 + + Female ND 20 + + Female ND 21 + + Female ND 22 + + Female ND 23 + + Female ND 24 - - Female ND 25 + + Female ND 26 + - Male <4 27 + - Male <4 28 + - Male <4 29 + - Male <4 30 + - Male <4 31 + - Male <4 32 + - Male <4 4 - - Male ND 7 - - Male ND 9 - - Male ND 10 - - Male ND 18 + - Male ND 51

Figures

Figure 1: Interpretation of the Idexx Feline Immunodeficiency Virus / Feline Leukemia Virus lateral flow assay (from the provided kit insert).

A B C

Figure 2: Lateral flow assay tests for blood components. (a) Feline Immunodeficiency Virus (FIV) positive serum, (b) FIV positive blood meal, and (c) negative rabbit blood.

Figure 3: Lateral flow assay results for negative-fed adult female Ornithodoros tartakovskyi ticks.

Figure 4: Lateral flow assay results for positive-fed adult female Ornithodoros tartakovskyi ticks (All are positive, except for Tick 164).

Figure 5: Lateral flow assay results for positive-fed adult male Ornithodoros tartakovskyi ticks.

Figure 6: Lateral flow assay results for negative-fed adult male Ornithodoros tartakovskyi ticks.

Figure 7: Sample volume (in µL) plotted for the competitive enzyme-linked immunosorbent assay positive and negative samples. Color distinguishes between male and female Ornithodoros tartakovskyi ticks. All ticks were fed antibody positive blood meals. All points have been skewed horizontally so all observations could be visualized.

A B C

Figure 8: Variation of sizes of adult female Ornithodoros tartakovskyi ticks. This image shows levels of engorgement. Sample volumes collected from the ticks in the image: (A) 2 µl, (B) 21 µl, (C) 30 µl.

A B

Figure 9: Fully engorged adult Ornithodoros tartakovskyi ticks. Engorged male (left) and engorged female (right). Sample volumes collected from the ticks in the image: (A) 2 µl and (B) 27 µl.

References

Baer, G. M. and R. G. McLean (1972). A new method of bleeding small and infant bats. Journal of Mammalogy 53(1): 231-232.

Balashov, Y. S. (1967). Blood-sucking ticks (Ixodoidea)-vectors of diseases of man and animals. Blood-sucking ticks (Ixodoidea)-vectors of diseases of man and animals.

Ben-Yakir, D. (1989). Quantitative studies of host immunoglobulin G in the hemolymph of ticks (Acari). Journal of Medical Entomology 26(4): 243-246.

Ben-Yakir, D., C. Fox, J. Homer and R. Barker (1987). Quantification of host immunoglobulin in the hemolymph of ticks. The Journal of Parasitology 73(3): 669-671.

CHINZEI, Y. and H. MINOURA (1987). Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata. Medical and Veterinary Entomology 1(4): 409- 416.

Coons, L. B., R. Rosell-Davis and B. I. Tarnowski (1986). Bloodmeal digestion in ticks. Morphology, physiology, and behavioral biology of ticks/editors, John R. Sauer and J. Alexander Hair.

Daszak, P., A. A. Cunningham and A. D. Hyatt (2000). Emerging infectious diseases of wildlife- -threats to biodiversity and human health. Science 287(5452): 443-449.

Haydon, D. T., S. Cleaveland, L. H. Taylor and M. K. Laurenson (2002). Identifying reservoirs of infection: a conceptual and practical challenge. Emerging Infectious Diseases 8(12): 1468- 1473.

Helversen, O. v., M. Volleth and J. Nunez (1986). A new method for obtaining blood from a small mammal without injuring the animal: use of Triatomid bugs. Experientia 42(7): 809-810.

Jasinskas, A., D. C. Jaworski and A. G. Barbour (2000). Amblyomma americanum: specific uptake of immunoglobulins into tick hemolymph during feeding. Experimental Parasitology 96(4): 213-221.

Jones, K. E., N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman and P. Daszak (2008). Global trends in emerging infectious diseases. Nature 451(7181): 990.

Mans, B. J. (2011). Evolution of vertebrate hemostatic and inflammatory control mechanisms in blood-feeding arthropods." Journal of Innate Immunity 3(1): 41-51.

Minoura, H., Y. Chinzei and S. Kitamura (1985). Ornithodoros moubata: host immunoglobulin G in tick hemolymph. Experimental Parasitology 60(3): 355-363.

Nava, S., A. A. Guglielmone and A. J. Mangold (2009). An overview of systematics and evolution of ticks. Frontiers in Bioscience 14(8): 2857-2877.

Smith, C. S., C. E. De Jong and H. E. Field (2010). Sampling small quantities of blood from microbats. Acta Chiropterologica 12(1): 255-258.

Stadler, A., C. K. Meiser and G. A. Schaub (2011). “Living Syringes”: Use of Hematophagous Bugs as Blood Samplers from Small and Wild Animals. Nature Helps. Springer: 243-271.

Thomsen, R. and C. C. Voigt (2006). Non-invasive blood sampling from primates using laboratory-bred blood-sucking bugs (Dipetalogaster maximus; Reduviidae, Heteroptera). Primates 47(4): 397-400.

Voigt, C. C., M. Faßbender, M. Dehnhard, G. Wibbelt, K. Jewgenow, H. Hofer and G. A. Schaub (2004). Validation of a minimally invasive blood-sampling technique for the analysis of 59

hormones in domestic rabbits, Oryctolagus cuniculus (Lagomorpha). General and Comparative Endocrinology 135(1): 100-107.

Wang, H. and P. Nuttall (1994). Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands. Parasitology 109(4): 525-530.

CHAPTER 5. GENERAL CONCLUSIONS AND AREAS OF FUTURE RESEARCH

Emerging infectious diseases are a global threat to both humans and animals and the

majority of these pathogens occur along the human-domestic animal-wildlife interface involving

complex interactions between multiple species. Surveillance, often based on serological data,

along this interface is paramount to early detection and intervention to curtail disease spread

(Gilbert et al., 2013). The most critical component of any immunological assay will likely

continue to be the antibody itself, therefore application of ticks as biological samplers and the

utility of this approach will involve further research on the effects of tick digestion and heme

processing as well as the duration of antibody persistence in the blood meal (Peruski and Peruski,

2003). The method described in this thesis is meant as a component of a surveillance process and the culmination of multiple supporting approaches to develop an overarching understanding of

the effects of a pathogen in a population. Understanding disease dynamics in host systems can

further define the mechanisms of transmission and maintenance within that system, which can be

then developed into better risk mitigation and control strategies.

A novel membrane feeding apparatus was created using a six-well laboratory plate with

well-inserts and sealing film as the membrane. Adult Ornithodoros tartakovskyi ticks, an argasid

species, readily fed to repletion on this apparatus. This system allows for increased containment,

ease of sorting individual ticks, easy replacement of blood meals, and the reduction of total

volume of blood required during feeding. This system will add to the repertoire of available

techniques used to limit the use of animals in the maintenance of laboratory tick colonies, as well

as the use in other tick-based research.

The detection of host antibodies in transmembrane-fed ticks was examined using two

immunoassay formats and, using these methods, it was shown that both LFA and cELISA are 61

viable options when detecting antibody status of individual animals exposed to the pathogen of

interest and fed on by the ticks. The limiting factor in detection was the size of the tick, which

determined the sample size and therefore the minimum, total antibody concentration tested per

sample. Future work is needed to evaluate the usefulness of ticks as an alternative, less-invasive

technique for blood collection for the purpose of serology. In regards to the use for the LFA and

cELISA, work would need to focus on the precision (repeatability of results) and accuracy

(comparison between tick collected samples and traditional, syringe and needle collected samples) of the sample collection method. The robustness of the technique would need to be

validated by investigating the influence of tick digestion of the blood meal, and antibody

contents, on the results of the assays.

As our study looked at the antibody presence immediately after feeding, future studies

should be conducted to determine the length of time host antibodies are detectable post-feeding

and the effects digestion may have on that detection. This window of detection will also

determine the future utility of this process as well as evaluating how reliable the target animal’s

antibody titer can be estimated. Studies utilizing ticks fed on animals with known antibody

concentrations followed by a field study involving animals with natural antibody titers and

determining the window of detection for antibodies would be needed to judge the future utility of this process.

A significant hurdle in utilizing ticks for xenodiagnosis or xenosurveillance is collection of the engorged arthropods. If target animals are handled, engorged ticks can be removed from

the animal instead of utilizing conventional venipuncture techniques for blood collection. If

target animals are not to be handled, engorged ticks can be collected directly from dens, nests, or

rock crevices under roosting sites. 62

One example that supports our hypothesis that collection of ticks for the purpose of surveillance is possible was published by Schuh et al (Schuh et al., 2016). In this study, 3125 adult and nymph argasid ticks (Ornithodoros faini) were collected from rock crevices near

Egyptian rousette bat (Rousettus aegyptiacus) roosting sites within Python Cave, Queen

Elizabeth National Park, Uganda. Their goal was to investigate the role this tick species could play in the enzootic transmission and maintenance of Marburgvirus. This location has an intimate association with historical human outbreaks of this filovirus and has been shown to have a 2.5% prevalence of active Marburgvirus infection in its chiropteran population (Fujita et al.

2009; Timen et al., 2009; Towner et al., 2009; Schuh et al., 2016). This study utilized Q-RT-PCR and found none of the samples positive for Marburgvirus-specific RNA (Schuh et al., 2016).

Ticks were individually collected and pooled into grinding vials and initially processed the same as described in our Chapter 4 methods. We propose that, depending on the goals of the study, samples like this could be divided to incorporate both antigen and antibody assays. Of course, sample volume could become a concern in this situation, but we postulate that more sensitive antibody assays could be applied, for instance multiplexed protein assays like the Luminex binding system have been shown to outperform conventional ELISA approaches (Peruski and

Peruski, 2003; Bossart et al., 2007; Hartmann et al., 2009; Uttamchandani et al., 2009). As the study reported in this thesis was designed as a proof-of-concept for the use of ticks for xenodiagnostics, further work and validation would be necessary for this theory to come to fruition as a field surveillance method.

References

Hartmann, M., J. Roeraade, D. Stoll, M. F. Templin and T. O. Joos (2009). Protein microarrays for diagnostic assays. Analytical and Bioanalytical Chemistry 393(5): 1407-1416.

Uttamchandani, M., J. L. Neo, B. N. Z. Ong and S. Moochhala (2009). Applications of microarrays in pathogen detection and biodefence. Trends in Biotechnology 27(1): 53-61.

APPENDIX: THEILERIA EQUI COMPETITIVE ELISA KIT WORKSHEET