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PREVALENCE OF POWASSAN VIRUS AND LYME DISEASE (BORRELIA
BURGDORFERI) IN IXODES SCAPULARIS COLLECTED FROM NEW JERSEY
AND PENNSYLVANIA BLACK BEARS (URSUS AMERICANUS)
By
Kristine N. Bentkowski, B.S.
King’s College
A Thesis Submitted in Partial Fulfillment of
The Requirements for the Degree of
Master of Science in Biology
To the office of Graduate and Extended Studies of
East Stroudsburg University of Pennsylvania
May 10, 2019
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ABSTRACT
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of
Master of Science in Biology to the office of Graduate and Extended Studies of East
Stroudsburg University of Pennsylvania
Student’s Name: Kristine N. Bentkowski
Title: Prevalence of Powassan Virus and Co-infection of Lyme Disease (Borrelia
burgdorferi) in Ixodes scapularis from New Jersey and Pennsylvania Black Bears (Ursus
americanus)
Date of Graduation: May 10, 2019
Thesis Chair: Joshua Loomis, Ph.D.
Thesis Member: Abdalla Aldras, Sc.D.
Thesis Member: Emily Rollinson, Ph.D.
Thesis Member: Nicole Chinnici, M.S.
Abstract
The blacklegged tick is the main vector for Lyme disease and Powassan virus
Lineage II (Deer Tick Virus) in the United States. The objective of this study was to identify
the prevalence of Powassan virus (DTV) and Lyme disease in adult and nymph blacklegged
ticks collected in New Jersey (2015-2018) and Pennsylvania (2017-2018). All ticks were
collected from lived trapped or hunter harvested black bears (Ursus americanus). A total of
2,713 ticks were collected, made up of four species. Only blacklegged ticks were analyzed in
this study. Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used
to amplify cDNA specific to the NS5 gene of POW Lineage II, and qPCR was used to
amplify the 16s-23s intergenic spacer region rDNA of Borrelia burgdorferi (Lyme disease).
A minimum infection rate (MIR) of 3.52% was determined for Powassan vitus and a MIR of
19.2% for Lyme disease. The findings in this study were similar to previous studies
conducted for Powassan and Lyme prevalence in Lyme endemic region.
Acknowledgments
I would like to acknowledge everyone that has helped me through my thesis.
From all my committee members, friends and family who were there to support,
encourage and guide me through this process.
I thank my graduate committee members Dr. Joshua Loomis, Dr. Emily
Rollinson, Dr. Abdalla Aldras and Nicole Chinnici for all their wisdom, help and
guidance through this process. Special thank you to Dr. Loomis for working through this
process together and figuring out everything that needed to be done to complete this
project and for being an amazing thesis chairperson. As well, thank you to Nicole for
encouraging me to apply to ESU for their graduate program and allowing to do my
research at the Northeast Wildlife DNA Laboratory.
Thank you to everyone at the NEWDL for their help and support throughout this
process. As well, to all the great friends who I have made who encouraged me to keep
going and helping get through the most stressful of times as we all worked towards out
Master’s degree. Especially Justin Clarke, Jon Adamski, Amanda Layden, Kacie Chern,
Eric, Sam and Ali Machrone who gave me a lot of support and created some great
friendships.
Lastly, I would like to thank my family and boyfriend. Thank you, mom, dad,
Ashley, Maggie and Matt, for all your encouragement and support you guys gave me
through all of this. I greatly appreciated it.
Table of Contents
List of Figures ............................................................................................................III
List of Tables ............................................................................................................ VI
Chapter I .................................................................................................................... 1
Introduction ......................................................................................................................1
Lyme Disease .......................................................................................................................................... 2
Immune Response.................................................................................................................................. 4
Lyme disease symptoms ........................................................................................................................ 6
Diagnosis and Treatment ....................................................................................................................... 7
Post Treatment Lyme Disease Syndrome .............................................................................................. 8
Powassan Virus .................................................................................................................................... 11
Vector ................................................................................................................................................... 13
Black Bears ........................................................................................................................................... 17
Study Objectives ................................................................................................................................... 19
Chapter II ................................................................................................................. 20
Materials and Methods .................................................................................................... 20
Study Area and Sample Collection ....................................................................................................... 20
Identification and Extraction ................................................................................................................ 23
Powassan virus (Lineage II) SYBR Assay Optimization ......................................................................... 25
RNA Analysis ......................................................................................................................................... 26
DNA analysis ......................................................................................................................................... 26
Statistical Analysis ................................................................................................................................ 28
Chapter III ................................................................................................................ 29
Results............................................................................................................................. 29
Tick Collections ..................................................................................................................................... 29
Optimization of Powassan Lineage II Assay ......................................................................................... 34
Disease and Statistical Analysis ............................................................................................................ 35
Powassan Virus .................................................................................................................................... 38
Borrelia burgdorferi.............................................................................................................................. 40
Co-infection .......................................................................................................................................... 42
County Data .......................................................................................................................................... 42
Powassan ......................................................................................................................................... 42
Borrelia burgdorferi......................................................................................................................... 44
Chapter IV ................................................................................................................ 46
Discussion........................................................................................................................ 46
Tick Collection Data .............................................................................................................................. 46
Powassan (DTV) Prevalence ................................................................................................................. 50
Borrelia burgdorferi Prevalence Data .................................................................................................. 53
Co-infection Prevalence Data............................................................................................................... 56
Conclusion ............................................................................................................................................ 57
Future Study ......................................................................................................................................... 58
Literature Cited ........................................................................................................ 60
I
Appendix A: Tick Collection Raw Data ............................................................................... 67
Appendix B: Powassan and Lyme Raw Data ...................................................................... 69
Appendix C: Statistical Raw Data ...................................................................................... 85
II
List of Figures
Figure
Page
Figure 1. Reported cases of Lyme disease in the United States (2017). Each blue dot
represents a confirmed case of Lyme disease. Endemic regions are illustrated within the
Northeast and Mid-west. Massachusetts surveillance method does not match the national
surveillance case definition set by the Council of State and Territorial Epidemiologist,
information on most Lyme disease cases are not sent to the CDC and are not represented
on this map. (Centers for Disease Control and Prevention).................................................4
Figure 2. Map of neuroinvaisve Powassan virus cases reported in each US state, 20082017. (Centers for Disease Control and Prevention) ........................................................12
Figure 3. Distribution of blacklegged ticks in the United States as of 2018 (Centers for
Disease Control and Prevention) .......................................................................................14
Figure 4. The two-year life cycle of the blacklegged tick. Broken down into when each
life stage is most prevalent and the main host each life stage feeds on. (Centers for
Disease and Control Prevention) .......................................................................................15
Figure 5. Sedated black bear cub, 2018 NJDFW research trapping (Photo credit Kristine
Bentkowski) .......................................................................................................................18
Figure 6. New Jersey counties where black bears were trapped or harvested, and ticks
were collected. Blue stripped counties had ticks analyzed for Powassan and Lyme. .......22
Figure 7. Pennsylvania counties where black bears were trapped or harvested, and ticks
were collected 2017-18. Blue counties ticks were collected in 2017 and pink counties
ticks were collected in 2018. ..............................................................................................23
III
Figure 8. Measurement of a female blacklegged tick using the scutal index (SI = b/a), a =
maximum width of scutum, b = length of tick from posterior to the basis capitulium
(Photo credit Kristine Bentkowski) ...................................................................................24
Figure 9. Examples of engorgement sizes of adult female blacklegged tick. Left to right:
Fully engorged, semi-engorged, semi-engorged, unengorged, unengorged (Photo credit
Kristine Bentkowski) .........................................................................................................25
Figure 10. Total number of ticks collected from 2015-18 in New Jersey by month .........31
Figure 11. Total adult I. scapularis collected in the month throughout the year from New
Jersey (2015-18).................................................................................................................32
Figure 12. Total number of ticks collected from 2017-18 in Pennsylvania by month ......33
Figure 13. Total number of adult I. scapularis collected in Pennsylvania in 2017-18 by
month .................................................................................................................................33
Figure 14. Total number of nymph and larval I. scapularis collected in Pennsylvania in
2017-18 by month ..............................................................................................................34
Figure 15. Plotted linear regression of the standard curve, using the log number of copies
of each dilution by the CT call from each triplicate ..........................................................35
Figure 16. Total number of I. scapularis female, male and nymph ticks analyzed (n=
1,277) from NJ (2015-18) and (PA 2017-18) ....................................................................38
Figure 17. Minimum infection rate (MIR) percent of adult I. scapularis with POW (DTV)
(n=1,213) from NJ (2015-18) and PA (2017-18)...............................................................39
IV
Figure 18. Minimum infection rate percent of nymph I. scapularis ticks with POW
(DTV) (n=64), from NJ and PA 2018 ................................................................................39
Figure 19. MIR (%) of adult I. scapularis ticks with B. burgdorferi (n=1,213) from NJ
(2015-18) and PA (2017-28) ..............................................................................................41
Figure 20. Minimum infection rate percent of nymph I. scapularis ticks with B.
burgdorferi (n=64), from NJ and PA 2018 ........................................................................41
V
List of Tables
Table
Page
Table 1. Average tick load of small to large animals identified by LoGiudice, Satefeld,
Scmidt & Keesing (2003) in Dutchess county, NY ...........................................................16
Table 2. Oligonucleotide primer used for PCR. SYBR green primer targeting the NS5
region of Powassan virus. Real-time primer targeting the 16S-23S intergenic spacer
region for Lyme (Borrelia species) and specific probe for Borrelia burgdorferi .............27
Table 3. Total number of blacklegged ticks analyzed from each county in New Jersey ...36
Table 4. Total number of blacklegged ticks analyzed from each county in Pennsylvania 37
Table 5. Total Powassan positive pools by year in each New Jersey county ....................43
Table 6. Total Powassan positive pools by year in each Pennsylvania county .................43
Table 7. Total B. burgdorferi positive tick pools by year in New Jersey counties ............44
Table 8. Total B. burgdorferi positive tick pools by year in Pennsylvania counties .........45
VI
Chapter I
Introduction
Tick-borne diseases affect thousands of people all over the globe every year.
Ticks have been vectors of disease for thousands of years and the spread of tick-borne
diseases has increased over the last decade, with over thirteen newly-recognized diseases
discovered over the last two decades48. The organisms that cause these diseases range
from bacterial to protozoan to viral, and each have their own unique transmission path
and can have various effects on those infected. The most common vector-borne disease in
the United States is Lyme disease, caused by the bacterium Borrelia burgdorferi. Lyme
disease is one of the top ten most recorded diseases in the United States but is not the
only tick-borne disease seen on the rise over the last decade33. Powassan virus is an
emerging tick-borne virus and is broken into two lineages, lineage I vectored by the
groundhog tick (Ixodes cookei) and lineage II (Deer Tick Virus) vectored by the
blacklegged tick (Ixodes scapularis)32. Powassan virus can be asymptomatic or can cause
encephalitis and severe neurological sequelae for those infected 36. The blacklegged tick is
the vector for many of these pathogens, including Lyme disease and Powassan virus, and
plays a large part in tick-borne disease transmission to humans. As the blacklegged tick
1
population continues to grow and expand, tick-borne diseases are becoming a
public health and safety crisis21,22. The increase in tick-borne diseases over the last
decade has sparked a nationwide concern for education on treatment and prevention.
Lyme Disease
Lyme disease, also known as Lyme borreliosis was first discovered in Lyme,
Connecticut in 1975 and since has had confirmed cases in North America, Europe and
Asia. Lyme disease is caused by several genospecies of the Borrelia burgdorferi sensu
lato group6. Of these, only two species of Borrelia bacteria, B. burgdorferi and Borrelia
mayonii, cause Lyme disease in the United States. Borrelia burgdorferi is the leading
cause of Lyme disease in the United States and Canada. In 2013, the Mayo Clinic
discovered B. mayonii, a new species of Borrelia that was causing Lyme disease like
symptoms in the upper Midwest37. Lyme disease is transmitted by the blacklegged tick
(Ixodes scapularis) in the Northeast and upper Midwest and by the western blacklegged
tick (I. pacificus) along the western coast of the U.S. The majority of Lyme disease cases
are reported during the late spring, summer and fall when blacklegged ticks are actively
seeking hosts.
Lyme disease is the fastest growing vector-borne disease in the United States. The
CDC estimates reports approximately 30,000 confirmed cases each year. With
accordance to the CDC and National Notifiable Diseases Surveillance System (NNDSS)
confirmed cases must present an erythema migran (EM) with a single primary lesion that
reaches greater than or equal to 5 cm in size across its largest diameter. The EM lesion is
accompanied by other acute symptoms, particularly fatigue, fever, headache, mildly stiff
2
neck, arthralgia, or myalgia. As well, a Physician must confirm laboratory evidence in the
form of a positive ELISA and western blot or a positive culture for B. burgdorferi. A
probable case is defined by the CDC when a Physician diagnoses a positive laboratory
test, but the patient lacks other evidence such as an erythema migran and no known
exposure in a high incidence state. In addition to strict diagnostic guidelines, studies have
shown only 10 percent of Lyme disease cases are reported. In 2008, Hinckley et, al.
surveyed 2.4 million specimens from laboratories throughout the U.S. Following
guidelines for testing Lyme disease recommended by the U.S. Public Health Service
Agencies and the Infectious Diseases Society of America, the estimated percentage of
true infection that year was upwards of 288,000 cases a year 33. Nelson et al. (2015),
evaluated the nationwide health insurance claims database from 2005-2010 identifying
patients with clinician diagnosed Lyme disease. Positive cases were determined using
ICD-9-CM codes for communicable diseases, along with a comprehensive analysis of the
positive predictive values (PPVs), and the case definition for Lyme disease by the CDC 57.
The ICD-9-CM codes are used by hospitals to assign diagnosis in patient charts. The
study determined based on clinical symptoms, there are approximately 329,000 cases of
Lyme disease each year1. In 2016, there were 36,429 confirmed and probable cases of
Lyme disease in the U.S. and 42,743 in 2017, an 8.5% increase4. Of these cases, 96% are
reported in 14 states located in the Northeast and upper Midwest35(Figure 1). The MidAtlantic states of Pennsylvania, New Jersey and New York have the highest number of
reported cases. In 2017, Pennsylvania reported 9,250 confirmed cases and New Jersey
reported 3,629 confirmed cases4.
3
Figure 1. Reported cases of Lyme disease in the United States (2017). Each blue dot
represents a confirmed case of Lyme disease. Endemic regions are illustrated within the
Northeast and Mid-west. Massachusetts surveillance method does not match the national
surveillance case definition set by the Council of State and Territorial Epidemiologist,
information on most Lyme disease cases are not sent to the CDC and are not represented
on this map. (Centers for Disease Control and Prevention)
Immune Response
Transmission time of Lyme disease from tick to host can be as short as 16 hours
or up to 48 hours15,44. Transmission of Lyme disease from vector to host causes the
bacterium to change its outer surface proteins to survive different environments. Within
the vector, B. burgdorferi resides and replicates in the midgut. During this time Outer
surface proteins are upregulated (OspA) allowing the bacterium to adhere to the tick’s
midgut until the tick feeds. As the tick attaches to a host and begins taking in a blood
meal, the OspA gene is downregulated and the OspC gene is expressed as a result of the
ticks midgut temperature and pH changes from the blood meal64,44.
4
The bacterium will than migrate from the tick’s midgut to the salivary glands; this
allows access for the bacterium to enter the new host. At the initial infection site B.
burgdorferi has several proteins that allow it to survive in the mammalian host and avoid
destruction. The OspC gene allows for the bacterium to survive the warmer temperatures
of the human body and basic pH level of the blood. The bacterium uses extra cellular
matrix (ECM) binding proteins DbpA and DbpB, to bind to decorin, and protein BBk32
to bind to fibronectin, as well as Bgp to bind to proteoglycans and P66 which further
binds to integrins64. These proteins may also play role in dissemination through
mammalian tissue and persistence in joints64. The OspC gene has been found to play a
possible role in dissemination by binding to human plasminogen44. These proteins at the
initial infection site are recognized by the innate immune system, recognized by
pathogen-associated molecular pattern (PAMP’s) and signal Toll-like receptors (TLR’s)
to release signals to the rest of the immune system. At the erythema migran lesion sites,
TLR2 has been found to play a specific role in releasing IFN, IL1 and IL6 cytotoxins52.
TLRs signal an influx of macrophages, dendritic cells and neutrophils to the initial
infection site. The immune system then produces pro-inflammatory cytokines such, TNF, IL-2, IL-6 and IFN’s. Production of cytokines recruit T-cells to the initial site of
infection which play an important role in activating complement and phagocytosis of the
bacterium during early localized infection. After several days of infection, the immune
system will begin to produce anti-inflammatory cytokine IL-10, this allows for surviving
bacteria to disseminate throughout the body66.
During early stage dissemination the surviving bacterium can travel through the
body hidden in tissue and being motile through viscous media found in the body. The
5
spirochete shape and flagella transverse the whole body protected under the outer
membrane, this helps the bacterium to penetrate host tissue and disseminate 64. As the
bacterium disseminates throughout the body the host immune system continues to
produce pro-inflammatory cytokines damaging tissue, joints, muscles and the heart due
to the inflammation caused by the host own immune system52.
Lyme disease symptoms
Lyme disease is a multisystem illness that can affect the skin, nervous system,
musculoskeletal system and the heart. Transmitted from a tick bite, symptoms may occur
3-30 days after exposure to the bacteria. There are three stages of symptomatology which
include: early localized, early disseminated and late disseminated Lyme disease.
In early localized (stage 1) Lyme disease, 60-90% of patients may develop a rash known
as an erythema migrans (EM)50. The rash tends to be localized in the area of the tick bite
and has the characteristic bulls-eye shape. Some patients may develop an uncharacteristic
rash that is patchy with no specific shape, and some patients may develop no rash at all
during their infection. Early Lyme disease may also include flu-like symptoms of fatigue,
malaise, fever, headache, arthralgias (joint pain), and myalgias (muscle pain)31. Lyme
disease symptoms for B. burgdorferi and B. mayonii infections are similar with additional
symptoms of B. mayonii including nausea, vomiting and diffuse rashes. Patients with B.
mayonii tend to have higher concentrations of bacteria circulating in the bloodstream 21.
If early localized Lyme disease goes untreated, patient symptoms may progress into the
early disseminated stage (stage 2). These symptoms can occur weeks or months after a
6
tick bite and symptoms may vary depending on the species of Borrelia causing the Lyme
disease infection. Early disseminated symptoms can include neck stiffness, facial palsy
(typically Bell’s palsy), lymphocytic meningitis, progressing into the loss of motor and
sensory function31. Lyme carditis may occur in this stage and cause an atrioventricular
block.
If Lyme disease is continued to be left untreated, it can persist into late
disseminated (stage 3) Lyme disease. This stage of Lyme disease can lead to severe
arthritis synovitis, severe neurological problems, such as memory loss and black outs,
and in rare cases cause encephalopathy31.
Diagnosis and Treatment
In endemic regions, clinicians use characteristic signs of Lyme disease described
and presented by patients for diagnosis. These characteristic signs include the patient
reporting a known tick bite, developing an EM rash, or developing other symptoms
common to early-localized Lyme disease. Not all patients develop an EM rash or
remember a tick bite. These patients will present symptoms similar to other diseases such
as fibromyalgia, multiple sclerosis and the flu. These patients may not receive the correct
treatment resulting in symptoms developing into early and late disseminated Lyme
disease30.
Current testing recommended by the CDC is a two-tier serological test. The first
step uses an ELISA (enzyme-linked immunosorbent assay) to detect IgG and IgM
antibodies against B. burgdorferi, if positive, the second test is an immunoblot (Western
7
blot) to measure IgG and IgM30,44. During early Lyme disease serological testing can be
inaccurate due to low antibody production and sensitivity from the serological test. If a
patient continues to have symptoms after a negative ELISA, they can be retested 2-4
weeks after the initial exposure. IgM can be detected 2-4 weeks after initial infection and
reaches peak antibody production at six weeks before the titer drops 66. Studies are being
conducting to develop new methods for diagnosing Lyme disease with higher sensitivity
and accuracy44.
Lyme disease can be treated with antibiotics. Doxycycline is the most commonly
used antibiotic to treat Lyme disease; however, other antibiotics such as amoxicillin, or
cefuroxime axetil can be used. Antibiotics are prescribed orally one to two times a day
for 14-30 days47.
Post Treatment Lyme Disease Syndrome
In most Lyme disease cases, patients clear the infection and no longer have
symptoms following a course of oral antibiotics. However, 10 to 20% of treated patients
continue to have symptoms for months to years following completion of their treatment 46.
This condition was previously referred to as Chronic Lyme Disease (CLD), and more
recently referred to as Post Treatment Lyme Disease Syndrome (PTLDS) or Post Lyme
disease Syndrome (PLDS)23. PTLDS received a case definition from the Infectious
Disease Society of America (IDSA) stating that the individual must have a documented
case of Lyme disease who has completed treatment but continues to show a relapse of
symptoms including fatigue, musculoskeletal pain, and complaints of cognitive
difficulties for a minimum of 6 months from treatment completion55.
8
The cause of PTLDS is unknown but there are many studies with potential
hypotheses as to the cause55. These theories include, the body is having an autoimmune
response as a result of Lyme disease, the antibiotic course fails to clear the Lyme disease
infection, the bacterium has the ability to change form and create biofilms during
environmental stress and a secondary infection by a different pathogen with similar
symptoms to Lyme disease55.
The first theory focuses on the idea that PTLDS is a delayed autoimmune
response to Lyme disease. Singh & Girschick (2004) reviewed the effects of T-cells on
inflammation in the joints during Lyme disease. They found elevated levels of Tlymphocytes in synovial fluid and peripheral blood in adults who were showing
symptoms of PTLDS58. Maccallini et al. (2018) evaluated the role of B cells and the
similarity between human enolase and Borrelia enolase. They found that human and
Borrelia enolase share a conformational B cell epitope which can cause a release of
autoantibodies against enolase. These antibodies have been seen in other autoimmune
diseases that affect the brain41. Further studies testing this theory must be conducted
using several case studies with known Lyme disease patients and those who are suspected
to have PTLDS41.
Lyme disease is typically treated with a course of oral antibiotics between 14-28
days and is supposed to clear the infection. Cameron, Johnson & Maloney (2016)
reviewed several studies conducted to determine the effectiveness of clearing Lyme
disease after a course of antibiotics and found that those treated during early localized
and early disseminated failed to bring 16% to 48% of the patients back to their pre-Lyme
health status. A observation trial found that 33% of patients treated with a three week
9
course of doxycycline continued to have symptoms three to six months post-treatment12.
A study conducted by Logigian, Kaplan & Steere (1990) observed patients with late
disseminated Lyme disease and found that 63% of patients treated with intravenous (IV)
ceftriaxone for 14 days showed improvement, 15% showed no health improvement and
22% showed initial improvement and relapsed with symptoms six months after
treatment39. Failure to clear the infection with an oral or IV course of antibiotics can lead
to these symptoms and be a possible cause for PTLDS. Patients were not tested for other
tick-borne diseases, lasting symptoms may have been caused by a TBD that could not be
treated with antibiotics.
Recent studies have found that the Lyme bacterium has the ability to change
under environmental stresses (pH, temperature, immune attack, nutrient starvation) and
form biofilms to protect itself. It was determined that B. burgdorferi has several forms,
spirochete (stationary phase and log phase spirochete), round body and an aggregated
microcolony that has the ability to form biofilms 17,28. A study conducted by Feng,
Tingting & Zhang (2018) determined that when studied in vitro B. burgdorferi can persist
in variant forms and protect itself from antibiotics and, in vivo mice, the microcolony
form and stationary phase caused more server inflammation in joints that log phase.
Antibiotics were able to destroy log phase spirochete and some round body forms of the
bacterium but failed to completely destroy stationary spirochete phase and the biofilm
aggregates17,28. These forms can prevent the destruction of all B. burgdorferi bacterium in
the body, leaving persistent bacteria behind and may play a role in PTLDS.
The last hypothesis states that PTLDS may be caused by a secondary infection
masked by Lyme disease. One of the main co-infections that has been hypothesized to
10
cause PTLDS is Powassan virus. Powassan virus 63 has similar symptoms to those present
in PTLDS such as fever, fatigue, myalgia, dizziness, confusion, memory loss and in
severe cases, encephalitis and death63. Deer tick virus is not a well-known or studied
virus but has been increasing in the U.S. over the last decade and can be hard to diagnosis
and detect29. Frost et al. (2017) tested 41 patients with known Lyme disease for Powassan
virus and identified 10 (4.1%) of those patients were also positive for Powassan virus.
Furthermore, Thomm et al. (2018) tested 106 patients for Powassan virus which have
been diagnosed with at least one tick-borne disease and identified 10 (9.4%) of these
patients were also positive for Powassan virus 63.
Powassan Virus
Powassan virus (POW) is an emerging tick-borne virus that is on the rise in North
America. It was first discovered in Powassan, Ontario in 1958 after a young boy died of
encephalitis49. POW has since been found in the Great Lake Region and along the
Northeast in the U.S., up into Canada and in the Primorsky region of Russia51. It is a
ssRNA Flavivirus, part of Flavividae family19. It is part of the tick-borne encephalitis
complex (TBC-E), and transmitted by the bite of an infected tick51,63. There are two
lineages of POW; lineage I was first discovered in the 1958 Powassan, Canada case and,
lineage II was discovered in 1996 and referred to as Deer Tick Virus (DTV). The two
lineages are associated with having different vertebrate reservoirs and vectors 62. Lineage
I transmission cycle is maintained by the woodchuck tick (Ixodes cookei) and medium
sized mammals such as the woodchuck (Marmota monax) whereas lineage II
transmission cycle is maintained by the blacklegged tick (Ixodes scapularis) and the
11
white-footed mouse (Peromyscus leucopus)51. Although they are two distinct lineages,
phylogenetic studies have found them to have 84% nucleotide similarity and an amino
acid similarity of 93%5.
POW lineage I is more aggressive with a higher possibility to be fatal than DTV.
DTV is less aggressive20 but studies over the last decade have found that DTV can cause
fatal encephalitis or lasting neurological effects similar to lineage I26,61,63. The CDC has
reported an increase in neuroinvasive POW cases from 6 in 2015 to 33 in 2017 60.
Neuroinvasive cases have been confirmed within 11 states from 2008 to 2017 (Figure 2).
Since 2008, NJ has had 5 cases and PA has had 7. In 2017, PA and NJ both had 4 cases
of confirmed POW, an increase from 0 diagnosed in 2016 in both states 60.
Figure 2. Map of neuroinvaisve Powassan virus cases reported in each US state, 20082017. (Centers for Disease Control and Prevention)
POW lineage II is typically asymptomatic, however it can cause life-threating
conditions such as encephalitis and meningitis to those infected 49. Symptoms occur
12
between 1-5 weeks after initial exposure by a tick bite53. They can range from headaches,
drowsiness, nausea and disorientation during early infection and can progress into
encephalitis, meningoencephalitis and coma during later stages of infection32. The
mortality rate can be as high as 15% for patients infected, and 50% of the surviving
patients are diagnosed with neurological sequelae53. Patients can be tested using their
CSF or serum targeting POW IgM antibodies29,53. Current serological testing consist of
using IgM ELISA, IgM immunofluorescence antibody assay (IFA), IgG ELISA and
conformation testing with a > 90% or > 50% plaque neutralization test (PRNT90 or
PRNT50) with a >4-fold increase in antibody titers from acute- and convalescent-phase
sera29,63. Based on the sensitivity of current testing protocols, there may be more
confirmed cases of POW then what is being reported 29,53. Confirming lineage I vs lineage
II POW requires using neutralization assays such as qPCR and sequencing following a
positive serological test49. Transmission of POW from vector to host is as fast as 15
minutes following attachment as the virus resides within the salivary glands of the tick18 .
Vector
There are hundreds of ticks worldwide which fall into one of three
families: Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae (ticks of South
Africa)27. The largest family Ixodidae, play a role in vectoring and transmitting a
majority of tick-borne diseases globally. Ixodes scapularis (blacklegged tick or deer tick)
is a medically-important tick as it contributes to many tick borne diseases in North
America38. The blacklegged tick is distributed up into southeastern Canada down the
13
northeast coast and as far west as Texas, Oklahoma and parts of North and South Dakota
(Figure 3).
The blacklegged tick is a 3-host tick with three life stages: larval, nymph and
adult, that lives a two-year life cycle, molting between each life stage38 (Figure 4). Larval
ticks hatch in May and are active until August, feeding on small rodents such as the
white-footed mouse (Peromyscus leucopus) and birds. Once a larval tick has fed to
engorgement, it will drop off its host and molt into a nymph and enter diapause until the
late spring and summer24. The nymph will quest for its second host which can be small to
medium-sized animals such as rodents, lagomorphs, ungulates, cats, dogs and humans.
Following engorgement, the nymph will molt into an adult. Adult females will use larger
animals as a host before copulating with adult males and laying their eggs in the early
spring38, laying up to 3,000 eggs.
Figure 3. Distribution of blacklegged ticks in the United States as of 2018 (Centers for
Disease Control and Prevention)
14
Figure 4. The two-year life cycle of the blacklegged tick. Broken down into when each
life stage is most prevalent and the main host each life stage feeds on. (Centers for
Disease and Control Prevention)
Blacklegged ticks acquire pathogens transovarially or transstadially depending on
the pathogen. Transovarial pathogens are able to be passed on from an infected mother
tick to the eggs. It is unknown if POW is transovarially passed down to the next
generation of an infected tick. A transstadial pathogen is picked up by a vector when
feeding on an infected reservoir. The bacterium that causes Lyme disease is a transstadial
pathogen that will continue to live within the tick once acquired from a reservoir40. A
reservoir host is able to survive living with a pathogen, without presenting symptoms or
infection.
15
The white footed-mouse (Peromyscus leucopus) is the reservoir for several tickborne pathogens that are vectored by the blacklegged tick. The most notable pathogen is
B. burgdorferi (Lyme disease), in addition to Anaplasma phagocytophilum
(Anaplasmosis), Babesia microti (Babesiosis), Borrelia miyamotoi and, DTV (Powassan
virus lineage II)16. Other rodents, such as chipmunks, squirrels, shrews and woodchucks
are competent reservoirs for tick-borne pathogens by the blacklegged tick10. Although
only a few animals are reservoir host, other large animals play a role in the distribution
and spread of infected ticks. Studies enumerating the number of ticks on black bears have
identified on average 400 ticks3. A study conducted by LoGiudice, Ostfeld, Schmidt &
Keesing (2003) identified the host diversity and community composition of ticks in
Dutchess County, New York seen in Table 1. 40. The average tick load on an animal can
play a large role in pathogen and tick distribution depending on the animal. The whitefooted mouse is a key reservoir host for many tick-borne pathogens and a high tick load
can increase infected ticks in an area. While, other larger animals such as the white-tailed
deer and black bear are not known reservoir host but can play crucial roles in carrying
large tick loads and distributing them into and out of urban and rural areas. This
mechanism of transport can result in introducing new tick species and diseases to an area
inhabited by humans.
Table 1. Average tick load of small to large animals identified by LoGiudice, Satefeld,
Scmidt & Keesing (2003) in Dutchess County, NY
Animal
White-footed mouse (Peromyscus leucopus)
Eastern chipmunk (Tamias striatus)
White-tailed deer (Odocoileus virginianus)
16
Average tick load
27.8
36.0
239
Short-tailed shrew (Blarina brevicauda)
Grey Squirrel (Sciurus carolinensis)
62.9
142
Black Bears
The American black bear (Ursus americanus) is medium-sized and historically
found in the United States, Canada and Mexico. Black bears have great mobility, are
generalist and have an omnivorous diet. This allows black bears to live in a wide range of
habitats, from swamps to semi-desserts and dense forests. Black bear males (called boars)
can weigh between 150 to 600lbs and females (called sows) can weigh from 150 to
400lbs8. Female black bears will typically have a home range between 2.5-10 square
miles, while males can have a much larger home range between 10-59 square miles9. In
the Northeast, they typically are black in color with a brown muzzle and can have a white
blaze on their chest. Some bears are an atypical cinnamon color. Black bears are strong
swimmers and good climbers due to their five toes and long curved claws 45. In the wild,
black bears can live up to 25 years. Although bears prefer to eat wild foods such as
acorns, skunk cabbage, and blueberries they will also eat from human garbage and bird
feeders. Merkle et al.(2013) surveyed black bears from 2009 and 2010 in Missoula,
Montana where bears were 80% more likely to choose urban grounds for food than in the
wild42. Black bears will do a wide range of damage to homes, sheds that have food stored
in them, destroy garbage cans and bird feeders.
Black bears typically begin to mate around the age of three, but can start as early
as two years old. Mating occurs late May through July. Females may enter their dens as
early as the end of October and males may enter their dens as late as mid-December.
During January, sows will give birth in their dens and can have a litter of one to five
17
cubs. Bears will begin to exit their dens in April, with the cubs following the sow for
about a year to year and half before they go off on their own8,45.
Figure 5. Sedated black bear cub, 2018 NJDFW research trapping (Photo credit Kristine
Bentkowski)
Black bear populations in NJ, PA and NY have been increasing over the last
decade. The New Jersey Division of Fish and Wildlife (NJDFW) has seen an expansion
of the black bear population from the northeast region of the state in 1995 to sightings in
every county to date45. Today in northwestern NJ, there are as many as three bears per
square mile45. In PA, the Pennsylvania Game Commission (PAGC) has also seen an
increase in the black bear population over the last decade. As a result of increased
populations, both states have had significant increases in nuisance bear reports. Nuisance
bears defined by the PAGC and NJDFW are black bears that are entering residential
areas, destroying farmland or homeowner property. To control and monitor black bear
18
populations, the PAGC and NJDFW conduct annual research trappings, and an annual
black bear harvest to control overpopulation (Figure 5). With the increase of interactions
between black bears and humans, there is an increasing public health concern for
zoonotic diseases. Very little research has been conducted to determine if black bears are
reservoirs to some tick-borne diseases, however, these mammals play an important role in
tick dispersal carrying ticks into residential areas.
Study Objectives
Monitoring the prevalence of Lyme disease and Powassan virus are important to
public health and safety. This will be the first study conducted in Pennsylvania and New
Jersey to determine the prevalence of Powassan virus and co-infection prevalence with
Lyme disease. The goal of this study was to determine the prevalence of Lyme disease
and Powassan virus in blacklegged ticks collected from black bears in New Jersey and
Pennsylvania from 2015-2018. The objectives were to:
1.
Determine the prevalence of Powassan virus (DTV) in blacklegged ticks from
bears in NJ and PA from 2015-208
2.
Determine the prevalence of Lyme disease in blacklegged ticks collected from
black bears in NJ and PA from 2015-2018
3.
Evaluate the co-infection rate of Powassan virus and Lyme disease in NJ and PA
19
Chapter II
Materials and Methods
Study Area and Sample Collection
From 2015-2018, ticks were collected from black bears (Ursus americanus) with
assistance from the New Jersey Division of Fish and Wildlife (NJDFW). Ticks were
collected from Hunterdon, Morris, Passaic, Sussex, and Warren Counties in New Jersey
(Figure 6). They were collected three to four times throughout the year during research
trapping and at the black bear hunt check stations. Biannual research trapping occurred
May through June and again in the fall from August through September. Research
trapping sites were selected based on bear activity, proximity to food sources such as
cornfields, acorn mass, and reported bear sightings. Black bears were captured using
Aldrich foot snares and culvert traps. Trained personnel used a mixture of ketamine and
xylazine (ZooPharm Inc, Windsor, CO) to anesthetize captured animals. Morphological
measurements were collected, and each animal was tagged, tattooed with corresponding
tag number, and sexed. To determine age, the premolar of yearling and adult bears was
removed. Ticks were also collected from hunter-harvested bears during NJDFWs hunting
season. Segment A of the bear hunt occurred for one week in October, and segment B
20
occurred for one week in December. Harvested black bears were brought into check
stations, where morphological measurements were collected, and tick searches were
completed.
During the annual research trapping and black bear harvest, a 5 to 10-minute tick
check focusing on the ears, around the eyes, neck, under the armpits and around the groin
region was conducted. Ticks were collected with forceps and placed into 2mL screw top
tubes labeled with the bear’s ID number, date and location of capture. Ticks were stored
in a cooler on ice until being brought back to the Northeast Wildlife DNA Lab
(NEWDL), where they were stored at -20˚C.
21
Figure 6. New Jersey counties where black bears were trapped or harvested, and ticks
were collected. Ticks collected from blue stripped counties were analyzed for Powassan
and B. burgdorferi.
Ticks were collected from black bears in Pennsylvania between 2017-2018 with
the assistance of the Pennsylvania Game Commission (PAGC) and collaborators at Penn
State University. In 2017, ticks were collected from Monroe and Pike Counties, and in
2018 from Centre, Clearfield, Clinton, Huntingdon, Lycoming, Potter, and Tioga
Counties (Figure 7). The PAGC collected ticks from nuisance and vehicle-strike bears
between September and October of 2017. In 2018 ticks were collected throughout the
year from June to December during the annual research trapping in the Fall and Spring,
the bear hunt in October and December and bears caught throughout the time period with
suspicion of mange infection from central Pennsylvania. Tick check methods were
22
designed by Hannah Greenberg from Pennsylvania State University from an ongoing
study to determine the abundance and distribution of ticks on American black bears in
Pennsylvania. Tick checks were conducted over 16 designated body regions on the black
bear with 4" X 4" standardized tick square. There was no time constraint on tick checks.
Ticks were placed in 2mL screw top tubes filled with 70% ethanol and had the black
bear’s age, sex, and date.
Figure 7. Pennsylvania counties where black bears were trapped or harvested, and ticks
were collected 2017-18. Blue counties ticks were collected in 2017 and pink counties
ticks were collected in 2018.
Identification and Extraction
All ticks were identified to species and life stage following Ward’s Guide to
North American Ticks (Ward’s, Rochester, NY). This was done by examining their
festoons, geographic location and scutum (shield) pattern, as each tick has their own
unique pattern or color. The body of the I. scapularis begins to engorge and stretch as it
feeds, while the hard scutum continues to keep its size and shape. A scutal index can be
used to measure the engorgement level of the tick and estimate duration of attachment.
The engorgement level of I. scapularis females and nymphs were determined using the
23
scutal index (SI = b/a) measuring the body length from the posterior edge to the basis
capitulum and the maximum width of the scutum (Figure 8). Based on the SI, estimated
engorgement hours were determined, and ticks were then labeled as unengorged (≤ 14
hours), semi-engorged (≤ 15-92 hours) or fully engorged (≥ 93 hours) (Figure 9).
A tick pool consisted of ticks of the same sex, life stage, engorgement level,
individual black bear and county. Bears that had more than five ticks of the same
engorgement, sex and life stage were pooled together but if the bear had less than five
ticks each tick was analyzed individually. For example, a bear with three ticks collected
from it had each tick analyzed alone, while a bear with five females of the same
engorgement had one pool containing all five ticks. Pools typically ranged from 1-5 ticks.
Ticks in the same pool were all placed into one tube for extraction of RNA and DNA and
analyzed as one sample.
Figure 8. Measurement of a female blacklegged tick using the scutal index (SI = b/a), a =
maximum width of scutum, b = length of tick from posterior to the basis capitulium
24
Figure 9. Examples of engorgement sizes of adult female blacklegged tick. Left to right:
Fully engorged, semi-engorged, semi-engorged, unengorged, unengorged (Photo credit
Kristine Bentkowski)
Powassan virus (Lineage II) SYBR Assay Optimization
To optimize a reverse transcriptase real-time PCR (RT- PCR) assay for Powassan
virus Lineage II, a standard curve analysis was performed. Using a synthetic positive,
serial 1:10 dilutions were created ranging from 10 -1 to 10-10. Each dilution was performed
in triplicate to validate accuracy and a negative control of nuclease free water was used to
confirm absence of contamination. A synthetic positive sample was created by
GENEWIZ (South Plainfield, NJ) with the NS5 target primers. PCR was performed in
25L reactions. Each reaction contained 0.2M of forward primer
5’gaagctgggtgagtttggag 3’ and 0.2M reverse primer 5’cctgagcaaccaaccaagat 3’ targeting
a 318 base pair region of the NS5 gene (Knox et al. 2017). The PCR protocol followed
manufacture guidelines with the modification of using 0.25L of SYBR enzyme mix
(Thermofisher, Waltham, MA). The 25L RT-PCR reaction consisted of 1X SYBR
Green Master Mix (Thermofisher, Waltham, MA), 1.0L premixed forward and reverse
primers, 6.25L Qiagen nuclease free water, 0.25L SYBR enzyme mix, and 5L of
synthetic positive. The standard curve was run on an Applied Biosystems StepOnePlus TM
25
PCR system. Thermal cycling conditions for RT-PCR was performed at 50C for 20
mins, 95C for 5 minutes, followed by 45 cycles of 94C for 10 seconds, 55C for 5
seconds and 60C for 25 seconds. A standard curve was created using the log dilution of
copies (ng) by the CT (cycle threshold) value. Each CT value was determined by how
many cycles it took for the fluorescent signal of the sample to cross the threshold.
RNA Analysis
RNA was extracted from tick pools following the Qiagen viral RNA extraction
protocol (Qiagen, Germantown, MD). The protocol was modified to include an overnight
incubation at room temperature. A blank was included in each analysis extraction to
confirm absence of contamination during the extraction process.
Samples were analyzed for Powassan virus using the optimized Powassan virus
Lineage II SYBR green assay. Specific primers were used to target the NS5 region of the
Powassan virus genome (Table 2). Each analysis was run with a positive to validate the
assay and negative to confirm the absence of contamination. All RT-PCR was conducted
on an Applied Biosystems StepOnePlusTM PCR system. Positive samples were identified
with CT values of 30-44 and a threshold of 1.725.
DNA analysis
DNA was extracted from tick pools along with RNA following the Qiagen viral
RNA extraction protocol (Qiagen, Germantown, MD) and modifications. A blank was
included in the extraction process to confirm the absence of contamination during
extraction. To identify the presence of Borrelia burgdorferi, DNA was amplified using a
26
TaqMan real-time PCR protocol. A specific primer and probe targeting the 16s-23s
intergenic spacer region of Borrelia burgdorferi was used (Table 2). A 25µL TaqMan
real-time PCR (qPCR) reaction containing 1X TaqMan Master Mix (Thermofisher,
Waltham, MA) was used for qPCR. The reaction was made up of 12.5L TaqMan master
mix, 5L Qiagen nuclease free water, 4.48L of premixed forward and reverse primer,
0.56L B. burgdorferi probe, and 2.5L of sample DNA. Thermal cycler conditions for
qPCR were performed at 50C for 2 minutes, an enzyme activation of 95C for 10
minutes, followed by 50 cycles at 95C for 15 seconds and 60C for 1 minute. Positive
samples were determined with CT values of 30-38 at a threshold of 0.047. A positive
control was run with each analysis to validate the assay and a negative control to confirm
the absence of contamination.
Table 2. Oligonucleotide primer used for PCR. SYBR green primer targeting the NS5
region of Powassan virus. Real-time primer targeting the 16S-23S intergenic spacer
region for Lyme (Borrelia species) and specific probe for Borrelia burgdorferi
Target
Organism
Powassan
virus (DTV)
Powassan
Virus
(DTV)
Gene
Target
Sequence (5’ → 3’)
Amplicon Size
(bp)
F 5’-AACATGATGGGAAAGAGAGAG-3’
NS5
318bp
R 5’ -CAGATCCTTCGGTACATGGAA-3’
Borrelia
species
16S-23S
intergenic
spacer
Borrelia
burgdorferi
Probe
F 5’-GCTGTAAACGATGCACACTTGGT-3’
R 5’-GGCGGCACACTTAACACGTTAG-3’
6FAM-TTCGGTACTAACTTTTAGTTAAQSY
27
69bp
Statistical Analysis
The minimum infection rate (MIR) was determined by dividing the total number
of positive individuals and pools by the total number of ticks analyzed. This value
assumes only one tick per pool was infected; therefore, it is a conservative estimate of
prevalence. The minimum infection rate calculated from the tested ticks was used as an
estimate of the minimum prevalence of infected ticks in New Jersey and Pennsylvania.
Prevalence rates were determined by life stage for adults and nymphs.
Statistical analysis was conducted using the statistical computing program
R34,56,67. A Chi-square test was used to determine if there was significance between the
proportion of tick species collected in New Jersey and Pennsylvania. A generalized linear
model (GLM) was used to test for the effect of engorgement status (unengorged, semiengorged or fully engorged) and life stage (nymph or adult) on the minimum infection
rate. All factors were treated as fixed. A GLM was also used to test for the effect of the
state (New Jersey/Pennsylvania) and year (2015-18 in New Jersey, 2017-28 in
Pennsylvania) on infection rate. Lastly, a GLM was used to test for effect of New Jersey
counties on infection rate. For all statistical analyses, the criterion for significance was
set to = 0.05.
28
Chapter III
Results
Tick Collections
Appendix A presents the total number of ticks collected from 2015-2018 by
collection season (Fall harvest and Fall or Spring research trapping), year, state, species
and life stage. Overall, four species of ticks were collected: Ixodes scapularis, Ixodes
cookei, Amblyomma americanum and Dermacentor variabilis. Larval, nymph and, adult
life stages were collected of I. scapularis were collected. All other tick species were
collected in their adult life stage. The most commonly collected tick was Ixodes
scapularis (n=2,119) (78.1%), followed by Dermacentor variabilis (n = 590) (21.7%),
Amblyomma americanum (n=2) (0.07%) and Ixodes cookei (n=2) (0.07%). The average
tick load per black was 6.33. The total number of ticks collected from New Jersey
between 2015-18 was 2,133 and in Pennsylvania from 2017-18 was 580. There was no
significant difference between New Jersey and Pennsylvania in the relative abundance of
tick species collected (chi-squared test; X2= 8, p = 0.2381, df = 6).
29
In New Jersey, four different tick species were collected throughout the year, with
the majority of collected ticks being adult I. scapularis in October and D. variabilis in
30
June. Overall the majority of ticks were collected in October when adult I.
scapularis are most active, then in June when I. scapularis nymphs and adult D.
variabilis are most active (Figure 10). Fewer ticks were collected in August, most likely
due to the hot, dry weather unfit for tick activity. Fig. 10 presents the total number of all
ticks collected in the month of October between 2015-18, in June 2015-18 and August
2015-18. There were 1,554 I. scapularis collected between 2015-18 in New Jersey with
98% being adults, 1.6% being nymphs and 0.1% being larval. The majority of adults
were collected each year in October (Figure 11).
1600
Total Ticks Collected
1400
1200
1000
800
600
400
200
0
October
June
August
Month
Figure 10. Total number of ticks collected from 2015-18 in New Jersey by month
31
Total I. scapularis Collected
600
500
400
300
200
100
0
October October June 2016 August October June 2018 August
2015
2016
2017
2017
2018
October
2018
Month and Year
Figure 11. Total adult I. scapularis collected in the month throughout the year from New
Jersey (2015-18)
Three tick species collected in Pennsylvania between 2017-18. The majority of
ticks in Pennsylvania were collected in May, June, and November, active months for
nymphs in the Spring and Summer, and adults in the Spring and Fall (Figure 12). The
majority of ticks collected were I. scapularis, with 90.6% consisting of adults, 5.3%
nymphs and 4.1% larval. Adult I. scapularis were collected through the majority of
months but were most prevalent in May, June and November during periods that I.
scapularis adults are known to be most active (Figure 13). Nymph I. scapularis were
collected in five months out of the year, with May, June and August having the highest
collection rate (Figure 14). Larval I. scapularis were collected four months out of the
year with August being the peak month for larval collection (Figure 14).
32
Total number of ticks collected
200
180
160
140
120
100
80
60
40
20
0
Month
Figure 12. Total number of ticks collected from 2017-18 in Pennsylvania by month
Total I. scapularis collected
160
140
120
100
80
60
40
20
0
Month anf Year
Figure 13. Total number of adult I. scapularis collected in Pennsylvania in 2017-18 by
month
33
Total Number of larval and nymph
collected
30
25
20
15
Nymph
Larval
10
5
0
March
May
June
July
August
Month
Figure 14. Total number of nymph and larval I. scapularis collected in Pennsylvania in
2017-18 by month
Optimization of Powassan Lineage II Assay
In order to standardize the Powassan virus Lineage II RT-PCR assay a standard
curve was created using a synthetic positive created by GENEWIZ (South Plainfield, NJ).
The synthetic positive control was created from the targeted NS5 primers used in the
assay. Using 10 fold serial dilutions ranging from 10-1 to 10-10 with a starting
concentration of 1.6711 copies/L were used and performed in triplicate to validate
accuracy. The 10-7 dilution was the last dilution to have a CT call for all three samples.
CT values from dilutions 10 -1 to 10-7 were inserted into Microsoft Excel and a linear
regression was created using the log of number of copies per each dilution. The slope
formula was determined to be y = -5.036x + 74.524, with an R2 of 0.9853 (Figure 15).
The CT cutoff value was determined to be 44.7 by plugging in the highest triplicate value
34
from the 10-7 dilution into the slope formula. A threshold generated by the Applied
Biosystems StepOnePlus standard curve was 1.725 for Powassan virus (DTV)
50
45
y = -5.036x + 74.524
R² = 0.9853
40
35
CT
30
25
20
15
10
5
0
5
6
7
8
9
10
11
12
13
Log of copy number
Figure 15. Plotted linear regression of the standard curve, using the log number of copies
of each dilution by the CT call from each triplicate
Disease and Statistical Analysis
Ixodes scapularis were selected for analysis from three counites in New Jersey
and nine counties in Pennsylvania because they are the known vectors of Powassan virus
Lineage II. Samples that were selected to be analyzed were separated by state, county and
year and each individual was accounted for (Table 3,Table 4). Counites in New Jersey
were picked according to which had the highest yield of I. scapularis collected.
Hunterdon County was also included as it has had a known human case of Powassan
virus. All I. scapularis nymph and adult ticks from Pennsylvania were analyzed due to
having only two years of ticks collected. A total of 1,277 blacklegged ticks were selected
35
to be analyzed for Powassan virus Lineage II and Lyme disease (Borrelia burgdorferi)
using the polymerase chain reaction (PCR). A total 595 samples consisting of 344
individuals and 251 pools were analyzed (Appendix B). Of these, 831 (64.9%) were
female, 384 (30.0%) were male and 64 (5.1%) were nymphs (Figure 16).
Table 3. Total number of blacklegged ticks analyzed from each county
in New Jersey
YEAR
Hunterdon
Sussex
Warren
Total
2015
26
23
21
90
2016
0
184
47
231
2017
0
109
91
200
2018
0
108
117
225
36
37
32
0
2017
2018
0
3
10
0
22
0
304
0
23
0
111
0
5
0
23
0
498
35
YEAR Monroe Pike Centre Clearfield Clinton Huntingdon Lycoming Potter Toga Total
Table 4. Total number of blacklegged ticks analyzed from each county in Pennsylvania
300
Number of Ticks
250
200
Female
150
Male
Nymph
100
50
0
NJ 2015 NJ 2016 NJ 2017 NJ 2018 PA 2017 PA 2018
State and Year
Figure 16. Total number of I. scapularis female, male and nymph ticks analyzed (n=
1,277) from NJ (2015-18) and (PA 2017-18)
Powassan Virus
The overall minimum infection (MIR) rate was determined for positive tick pools
for Powassan virus as 3.52%. The overall minimum infection rate was determined for
adult blacklegged ticks positive for Powassan virus as 3.54%. The MIR of adults I.
scapularis infected in New Jersey ranged from 0-3.0% between 2015-18, with 2017
having the highest MIR with 3.0%. The MIR in northeast Pennsylvania counties was
5.7%, compared to 4.0% in central Pennsylvania counties (Figure 17). The minimum
infection rate was determined for nymph blacklegged ticks positive for Powassan virus in
New Jersey 0%, and Pennsylvania 3.92% for 2018 (Figure 18).
38
6.0%
MIR % Positive
5.0%
4.0%
3.0%
2.0%
1.0%
0.0%
NJ 2015
NJ 2016
NJ 2017
NJ 2018
PA 2017
PA 2018
State and Year
Figure 17. Minimum infection rate (MIR) percent of adult I. scapularis with POW (DTV)
(n=1,213) from NJ (2015-18) and PA (2017-18)
5%
4%
MIR % Positive
4%
3%
3%
2%
2%
1%
1%
0%
NJ 2018
PA 2018
State and Year
Figure 18. Minimum infection rate percent of nymph I. scapularis ticks with POW
(DTV) (n=64), from NJ and PA 2018
39
Ixodes scapularis nymphs and adults did not differ in Powassan infection
prevalence (GLM df = 2, p = 0.897). Powassan prevalence also did not vary with
engorgement state in either adults (df = 3, p = 0.910) or nymphs (df = 1, p = 0.976).
There was no significant difference between males and females in Powassan infection (df
= 1, p = 0.836). There was no significant difference between New Jersey and
Pennsylvania overall in Powassan infection (df = 1, p = 0.852). Appendix C consist of
full statistical tables.
Borrelia burgdorferi
The overall minimum infection rate was determined as 19.2% for ticks positive
for B. burgdorferi. The overall minimum infection rate was determined as 19.8%. for
adult blacklegged ticks positive for B. burgdorferi. The MIR of adults I. scapularis
infected in New Jersey ranged from 17.7-23.0% between 2015-18, increasing each year
(Figure 19). The MIR in northeast Pennsylvania counties was 28.5%, compared to 18.1%
in central Pennsylvania counties. The minimum infection rate was determined for nymph
blacklegged ticks positive for B. burgdorferi in New Jersey 0%, and Pennsylvania 7.84 %
for 2018 (Figure 20).
40
30.0%
MIR % Positive
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
NJ 2015
NJ 2016
NJ 2017
NJ 2018
PA 2017
PA 2018
State and Year
Figure 19. MIR (%) of adult I. scapularis ticks with B. burgdorferi (n=1,213) from NJ
(2015-18) and PA (2017-28)
9%
8%
MIR% Positive
7%
6%
5%
4%
3%
2%
1%
0%
NJ 2018
PA 2018
State and Year
Figure 20. Minimum infection rate percent of nymph I. scapularis ticks with B.
burgdorferi (n=64), from NJ and PA 2018
41
I. scapularis nymphs and adults did not differ in Borrelia burgdorferi infection
prevalence (GLM df = 2, p = 0.725). B. burgdorferi prevalence also did not vary with
engorgement state in either adults (df = 3, p = 0.799) or nymphs (df = 2, p = 0.815).
There was no significant difference between males and females in B. burgdorferi
infection (df = 1, p = 0.945). There was no significant difference between New Jersey
and Pennsylvania in B. burgdorferi infection (df = 1, p = 0.986).
Co-infection
Overall of the 45 positive Powassan ticks, 25 of them were co-infected with B.
burgdorferi (55.5%). Of these, 23 were adults (53.4%) and the two Powassan positive
nymph pools were both co-infected with B. burgdorferi (100%). New Jersey had 11 coinfected tick pools and Pennsylvania had 14. There were 12 co-infected female pools, 11
co-infected male pools and 2 co-infected nymph pools. The majority of co-infected ticks
came from central Pennsylvania in 2018, compared to New Jersey 2015-18 and
Pennsylvania 2017.
County Data
Powassan
Positive pool samples of Powassan virus in New Jersey counties were consistent
over the three years 2016-18. Sussex County had the most positive tick pools compared
to Warren or Hunterdon Counties (Table 5). No counites in 2015 had a positive pool for
42
Powassan virus. Northeast Pennsylvania presented a lower positive pool sample of
Powassan virus than central Pennsylvania (Table 6). Clinton County in central
Pennsylvania had the highest amount of positive pools of Powassan virus throughout
New Jersey or Pennsylvania with 16 positive tick pools.
New Jersey did not differ in Powassan infection prevalence between 2015- 2018
(GLM df = 2, p = 0.986). Pennsylvania did not differ in Powassan infection prevalence
between 2017 and 2018 (df = 1, p = 0.906). There was no significant difference between
Sussex and Warren county (df = 1, p = 0.917), Hunterdon county was excluded due to no
positive ticks. No statistical test was run for Pennsylvania counties due to uneven
distribution of ticks collected between counites.
Table 5. Total Powassan positive pools by year in each New Jersey county
COUNTY
HUNTERDON
SUSSEX
WARREN
TOTAL
2015
0
0
0
0
2016
0
4
2
6
2017
0
5
1
6
2018
0
4
1
5
Table 6. Total Powassan positive pools by year in each Pennsylvania county
COUNTY
PIKE
MONROE
CENTRE
CLEARFIELD
CLINTON
HUNTINGDON
LYCOMING
POTTER
2017
0
2
0
0
0
0
0
0
2018
0
0
0
2
16
1
5
0
43
TIOGA
TOTAL
0
2
2
26
Borrelia burgdorferi
The amount of positive tick pools overall and in Sussex County fluctuated over
the years. In contrast, Warren had a continuous increase of positive tick pools between
2015-18 (Table 7). Northeast Pennsylvania had fewer positive pools than central
Pennsylvania for B. burgdorferi (Table 8). Overall, Clinton County had the most positive
tick pools in Pennsylvania and New Jersey for B. burgdorferi.
New Jersey did not differ in B. burgdorferi infection prevalence between 2015
and 2018 (GLM df = 3, p = 0.885). Pennsylvania did not have a significant difference in
B. burgdorferi infection prevalence between 2017 and 2018 (df = 1, p = 0.955). There
was no significant difference between counties in New Jersey 2015-18 (d=3, p = 0. 720).
Statistical analysis were not conducted for Pennsylvania counties due to uneven
distribution of tick collection.
Table 7. Total B. burgdorferi positive tick pools by year in New Jersey counties
COUNTY
HUNTERDON
SUSSEX
WARREN
TOTAL
2015
4
6
6
16
2016
0
36
9
45
2017
0
27
14
41
44
2018
0
25
22
47
Table 8. Total B. burgdorferi positive tick pools by year in Pennsylvania counties
PIKE
MONROE
CENTRE
CLEARFIELD
CLINTON
HUNTINGDON
LYCOMING
POTTER
TIOGA
TOTAL
2017
0
10
0
0
0
0
0
0
0
10
2018
0
0
0
2
55
3
19
1
6
86
45
Chapter IV
Discussion
Powassan virus is an emerging tick-borne virus that has the potential to be
debilitating and deadly to those infected. Lyme disease is one of the top vector-borne
diseases in the United States and has been increasing in the amount of cases reported
every year1. Conducting prevalence studies is an important tool to understanding tickborne diseases in areas with high human and tick interactions. This is the first study
identifying the prevalence of Powassan virus (DTV) and Lyme disease (Borrelia
burgdorferi) co-infection in Ixodes scapularis collected from black bears in New Jersey
and Pennsylvania.
Tick Collection Data
Tick distribution in the United States has been changing over the last decade.
Ticks not established in regions are becoming established and populations of ticks in
established regions are on the rise59. In 2014-2018, the distribution of I. scapularis has
increased from the Northeast and Great Lake regions down south further into Texas and
46
west into the eastern part of Nebraska and Kansas. Eisen, Eisen & Beard (2017)
examined the county-scale distribution of I. scapularis and noted that it had been
documented in 1,420 of the 3,100 continental US counties as of 2016. This represented
an increase of 44.7% when compared to the previous county-scale distribution map
created in 199823. Other tick species such as Amblyomma americanum (Lone star tick)
have been increasing in geographic distribution. The Lone star ticks distribution has
increased from established areas in southern states to newly established areas as far north
as New York and Maine25.
Several factors play a key role in the increased distribution of tick populations in
the United States, with the two main hypotheses focused around climate change and
habitat fragmentation. To survive, the majority of ticks need moderately warm
temperatures. In fact, I. scapularis cannot survive temperatures over 40C or under
-10C59. Additionally, ticks need a moderately humid climate to survive, as too not dry
out or become overly saturated24. Climate change in the U.S. has caused warmer winters
and wetter summers. This has allowed ticks to have a higher survival rate over the winter
and summer24. In Warren County, NJ the average temperature in June 2015 was 24.6C
and in June 2018 increased to 24.8C. June of 2015 in Warren county had a high average
precipitation of 7.73 inches compared to June 2018 which had an average precipitation of
3.43 inches. In Monroe County the average temperature in June 2015 was 25.0C and in
June of 2018 it was 26.0C with an average precipitation of 10.95 inches in 2015 and
3.62 inches in 2018. In Clinton County the average temperature was 25.6C in 2015 and
25.2C in 2018 with an above average precipitation in 2015 of 8.36 inches and an
47
average precipitation of 5.28 inches in 2018. Between 2015 and 2018 in these three
counties temperatures tended to increase and had lower precipitation each year. The high
precipitation in 2015 may of caused the tick population that year to become overly
saturated with water, while in 2018 the precipitation was close to the known average for
each county and could of created a more stable humid for ticks to survive in 14.
Changes due to human growth have not just impacted the climate but wildlife
habitat too. Human growth and expansion have increased forest fragmentation and
changed the micro-habitat of wildlife. Allan, Keesing & Ostfeld (2003) conducted a study
to determine if forest fragmentation increased the white-footed mouse (Peromyscus
leucopus) population and if it affected the infection rate of B. burgdorferi in larval and
nymph I. scapularis. They concluded that in highly fragmented areas, the white-footed
mouse population increased, possibly due to low predator abundance and low
competition of resources. As the white-footed mouse is the main reservoir for many tickborne pathogens, they noted larval and nymph infection rates of B. burgdorferi increased
in highly fragmented areas2. Furthermore, forest fragmentations create edge habitats that
are suitable for high populations of white-tailed deer (Odocoileus virginianus), a
favorable host for I. scapularis adults to feed and reproduce on11. Forest fragmentations
have increased the risk of human-tick interactions as many of these forest fragmentations
border homes11 and increased populations of ticks are present in them.
In this study, Lone star ticks were collected from black bears in 2018 in northern
New Jersey and northeast and central Pennsylvania. Lone star ticks were not found on
black bears in previous years in either states. Previous studies conducted by Zolink et al.
(2015) and Chern, Bird & Frey (2016) collected ticks from black bears in New Jersey and
48
found the majority of ticks to be adult D. variabilis and adult and nymph I.
scapularis13,68. Although, nymph and larval I. scapularis were only found in 2017 and
2018 on black bears during this study. The collection of larval and nymph I. scapularis
from black bears could be due to an increase in the nymph and larval populations in 2017
and 2018 and choosing a larger mammal to feed on due to accessibility of feeding space.
Ticks used in this study from 2015 and 2016 were collected by previous students and all
nymphs collected those years may have been used for other studies. Lastly, another
reason could be due to a more thorough search on the black bears by collectors, looking
for all life stages of ticks, and not just the large adults easily visible to the eye. Black
bears were combed over in 10-15-minute increments, a short period of time to search for
ticks. The average bear had 6.7 ticks collected from it, with the low being 0 and the
highest being 44 ticks. Al-Warid et al. (2017) determined an average tick community
composition of 400 ticks on black bears in Missouri. Al-Warid did not state if there was a
time restraint for each tick search. A longer search duration may yield a higher average of
ticks on black bears in New Jersey and Pennsylvania and may more accurately describe
the community composition of ticks on these bears.
Black bears may play an important role in the dilution of tick-borne diseases in an
area. Black bears, like white-tailed deer, are not known reservoirs of many tick-borne
pathogens but can host many ticks on their bodies. Huang et al. (2019) conducted a study
on Block Island, RI and determined when nymphs and larval I. scapularis fed on whitetailed deer, the infection rate of B. burgdorferi in I. scapularis decreased the next year.
Black bears may also help to decrease the infection rate of I. scapularis if larval and
nymphs begin to feed on them as their first and second meals. Black bear populations in
49
the New York, New Jersey and Pennsylvania are on the rise and if they are becoming a
primary host for larval, nymph and adult I. scapularis the infection rate of tick-borne
disease may decrease in high-density black bear areas. It is unknown if black bears are
reservoirs for Powassan virus and is unclear at this time if they play a role the
transmission cycle.
Powassan (DTV) Prevalence
Few prevalence studies for Powassan virus lineage I and/or lineage II have been
conducted over the last decade. A study conducted by Brackney et al. (2008) determined
that 1.3% of I. scapularis adults tested were DTV positive in Wisconsin. Another study
conducted in 2011-12 by Knox et al (2017) analyzed four quadrants of Wisconsin and
determined a MIR range for DTV between the four quadrants to be 1.56-4.62%.
Anderson and Armstrong (2012) analyzed adult and nymph I. scapularis in Connecticut
and found 0.8% to 1.6% DTV-positive tick pools in Bridgeport and 0.4% to 3.9% DTVpositive pools in North Branford. A study conducted by Dupuis II et al. (2013) analyzed
adult and nymph I. scapularis collected from several counties in Hudson Valley, NY
between 2007 and 2012 and determined a Maximum Likelihood Estimate (MLE) range to
be 0.2-6.0% for adults. Results from this study were similar to those found in previous
studies, with a minimum infection rate (MIR) prevalence ranging from 0.0-3.0% in New
Jersey (2015-18) and 4.0-5.7%% in Pennsylvania (2017-18). As the MIR was calculated
to determine the prevalence of tick pools in New Jersey and Pennsylvania, the true
prevalence rate of each state may be higher, as it was assumed only one tick was positive
for Powassan virus in a pool and not multiple ticks in the pool being positive.
50
The prevalence of Powassan virus (DTV) in Warren and Sussex County, New
Jersey appear to be stable, as each year the amount of positive pools either increased by
one or decreased by one. It is unclear what the true prevalence of Powassan virus (DTV)
is in Hunterdon County, as only one year had tick pool samples analyzed and zero were
positive. The stability of Powassan virus (DTV) in Pennsylvania could not be determined
by this study for reported counties as each county was only analyzed for one year.
Although it was not found to be statistically significant Pennsylvania did have a higher
prevalence rate than New Jersey in 2018 with 2.2% and 4.0% in Pennsylvania. These
results could possibly be biased due to the sample size difference between the two states
in 2018, with 102 more sample pools analyzed in Pennsylvania than New Jersey. Other
biases may be seen in the results from Pennsylvania 2017 due to a small sample size of
only 26 tick pools. An uneven number of ticks were collected from counties in
Pennsylvania, creating an uneven distribution of tick pools per county. This could create
biased results for counties with small tick pools such as Potter (5 ticks pools) and Centre
(4 tick pools) compared to Clinton which had 203 tick pools. Although several of the
counties had small sample sizes, Powassan positive tick pools came from eight of the
twelve counties analyzed. This indicates a risk in several counties in New Jersey and
Pennsylvania for humans to come into contact with Powassan infected ticks.
All positive pools for Powassan in New Jersey came from ticks collected in
October, while the majority of positive ticks in Pennsylvania were collected in May and
June. One possible explanation for this difference is that the population of the whitefooted mouse, the primary reservoir of Powassan virus (DTV), may be different in New
Jersey and Pennsylvania. If there are more white-footed mice in one area than another
51
ticks have a greater opportunity to feed on these mice as their first or second meal. This
could cause some ticks to be infected earlier in the year and others to be infected later in
the year. The majority of ticks analyzed were adults and 43 of the 45 positive tick pools
were composed of adults. In New Jersey, adult I. scapularis ticks are most active in the
Fall and as noted the majority of Powassan positive ticks were collected in October. The
Fall months, notably October, in New Jersey are potential high-risk months for Powassan
virus infection from I. scapularis. In Pennsylvania, ticks had the highest rates of
Powassan infection in May and June. Although adults are not typically the most active in
the Spring and Summer, they can be found questing for a host and will attach to a host if
they find one. Nymphs are most active during this season and with two positive nymph
pools, there is a risk that Powassan infection is present in these months in Pennsylvania.
A concern with nymph ticks being positive for infection is the fact they are very small,
and the majority of humans will not know to check or may miss them during a tick check,
giving these ticks ample time to feed and transmit tick-borne diseases. As Powassan virus
can transmit within 15 minutes adults and nymphs are both likely to be able to transmit
the virus even if the tick is found within a short amount of time of attaching and feeding
before being removed.
There was no significant effect of gender, life stage, or engorgement on Powassan
infection of I. scapularis. This indicates there is no difference in the chance of
contracting Powassan virus from a I. scapularis depending on its sex, life stage or
engorgement level. There is also no significant difference in Powassan infection between
2015-18 in New Jersey or 2017-18 in Pennsylvania. Lastly, there was no significant
difference between the overall positive pools in New Jersey and Pennsylvania.
52
Powassan virus (DTV) cases have been increasing over the last decade with 6 human
cases reported in the US in 2015, 21 reported in 2016, and 33 reported in 2018.
Prevalence research has been sparse for Powassan virus over the last decade, although it
is on the rise. Most cases of Powassan virus have occurred in the Northeast and Great
Lakes regions of the U.S., which are in Lyme endemic regions 63. It is important for states
with confirmed Powassan virus (DTV) cases to monitor the tick population to better
understand the prevalence rate in high-risk areas for infection.
It is unclear how Powassan virus interacts with other tick-borne diseases, such as
Lyme disease, or why some individuals are asymptomatic to the virus and others develop
severe symptoms. This study did find I. scapularis adults and nymphs capable of being
co-infected with Powassan virus and B. burgdorferi. Further research needs to be
conducted on Powassan virus to better understand prevalence, transmission effects in
presence of other tick-borne diseases and its ability to cause disease in those infected.
Borrelia burgdorferi Prevalence Data
Previous studies have been conducted on the prevalence of B. burgdorferi in I.
scapularis ticks in Lyme disease endemic regions of the U.S.. Courtney et al. (2003)
conducted a prevalence study in Northwestern and Southeastern Pennsylvania and
determined a prevalence of 61.6% and 13.1%, respectively. A study conducted by Steiner
et al. (2008) collected adult I. scapularis from Indiana, Maine, Pennsylvania and
Wisconsin and found prevalence rates that ranged between to 35% and 70%. In central
New Jersey, Schulze et al. (2005) analyzed adult I. scapularis and determined an
infection prevalence for B. burgdorferi to be 50.3%. Prusinski et al. (2014) collected I.
53
scapularis adults and nymphs from eight New York Counties from 2003 to 2006 and
determined an overall prevalence rate of B. burgdorferi 14.4% in adults and 45.7% in
nymphs.
The current study determined a MIR prevalence range of 17.7-22.6% in New
Jersey 2015-18 for B. burgdorferi. The MIR prevalence in Pennsylvania in 2017 and
2018 was 28.5% and 18.9%. These findings are comparable to other previous studies
conducted for B. burgdorferi prevalence in Lyme endemic regions. In a thesis study
conducted by Bird (2014) found a MIR of 0% of I. scapularis larval pools positive and
41% adult I. scapularis pools positive for B. burgdorferi7. The 9.6% difference between
2017 and 2018 could be caused by the large sample size difference between 2017 (26 tick
pools) and 2018 (203 tick pools). Additionally, different regions of Pennsylvania may
have different prevalence rates of Lyme disease, as seen with 2017 ticks collected from
the Northeast region of Pennsylvania and 2018 ticks collected from the Central region of
Pennsylvania.
Overall, out of 245 positive pools 98.3% were adults and 1.63% were nymphs.
Similar to Powassan, the majority of positive B. burgdorferi tick pools in New Jersey
were ticks collected in October. These may be due to the increased number of adult I.
scapularis out questing during this time of the year. Female adult I. scapularis accounted
for 71.8% of positive ticks pool, males made up 26.5% of the positive pools and nymphs
made up 1.63% positive pools. The Fall is a high-risk season for Lyme disease with large
populations adults questing, especially females in New Jersey. Pennsylvania had the
majority of B. burgdorferi positive tick pools from ticks collected in May (23 positive
tick pools), June (22 positive tick pools) and November (17 positive tick pools). All other
54
positive tick pools were from ticks collected in March, July, August, and December. Risk
for Lyme disease in Pennsylvania is high throughout the Spring, Summer and Fall due to
the high percentage of nymphs questing in the Spring and Summer and the adults
questing in the Fall. Human contact with ticks increases in the Spring, Summer, and Fall
as the warmer weather increases outdoor activities such as hiking and camping increase.
There was no significant difference between life stage, or engorgement and B.
burgdorferi positive ticks. There is no difference in chance of contracting B. burgdorferi
from a I. scapularis depending on its life stage or engorgement level. There was no
significant difference between 2015-18 positive tick pools in New Jersey or 2017-18 in
Pennsylvania. There was no significant difference between the amount of overall positive
tick pools between New Jersey and Pennsylvania.
As nymph ticks play a crucial role in causing Lyme disease infections in humans,
this study is not consistent with other studies with regards to prevalence rates in nymph I.
scapularis ticks. This can be due to the small sample size of nymphs. Also, the MIR was
calculated for prevalence and these numbers may be underestimating the true percentage
of positives ticks, as it was assuming only one out of the number of ticks in the pool was
positive for adult and nymph pools analyzed.
The CDC reported 12,801 confirmed cases of Lyme disease in 1997. Just20 years
later, the CDC reported 29,513 confirmed cases and 13,230 probable cases. The rise of
Lyme disease in the Northeastern and Midwest United States is a cause for alarm and a
need for continuous prevalence and surveillance studies to monitor high risk.
55
Co-infection Prevalence Data
I. scapularis is the vector to many different pathogens and is able to harbor more
than one pathogen at once. Many of these pathogens such as B. burgdorferi and
Powassan virus (DTV) cause infection in humans. Studies that have examined the ability
of I. scapularis to have co-infections and found them to be able to harbor up to four or
more pathogens65. Studies have focused on the infection rate and co-infection rate of B.
burgdorferi with the other tick-borne pathogens that include Anaplasma
phagocytophilum, Babesia microti, Powassan virus (DTV), and Borrelia miyamotoi65.
Many of these pathogens share the reservoir host of small mammals, specifically the
white-footed mouse (Peromyscus leucopus)16. Larval and nymphs can become infected
when they feed off of the reservoir host during their first or second meal. The larval and
nymph ticks will continue to carry these pathogens between molting to the next life stage
and can transmit the pathogen(s) to its next host during feeding.
Tokarz et al. (2010) conducted a study in New York where 70% of I. scapularis
had one pathogen and 30% had a polymicrobial infection. Specifically, they found that
24% were co-infected with B. burgdorferi/A. phagocytophilum, 31% were co-infected
with B. burgdorferi/Babesia microti, and five ticks were Powassan virus (DTV) positive,
with two being co-infected with Powassan (DTV)/B. burgdorferi (40%). Knox et al.
(2017) analyzed adult I. scapularis in Wisconsin and found that eight were positive for
Powassan virus (DTV) and four of the eight were co-infected Powassan (DTV)/B.
burgdorferi (50%).
This current study had similar co-infection rates to Knox et al. (2017) and Tokarz
et al. (2010) in adult I. scapularis, with 53.4% Powassan virus (DTV) positive adults
56
being co-infected with B. burgdorferi. Frost et al. (2015) tested patients with one known
tick-borne disease for Powassan and 17.1% of patients showed serological evidence that
they were infected with both B. burgdorferi and Powassan virus. Three patients (7.3%)
were laboratory confirmed (PCR) to have a Powassan and B. burgdorferi co-infection.
Co-infections increase the difficulty for accurately diagnosing and treating tickborne diseases43. Pathogens can range from bacterial to protozoan to viral and need
different treatment methods. Symptoms of one tick-borne disease can be masked by
another when a co-infection occurs. This allows one pathogen to be treated but the other
to continue causing symptoms and illness. Some studies, such as those conducted by
Thomm et al (2018), have suggested Post Treatment Lyme Disease Syndrome (PTLDS)
may be caused by an undiagnosed co-infection. Thomm et al (2018) has worked to
develop and validate a serological test panel for the detection of Powassan virus and has
suggested those who have been treated for Lyme disease but have persistent symptoms
consistent with PTLDS should be tested for Powassan virus due to a possible coinfection. With co-infections as high as 28% in I. scapularis ticks in Lyme disease
endemic regions in the U.S., surveillance studies are important to monitor co-infection
rates in these areas.
Conclusion
The overall increase in tick populations and distribution can have severe impacts
on human infection and tick-borne diseases. Ticks established in new areas increase the
risk of tick-borne disease brought into areas that they were not present in before. The
increased distribution of I. scapularis may be a possible cause of the increase in Lyme
57
disease infections in the U.S. and may lead to an increase of other tick-borne diseases
such as Powassan virus (DTV). Lone star ticks are known to carry the tick-borne
pathogens Ehrlichia chaffeensis, E. ewingii, Borrelia lonestari (STARI), and tularemia.
Human and pet populations in New Jersey and Pennsylvania can be at risk of being bitten
by Lone star ticks and becoming infected with these diseases, as they are now known to
inhabit these areas.
Tick surveillance studies such as this one help to determine the prevalence rate
and high-risk areas for tick-borne diseases. Pennsylvania and New Jersey are the top two
states for Lyme disease cases in the US 4. This is the first study to determine the MIR of
Powassan virus in New Jersey and Pennsylvania and found the MIR to be similar to rates
determined in previous studies. This indicates that these states are at risk for human tickborne infections and should be continued to be monitored for human, pet and wildlife
health. Tick surveillance is important to continue to monitor populations of new and
established ticks and diseases for the safety and health of those living in tick populated
areas.
Future Study
As this was the first study in New Jersey or Pennsylvania to survey the prevalence
of Powassan virus (DTV) and co-infection with Lyme disease (B. burgdorferi), there is
ample opportunity to continue monitoring the prevalence rates in these states. With
Powassan virus, as an emerging tick-borne virus capable of causing deadly disease in
humans, it is important to monitor the prevalence rate of it to determine if risk to humans
increases. This allows for physicians to be aware should a patient exhibit symptoms of it.
58
Additionally, only three of the twenty-one counties in New Jersey had ticks analyzed for
Powassan virus (DTV), leaving a need for other counties in New Jersey to be analyzed in
future work. Pennsylvania had nine out of sixty-seven counites analyzed with uneven tick
samples from each county, future studies can conduct more thorough analysis of counties.
Blacks bears in this study had several different ticks’ species and life stages
attached to them. Future studies can conduct a more thorough search on black bears to
better determine what the tick community composition consists of on black bears in New
Jersey and Pennsylvania. Another study can be done using serological test (ELISA, IFA
or PRNT50 or 90) with blood to determine if black bears have the capability to be reservoirs
of Powassan virus, Lineage I or Lineage II, as they were found to be host to I. scapularis
and I. cookei.
There has not been a study conducted to determine the prevalence rate of infected
white-footed mice with Powassan virus (DTV) in Pennsylvania or New Jersey. As the
reservoir host to many tick-borne pathogens, observing the prevalence of these pathogens
in these mice can help to determine the prevalence rate of DTV and the chances of ticks
obtaining the infection when feeding on wild mice.
59
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Appendix A: Tick Collection Raw Data
Tick Collection New Jersey 2015
County
Ixodes scapularis
Adult
Hunterdon
25
Morris
Passaic
Sussex
Warren
Total
6
0
23
51
105
Tick Collection New Jersey 2016
Ixodes scapularis
Dermacentor variabilis
County
Hunterdon
Morris
Passaic
Sussex
Warren
Total
County
Morris
Passaic
Sussex
Warren
Total
Adult
0
28
10
309
54
401
Adult
0
0
14
48
145
207
Tick Collection New Jersey 2017
Ixodes scapularis
Dermacentor
variabilis
Adult
Nymph
Larval
Adult
54
0
0
0
20
0
0
0
262
0
0
1
93
5
1
72
429
5
1
73
67
Ixodes
cookie
Adult
0
0
0
1
1
County
Morris
Passaic
Sussex
Warren
Total
Tick Collection New Jersey 2018
Ixodes scapularis
Dermacentor Ixodes Amblyomma
variabilis
cookei americanum
Adult Nymph Larval
Adult
Adult
Adult
19
0
0
1
0
0
12
1
0
0
0
0
199
6
0
3
0
0
149
9
1
121
1
1
379
16
1
125
1
1
Tick Collection Pennsylvania 2017
County
Ixodes scapularis
Adult
Lackawanna
1
Monroe
35
Pike
3
Total
39
County
Centre
Clearfield
Clinton
Huntingdon
Lycoming
Potter
Tioga
Total
Tick Collection Pennsylvania 2018
Ixodes scapularis
Dermacentor
variabilis
Adult
Nymph
Larval
Adult
1
9
0
0
19
3
1
0
276
28
22
1
23
0
0
0
102
9
0
13
3
2
0
0
23
0
0
0
447
51
23
14
68
Amblyomma
americanum
Adult
0
0
1
0
0
0
0
1
Appendix B: Powassan and Lyme Raw Data
ID
Bear ID
Sex
Pooled
Ticks
Engorgement
POW
Lyme
Year
County
State
1
86
F
1
Semi
-
-
2017
Sussex
NJ
2
86
M
1
Un
-
-
2017
Sussex
NJ
3
86
F
1
Un
-
-
2017
Sussex
NJ
4
92
F
1
Un
-
-
2017
Sussex
NJ
5
111
F
1
Un
-
+
2017
Sussex
NJ
6
111
M
1
Un
-
-
2017
Sussex
NJ
7
291
F
1
Un
-
-
2017
Sussex
NJ
8
453
F
1
Semi
-
-
2017
Sussex
NJ
9
453
F
1
Semi
-
-
2017
Sussex
NJ
10
453
F
1
Fully
-
-
2017
Sussex
NJ
11
10
F
1
Semi
-
-
2017
Sussex
NJ
12
10
F
5
Semi
-
+
2017
Sussex
NJ
13
32
F
1
Semi
-
+
2017
Sussex
NJ
14
32
F
5
Semi
-
-
2017
Sussex
NJ
15
605
F
5
Semi
-
+
2017
Sussex
NJ
16
318
F
1
Fully
-
-
2017
Sussex
NJ
17
318
F
1
Semi
-
-
2017
Sussex
NJ
18
318
F
1
Un
-
+
2017
Sussex
NJ
19
106
M
1
Un
-
-
2017
Sussex
NJ
20
106
M
1
Un
-
-
2017
Sussex
NJ
21
103
F
1
Semi
-
+
2017
Sussex
NJ
22
69
F
1
Un
-
+
2017
Warren
NJ
23
69
F
1
Un
-
+
2017
Warren
NJ
24
69
M
1
Un
-
-
2017
Warren
NJ
25
41
F
1
Semi
-
+
2017
Warren
NJ
26
41
F
1
Semi
-
-
2017
Warren
NJ
27
41
F
1
Semi
-
+
2017
Warren
NJ
28
41
F
1
Semi
-
+
2017
Warren
NJ
29
41
M
1
Un
-
-
2017
Warren
NJ
30
296
F
5
Semi
-
-
2017
Warren
NJ
31
9273
F
1
Semi
-
-
2018
Warren
NJ
32
9273
F
1
Un
-
+
2018
Warren
NJ
33
9273
M
1
Un
-
-
2018
Warren
NJ
34
10202
F
1
Semi
-
+
2018
Warren
NJ
69
35
10202
F
1
Semi
-
+
2018
Warren
NJ
36
10202
F
1
Un
-
-
2018
Warren
NJ
37
10202
F
1
Un
-
-
2018
Warren
NJ
38
10202
M
2
Un
-
+
2018
Warren
NJ
39
9473
F
4
Semi
-
+
2018
Warren
NJ
40
9473
F
1
Fully
-
-
2018
Warren
NJ
41
9473
M
1
Un
-
-
2018
Warren
NJ
42
9194
F
1
Fully
-
-
2018
Sussex
NJ
43
9194
N
1
Fully
-
-
2018
Sussex
NJ
44
10041
M
1
Un
-
-
2018
Sussex
NJ
45
10041
N
1
Semi
-
-
2018
Sussex
NJ
46
10041
F
1
Semi
-
-
2018
Sussex
NJ
47
6573
M
1
Un
-
-
2018
Warren
NJ
48
6573
F
5
Un
-
+
2018
Warren
NJ
49
10003
M
1
Un
-
+
2018
Sussex
NJ
50
10003
F
1
Semi
-
-
2018
Sussex
NJ
51
10003
F
1
Semi
-
-
2018
Sussex
NJ
52
10003
F
1
Semi
-
-
2018
Sussex
NJ
53
10004
F
1
Semi
-
-
2018
Sussex
NJ
54
9149
N
1
Un
-
-
2018
Sussex
NJ
55
9887
F
1
Semi
-
-
2018
Sussex
NJ
56
9198
M
1
Un
-
+
2018
Sussex
NJ
57
9198
F
1
Semi
-
-
2018
Sussex
NJ
58
9848
F
1
Fully
-
-
2018
Sussex
NJ
59
9848
M
1
Un
-
-
2018
Sussex
NJ
60
10204
F
1
Un
-
+
2018
Warren
NJ
61
10204
F
1
Semi
-
+
2018
Warren
NJ
62
10204
F
1
Semi
-
+
2018
Warren
NJ
63
9776
F
1
Fully
-
-
2018
Sussex
NJ
64
9848
M
4
Un
-
+
2018
Sussex
NJ
66
19
F
1
Un
-
-
2018
Sussex
NJ
67
19
F
1
Un
-
-
2018
Sussex
NJ
68
7900
M
5
Un
-
-
2018
Warren
NJ
69
7900
F
3
Un
-
-
2018
Warren
NJ
70
7900
F
1
Semi
-
-
2018
Warren
NJ
71
7900
N
1
Fully
-
-
2018
Warren
NJ
72
9848
N
1
Fully
-
-
2018
Warren
NJ
73
9848
F
1
Un
-
-
2018
Warren
NJ
70
74
9848
F
3
Semi
-
-
2018
Warren
NJ
75
9848
F
4
Semi
-
-
2018
Warren
NJ
76
8814
F
1
Fully
-
-
2018
Warren
NJ
77
8844
M
5
Un
-
+
2018
Warren
NJ
78
8844
F
4
Semi
-
-
2018
Warren
NJ
79
8844
F
4
Semi
-
-
2018
Warren
NJ
80
8844
F
2
Fully
-
-
2018
Warren
NJ
81
8844
N
3
Fully
-
-
2018
Warren
NJ
82
295
M
3
Un
-
+
2017
Warren
NJ
83
632
F
1
Fully
-
-
2015
Warren
NJ
84
632
F
1
Fully
-
+
2015
Warren
NJ
85
631
F
1
Semi
-
-
2015
Warren
NJ
86
631
F
1
Semi
-
-
2015
Warren
NJ
87
631
F
1
Fully
-
+
2015
Warren
NJ
88
631
M
1
Un
-
-
2015
Warren
NJ
89
633
F
3
Semi
-
-
2015
Hunterdon
NJ
90
633
F
4
Semi
-
+
2015
Hunterdon
NJ
91
633
F
1
Fully
-
-
2015
Hunterdon
NJ
92
633
M
1
Un
-
-
2015
Hunterdon
NJ
93
630
F
1
Semi
-
+
2015
Warren
NJ
94
616
F
1
Semi
-
-
2015
Hunterdon
NJ
95
616
F
1
Semi
-
-
2015
Hunterdon
NJ
96
628
F
1
Semi
-
-
2015
Warren
NJ
97
628
F
1
Semi
-
-
2015
Warren
NJ
98
628
F
1
Fully
-
-
2015
Warren
NJ
99
628
F
1
Un
-
-
2015
Warren
NJ
100
628
M
1
Un
-
-
2015
Warren
NJ
101
627
M
1
Un
-
-
2015
Warren
NJ
102
627
F
1
Semi
-
-
2015
Warren
NJ
103
627
F
1
Semi
-
-
2015
Warren
NJ
104
625
F
1
Fully
-
-
2015
Warren
NJ
105
625
F
5
Semi
-
+
2015
Warren
NJ
106
625
M
3
Un
-
+
2015
Warren
NJ
107
624
F
1
Semi
-
-
2015
Warren
NJ
108
624
F
1
Un
-
-
2015
Warren
NJ
109
623
F
1
Semi
-
-
2015
Warren
NJ
110
623
F
1
Semi
-
-
2015
Warren
NJ
111
623
F
1
Un
-
-
2015
Warren
NJ
71
112
622
F
4
Fully
-
-
2015
Warren
NJ
113
622
F
3
Semi
-
-
2015
Warren
NJ
114
622
M
1
Un
-
-
2015
Warren
NJ
115
619
F
2
Semi
-
+
2015
Hunterdon
NJ
116
619
F
3
Fully
-
-
2015
Hunterdon
NJ
117
619
M
3
Un
-
-
2015
Hunterdon
NJ
118
618
F
1
Semi
-
+
2015
Hunterdon
NJ
119
618
F
1
Fully
-
+
2015
Hunterdon
NJ
120
618
M
1
Un
-
-
2015
Hunterdon
NJ
121
617
F
1
Fully
-
-
2015
Hunterdon
NJ
122
617
F
1
Fully
-
-
2015
Hunterdon
NJ
123
617
F
1
Fully
-
-
2015
Hunterdon
NJ
124
617
M
1
Un
-
-
2015
Hunterdon
NJ
125
21
F
1
Fully
-
-
2015
Warren
NJ
126
20
F
1
Fully
-
-
2015
Warren
NJ
127
15
F
1
Semi
-
+
2015
Warren
NJ
128
15
F
1
Semi
-
-
2015
Warren
NJ
129
41
F
1
Fully
-
-
2015
Sussex
NJ
130
39
F
1
Semi
-
-
2015
Sussex
NJ
131
39
F
1
Semi
-
-
2015
Sussex
NJ
132
35
F
1
Fully
-
-
2015
Sussex
NJ
133
34
F
1
Fully
-
+
2015
Sussex
NJ
134
34
M
1
Un
-
-
2015
Sussex
NJ
135
31
F
1
Fully
-
-
2015
Sussex
NJ
136
31
M
1
Un
-
-
2015
Sussex
NJ
137
30
F
1
Fully
-
+
2015
Sussex
NJ
138
30
M
1
Un
-
-
2015
Sussex
NJ
139
28
F
3
Fully
-
+
2015
Sussex
NJ
140
28
F
1
Semi
-
-
2015
Sussex
NJ
141
28
M
1
Un
-
+
2015
Sussex
NJ
142
22
F
1
Un
-
+
2015
Sussex
NJ
143
22
F
1
Fully
-
-
2015
Sussex
NJ
144
18
F
1
Semi
-
-
2015
Sussex
NJ
145
18
M
1
Un
-
-
2015
Sussex
NJ
146
13
F
1
Semi
-
-
2015
Sussex
NJ
147
13
M
1
Un
-
-
2015
Sussex
NJ
148
12
F
1
Fully
-
+
2015
Sussex
NJ
149
11
F
1
Semi
-
-
2015
Sussex
NJ
72
150
2
M
3
Un
-
-
2016
Sussex
NJ
151
2
F
4
Semi
-
+
2016
Sussex
NJ
152
2
F
4
Semi
-
-
2016
Sussex
NJ
153
2
F
1
Fully
-
+
2016
Sussex
NJ
154
3
M
4
Un
-
-
2016
Sussex
NJ
155
3
F
1
Un
-
-
2016
Sussex
NJ
156
3
F
5
Semi
-
-
2016
Sussex
NJ
157
3
F
4
Semi
-
+
2016
Sussex
NJ
158
3
F
2
Fully
-
+
2016
Sussex
NJ
159
4
F
1
Semi
-
-
2016
Sussex
NJ
160
5
M
1
Un
-
-
2016
Sussex
NJ
161
5
M
1
Un
-
-
2016
Sussex
NJ
162
5
F
1
Semi
-
+
2016
Sussex
NJ
163
5
F
1
Semi
-
-
2016
Sussex
NJ
164
6
M
1
Un
-
-
2016
Sussex
NJ
165
6
F
1
Semi
-
-
2016
Sussex
NJ
166
6
F
1
Semi
-
-
2016
Sussex
NJ
167
6
F
1
Semi
-
+
2016
Sussex
NJ
168
8
F
1
Semi
+
+
2016
Sussex
NJ
169
8
F
1
Semi
-
+
2016
Sussex
NJ
170
8
F
1
Semi
-
-
2016
Sussex
NJ
171
8
F
1
Semi
-
-
2016
Sussex
NJ
172
9
M
1
Un
-
-
2016
Sussex
NJ
173
9
F
1
Semi
-
-
2016
Sussex
NJ
174
10
M
4
Un
-
+
2016
Sussex
NJ
175
10
F
2
Un
-
+
2016
Sussex
NJ
176
10
F
4
Semi
-
+
2016
Sussex
NJ
177
10
F
4
Semi
-
+
2016
Sussex
NJ
178
11
M
5
Un
-
-
2016
Sussex
NJ
179
11
F
5
Un
-
+
2016
Sussex
NJ
180
11
F
5
Semi
-
-
2016
Sussex
NJ
181
11
F
1
Fully
-
-
2016
Sussex
NJ
182
13
M
3
Un
-
+
2016
Sussex
NJ
183
13
F
5
Semi
-
+
2016
Sussex
NJ
184
13
F
5
Semi
-
-
2016
Sussex
NJ
185
13
F
3
Fully
-
-
2016
Sussex
NJ
186
14
M
2
Un
-
-
2016
Sussex
NJ
187
14
F
3
Semi
-
+
2016
Sussex
NJ
73
188
15
M
1
Un
+
-
2016
Sussex
NJ
189
15
F
3
Un
-
+
2016
Sussex
NJ
190
15
F
2
Semi
-
+
2016
Sussex
NJ
191
17
M
3
Un
-
+
2016
Sussex
NJ
192
17
F
5
Semi
-
-
2016
Sussex
NJ
193
20
M
7
Un
-
+
2016
Sussex
NJ
194
20
F
1
Un
-
-
2016
Sussex
NJ
195
20
F
5
Semi
-
+
2016
Sussex
NJ
196
20
F
5
Semi
-
+
2016
Sussex
NJ
197
20
F
5
Semi
-
+
2016
Sussex
NJ
198
22
M
2
Un
-
-
2016
Sussex
NJ
199
22
F
4
Semi
-
-
2016
Sussex
NJ
200
16
M
2
Un
-
-
2016
Warren
NJ
201
16
F
2
Un
+
+
2016
Warren
NJ
202
16
F
5
Semi
-
+
2016
Warren
NJ
203
30
M
3
Un
-
-
2016
Warren
NJ
204
30
F
3
Un
-
+
2016
Warren
NJ
205
30
F
4
Semi
-
+
2016
Warren
NJ
206
154
F
1
Fully
-
+
2016
Warren
NJ
207
162
M
2
Un
-
+
2016
Warren
NJ
208
162
F
4
Semi
-
+
2016
Warren
NJ
209
167
M
2
Un
-
-
2016
Warren
NJ
210
167
F
1
Un
-
-
2016
Warren
NJ
211
167
F
3
Semi
-
+
2016
Warren
NJ
212
169
M
1
Un
+
-
2016
Warren
NJ
213
169
F
3
Semi
-
-
2016
Warren
NJ
214
169
F
2
Fully
-
-
2016
Warren
NJ
215
8819
M
1
Un
-
-
2016
Warren
NJ
216
8819
F
1
Semi
-
-
2016
Warren
NJ
217
8819
F
1
Fully
-
-
2016
Warren
NJ
218
8819
F
1
Fully
-
-
2016
Warren
NJ
219
9209
F
1
Semi
-
-
2016
Warren
NJ
220
9553
M
1
Un
-
-
2016
Warren
NJ
221
9553
M
1
Un
-
-
2016
Warren
NJ
222
9553
F
1
Fully
-
-
2016
Warren
NJ
223
9553
F
1
Fully
-
+
2016
Warren
NJ
224
28
M
3
Un
-
-
2016
Sussex
NJ
225
28
F
1
Un
-
-
2016
Sussex
NJ
74
226
28
F
5
Semi
-
+
2016
Sussex
NJ
227
29
M
3
Un
-
+
2016
Sussex
NJ
228
29
F
3
Un
-
+
2016
Sussex
NJ
229
31
M
1
Un
+
+
2016
Sussex
NJ
230
31
F
1
Un
-
+
2016
Sussex
NJ
231
31
F
4
Semi
-
+
2016
Sussex
NJ
232
144
M
3
Un
+
+
2016
Sussex
NJ
233
144
F
5
Semi
-
-
2016
Sussex
NJ
234
145
M
1
Un
-
-
2016
Sussex
NJ
235
145
M
1
Un
-
-
2016
Sussex
NJ
236
145
F
1
Un
-
-
2016
Sussex
NJ
237
145
F
1
Semi
-
+
2016
Sussex
NJ
238
145
F
1
Semi
-
-
2016
Sussex
NJ
239
147
F
1
Semi
-
-
2016
Sussex
NJ
240
147
F
1
Semi
-
+
2016
Sussex
NJ
241
148
M
1
Un
-
-
2016
Sussex
NJ
242
148
F
1
Un
-
+
2016
Sussex
NJ
243
148
F
3
Semi
-
-
2016
Sussex
NJ
244
149
M
1
Un
-
-
2016
Sussex
NJ
245
149
F
1
Un
-
-
2016
Sussex
NJ
246
149
F
1
Semi
-
-
2016
Sussex
NJ
247
249
F
1
Semi
-
+
2016
Sussex
NJ
248
379
F
1
Semi
-
+
2016
Sussex
NJ
249
379
F
1
Semi
-
+
2016
Sussex
NJ
250
91
M
4
Un
-
-
2017
Warren
NJ
251
91
F
1
Un
-
-
2017
Warren
NJ
252
91
F
5
Semi
-
+
2017
Warren
NJ
253
91
F
5
Semi
-
+
2017
Warren
NJ
254
91
F
3
Semi
-
-
2017
Warren
NJ
255
105
M
2
Un
-
-
2017
Warren
NJ
256
105
F
2
Semi
-
+
2017
Warren
NJ
257
105
F
1
Fully
-
-
2017
Warren
NJ
259
110
M
7
Un
-
+
2017
Warren
NJ
260
110
F
2
Un
+
+
2017
Warren
NJ
261
110
F
3
Semi
-
+
2017
Warren
NJ
262
6
M
5
Un
-
-
2017
Warren
NJ
263
6
F
1
Un
-
-
2017
Warren
NJ
264
6
F
5
Semi
-
+
2017
Warren
NJ
75
265
6
F
5
Semi
-
+
2017
Warren
NJ
266
6
F
5
Semi
-
-
2017
Warren
NJ
267
6
F
5
Semi
-
-
2017
Warren
NJ
268
6
F
5
Semi
-
-
2017
Warren
NJ
269
6
F
5
Semi
-
-
2017
Warren
NJ
270
90
M
1
Un
-
-
2017
Sussex
NJ
271
90
F
2
Un
-
+
2017
Sussex
NJ
272
90
F
2
Semi
-
+
2017
Sussex
NJ
273
93
M
4
Un
-
+
2017
Sussex
NJ
274
93
F
1
Un
+
-
2017
Sussex
NJ
275
93
F
1
Semi
-
+
2017
Sussex
NJ
276
94
M
1
Un
-
+
2017
Sussex
NJ
277
94
F
1
Semi
-
+
2017
Sussex
NJ
278
94
F
1
Fully
-
+
2017
Sussex
NJ
279
30
M
1
Un
-
-
2017
Warren
NJ
280
30
F
1
Semi
-
-
2017
Warren
NJ
281
30
F
1
Semi
-
-
2017
Warren
NJ
282
30
F
1
Semi
-
-
2017
Warren
NJ
283
101
M
1
Un
-
-
2017
Sussex
NJ
284
101
F
1
Semi
-
-
2017
Sussex
NJ
285
101
F
1
Semi
-
-
2017
Sussex
NJ
286
104
F
1
Un
-
-
2017
Sussex
NJ
287
9
M
1
Un
-
+
2017
Sussex
NJ
288
9
F
6
Semi
-
-
2017
Sussex
NJ
289
9
F
1
Fully
-
+
2017
Sussex
NJ
290
55
F
1
Semi
-
-
2017
Sussex
NJ
291
55
F
1
Semi
-
+
2017
Sussex
NJ
292
55
F
1
Semi
-
-
2017
Sussex
NJ
293
55
F
1
Semi
-
+
2017
Sussex
NJ
294
64
M
1
Un
+
-
2017
Sussex
NJ
295
64
F
2
Un
-
-
2017
Sussex
NJ
296
64
F
2
Semi
-
-
2017
Sussex
NJ
297
67
M
2
Un
-
+
2017
Sussex
NJ
298
67
F
2
Un
-
+
2017
Sussex
NJ
299
67
F
2
Semi
-
-
2017
Sussex
NJ
300
68
M
1
Un
-
+
2017
Sussex
NJ
301
252
M
1
Un
+
-
2017
Sussex
NJ
302
252
F
1
Semi
-
-
2017
Sussex
NJ
76
303
252
F
1
Semi
-
-
2017
Sussex
NJ
304
302
M
1
Un
-
-
2017
Sussex
NJ
305
302
F
1
Un
-
-
2017
Sussex
NJ
306
302
F
1
Un
-
-
2017
Sussex
NJ
307
302
F
1
Fully
-
-
2017
Sussex
NJ
308
306
F
1
Un
+
+
2017
Sussex
NJ
309
306
F
1
Un
-
-
2017
Sussex
NJ
310
608
M
4
Un
-
-
2017
Sussex
NJ
311
608
F
1
Un
+
-
2017
Sussex
NJ
312
608
F
4
Semi
-
+
2017
Sussex
NJ
313
608
F
5
Fully
-
+
2017
Sussex
NJ
314
608
F
5
Fully
-
-
2017
Sussex
NJ
315
299
F
1
Un
-
+
2017
Sussex
NJ
316
299
F
1
Fully
-
+
2017
Sussex
NJ
317
299
F
1
Un
-
+
2017
Sussex
NJ
318
304
F
1
Un
-
-
2017
Sussex
NJ
319
304
F
1
Un
-
+
2017
Sussex
NJ
320
14
M
1
Un
+
-
2017
Monroe
PA
321
38
F
1
Semi
-
-
2017
Monroe
PA
322
37
M
1
Un
-
+
2017
Monroe
PA
323
37
M
1
Un
-
+
2017
Monroe
PA
324
37
F
1
Semi
-
-
2017
Monroe
PA
325
37
F
1
Semi
-
-
2017
Monroe
PA
326
37
F
1
Semi
-
+
2017
Monroe
PA
327
44
F
1
Un
+
+
2017
Monroe
PA
328
44
F
1
Semi
-
-
2017
Monroe
PA
329
44
F
1
Semi
-
-
2017
Monroe
PA
330
44
M
1
Un
-
-
2017
Monroe
PA
331
55
F
1
Fully
-
-
2017
Pike
PA
332
55
M
1
Un
-
-
2017
Pike
PA
333
43
F
1
Fully
-
-
2017
Pike
PA
334
1723665
M
2
Un
-
-
2017
Monroe
PA
335
1723665
F
2
Semi
-
+
2017
Monroe
PA
336
1723665
F
1
Fully
-
-
2017
Monroe
PA
337
1723667
M
1
Un
-
+
2017
Monroe
PA
338
1723667
F
4
Semi
-
+
2017
Monroe
PA
339
1723667
F
1
Fully
-
-
2017
Monroe
PA
340
1723668
M
2
Un
-
-
2017
Monroe
PA
77
341
1723668
F
2
Un
-
+
2017
Monroe
PA
342
172366
F
1
Semi
-
-
2017
Monroe
PA
343
172366
M
2
Un
-
-
2017
Monroe
PA
344
172366
F
1
Un
-
+
2017
Monroe
PA
345
172366
F
2
Semi
-
+
2017
Monroe
PA
346
51100
F
1
Un
-
-
2018
Clinton
PA
347
51100
N
1
Fully
-
-
2018
Clinton
PA
348
52030
F
1
Un
-
-
2018
Lycoming
PA
349
35998
M
5
Un
-
+
2018
Clinton
PA
350
35998
M
5
Un
-
+
2018
Clinton
PA
351
35998
F
1
Un
-
+
2018
Clinton
PA
352
35998
F
2
Semi
-
-
2018
Clinton
PA
353
35998
F
2
Fully
-
-
2018
Clinton
PA
354
27518
F
1
Un
-
-
2018
Clinton
PA
355
27518
F
1
Un
+
-
2018
Clinton
PA
356
27518
F
1
Semi
-
-
2018
Clinton
PA
357
51039
M
5
Un
-
+
2018
Clinton
PA
358
51039
M
5
Un
-
+
2018
Clinton
PA
359
51039
M
3
Un
-
+
2018
Clinton
PA
360
51039
F
5
Semi
-
+
2018
Clinton
PA
361
51039
F
5
Semi
-
+
2018
Clinton
PA
362
51039
F
5
Semi
-
+
2018
Clinton
PA
363
51039
F
1
Fully
-
+
2018
Clinton
PA
364
48076
N
1
Fully
-
-
2018
Lycoming
PA
365
48076
F
1
Semi
+
-
2018
Lycoming
PA
366
48076
F
1
Semi
-
-
2018
Lycoming
PA
367
51660
F
1
Un
-
+
2018
Potter
PA
368
51049
M
1
Un
-
-
2018
Clinton
PA
369
51049
F
1
Un
+
-
2018
Clinton
PA
370
51049
F
1
Semi
-
+
2018
Clinton
PA
371
52028
F
1
Un
-
+
2018
Clinton
PA
372
52028
F
4
Semi
-
+
2018
Clinton
PA
373
52028
F
1
Fully
-
+
2018
Clinton
PA
374
51032
M
5
Un
-
+
2018
Clinton
PA
375
51032
M
5
Un
+
+
2018
Clinton
PA
376
51032
M
2
Un
-
+
2018
Clinton
PA
377
51032
F
3
Un
+
+
2018
Clinton
PA
378
51032
F
3
Semi
-
+
2018
Clinton
PA
78
379
51032
F
2
Fully
-
+
2018
Clinton
PA
380
27516
M
5
Un
-
+
2018
Clinton
PA
381
27516
M
5
Un
+
+
2018
Clinton
PA
382
27516
M
1
Un
-
-
2018
Clinton
PA
383
27516
F
1
Un
-
-
2018
Clinton
PA
384
27516
F
5
Semi
-
+
2018
Clinton
PA
385
27516
F
5
Semi
-
+
2018
Clinton
PA
386
27516
F
1
Fully
-
+
2018
Clinton
PA
387
27516
F
1
Fully
-
-
2018
Clinton
PA
388
27516
F
1
Fully
-
-
2018
Clinton
PA
389
27516
N
1
Fully
-
-
2018
Clinton
PA
390
41630
M
5
Un
-
+
2018
Tioga
PA
391
41630
M
1
Un
+
+
2018
Tioga
PA
392
41630
F
5
Un
+
+
2018
Tioga
PA
393
41630
F
2
Semi
-
+
2018
Tioga
PA
394
41638
M
4
Un
-
+
2018
Tioga
PA
395
41638
F
5
Semi
-
+
2018
Tioga
PA
396
41638
F
1
Fully
-
-
2018
Tioga
PA
397
23095
F
1
Un
-
-
2018
Lycoming
PA
398
23095
N
1
Fully
-
+
2018
Lycoming
PA
399
51047
M
1
Un
-
-
2018
Clinton
PA
400
51047
F
1
Semi
+
-
2018
Clinton
PA
401
33138
M
2
Un
-
+
2018
Clinton
PA
402
33138
F
2
Un
-
-
2018
Clinton
PA
403
33138
F
1
Fully
-
-
2018
Clinton
PA
404
51942
M
1
Un
-
-
2018
Clinton
PA
405
51942
F
1
Un
-
+
2018
Clinton
PA
406
41688
N
1
Fully
-
-
2018
Potter
PA
407
33142
M
3
Un
-
+
2018
Clinton
PA
408
33142
F
3
Semi
-
-
2018
Clinton
PA
409
33142
F
1
Fully
-
-
2018
Clinton
PA
410
35414
M
2
Un
-
-
2018
Clinton
PA
411
35414
F
1
Un
-
+
2018
Clinton
PA
412
35414
F
2
Semi
-
+
2018
Clinton
PA
413
51636
F
1
Fully
-
+
2018
Clinton
PA
414
51172
F
1
Semi
-
+
2018
Clinton
PA
415
51172
F
1
Un
-
+
2018
Clinton
PA
416
51172
F
1
Un
-
-
2018
Clinton
PA
79
417
51172
F
1
Fully
-
-
2018
Clinton
PA
418
51949
M
4
Un
+
+
2018
Clinton
PA
419
51949
F
2
Un
-
+
2018
Clinton
PA
420
1
M
3
Un
+
+
2018
Warren
NJ
421
1
F
2
Semi
-
-
2018
Warren
NJ
422
2
M
1
Un
-
+
2018
Warren
NJ
423
2
F
6
Semi
-
+
2018
Warren
NJ
424
2
F
3
Un
-
+
2018
Warren
NJ
425
2
F
1
Fully
-
+
2018
Warren
NJ
426
2
N
2
Fully
-
-
2018
Warren
NJ
427
3
M
1
Un
-
-
2018
Warren
NJ
428
3
F
5
Semi
-
-
2018
Warren
NJ
429
3
F
5
Semi
-
+
2018
Warren
NJ
430
4
M
1
Un
-
-
2018
Sussex
NJ
431
4
F
2
Un
-
-
2018
Sussex
NJ
432
4
F
1
Semi
-
+
2018
Sussex
NJ
433
4
F
1
Fully
-
-
2018
Sussex
NJ
434
44
M
3
Un
-
+
2018
Sussex
NJ
435
44
F
2
Un
+
+
2018
Sussex
NJ
436
44
F
3
Semi
-
+
2018
Sussex
NJ
437
6
F
3
Un
+
+
2018
Sussex
NJ
438
6
F
5
Semi
-
-
2018
Sussex
NJ
439
7
M
5
Un
-
+
2018
Warren
NJ
440
7
F
2
Un
-
+
2018
Warren
NJ
441
7
F
2
Semi
-
+
2018
Warren
NJ
442
7
N
1
Fully
-
-
2018
Warren
NJ
443
8
M
1
Un
+
+
2018
Sussex
NJ
444
8
F
1
Un
-
+
2018
Sussex
NJ
445
8
F
3
Semi
-
+
2018
Sussex
NJ
446
8
N
1
Semi
-
-
2018
Sussex
NJ
447
9
M
1
Un
-
-
2018
Warren
NJ
448
9
F
3
Un
-
+
2018
Warren
NJ
449
9
F
6
Semi
-
+
2018
Warren
NJ
450
9
F
5
Semi
-
+
2018
Warren
NJ
451
45
M
5
Un
-
-
2018
Sussex
NJ
452
45
F
5
Un
-
+
2018
Sussex
NJ
453
45
F
2
Un
-
-
2018
Sussex
NJ
454
45
F
1
Semi
-
+
2018
Sussex
NJ
80
455
45
F
2
Fully
-
+
2018
Sussex
NJ
456
56
F
1
Fully
-
-
2018
Warren
NJ
457
15
M
5
Un
-
+
2018
Sussex
NJ
458
15
F
6
Semi
-
+
2018
Sussex
NJ
459
15
F
6
Semi
-
+
2018
Sussex
NJ
460
14
M
4
Un
-
+
2018
Sussex
NJ
461
14
F
4
Un
-
+
2018
Sussex
NJ
462
14
F
4
Semi
-
+
2018
Sussex
NJ
463
13
M
1
Un
-
+
2018
Sussex
NJ
464
13
F
4
Semi
-
+
2018
Sussex
NJ
465
13
F
4
Semi
-
+
2018
Sussex
NJ
466
13
F
2
Fully
+
+
2018
Sussex
NJ
467
44
F
1
Fully
-
+
2018
Sussex
NJ
468
56
N
1
Un
-
-
2018
Sussex
NJ
469
33168
F
1
Un
-
-
2018
Clinton
PA
470
33168
F
1
Semi
-
-
2018
Clinton
PA
471
51357
F
1
Semi
-
-
2018
Potter
PA
472
51357
F
1
Fully
-
-
2018
Potter
PA
473
51357
N
1
Semi
-
-
2018
Potter
PA
474
35676
M
3
Un
-
+
2018
Clinton
PA
475
35676
F
4
Un
-
+
2018
Clinton
PA
476
35676
F
2
Semi
-
+
2018
Clinton
PA
477
35676
F
3
Fully
-
-
2018
Clinton
PA
478
51370
N
3
Semi
-
+
2018
Clinton
PA
479
52026
N
2
Un
-
-
2018
Centre
PA
480
52026
N
5
Semi
-
-
2018
Centre
PA
481
52026
N
2
Semi
-
-
2018
Centre
PA
482
52026
F
1
Fully
-
-
2018
Centre
PA
483
35918
N
1
Un
-
-
2018
Clinton
PA
484
35918
N
1
Semi
-
-
2018
Clinton
PA
485
35918
N
1
Fully
-
-
2018
Clinton
PA
486
35918
N
1
Fully
-
-
2018
Clinton
PA
487
41479
N
2
Un
-
-
2018
Lycoming
PA
488
41479
N
5
Semi
-
-
2018
Lycoming
PA
489
41479
F
2
Semi
-
+
2018
Lycoming
PA
490
51486
M
5
Un
-
+
2018
Clinton
PA
491
51486
M
4
Un
-
+
2018
Clinton
PA
492
51486
F
1
Un
-
-
2018
Clinton
PA
81
493
51486
F
3
Semi
-
-
2018
Clinton
PA
494
51486
F
2
Fully
-
-
2018
Clinton
PA
495
51366
N
3
Un
+
+
2018
Clearfield
PA
496
36361
N
2
Un
-
-
2018
Clinton
PA
497
35017
N
5
Un
-
-
2018
Clinton
PA
498
35017
N
5
Semi
+
+
2018
Clinton
PA
499
35017
N
6
Fully
-
-
2018
Clinton
PA
500
35017
M
1
Un
+
-
2018
Clinton
PA
501
35017
F
2
Un
-
-
2018
Clinton
PA
502
51234
M
1
Un
-
-
2018
Clinton
PA
503
51234
M
5
Un
+
+
2018
Clinton
PA
504
51234
F
4
Un
-
+
2018
Clinton
PA
505
51234
F
2
Semi
-
+
2018
Clinton
PA
506
51234
F
1
Fully
-
-
2018
Clinton
PA
507
51034
M
4
Un
-
+
2018
Clinton
PA
508
51034
F
4
Un
-
+
2018
Clinton
PA
509
51034
F
5
Semi
-
-
2018
Clinton
PA
510
51034
F
5
Semi
-
-
2018
Clinton
PA
511
51034
F
2
Fully
+
-
2018
Clinton
PA
512
35017
M
5
Un
+
-
2018
Clinton
PA
513
35017
M
5
Un
-
-
2018
Clinton
PA
514
35017
M
5
Un
-
+
2018
Clinton
PA
515
35017
F
5
Un
+
+
2018
Clinton
PA
516
35017
F
2
Semi
-
-
2018
Clinton
PA
517
35017
F
4
Semi
-
-
2018
Clinton
PA
518
35017
F
1
Fully
-
-
2018
Clinton
PA
519
1805571
M
1
Un
-
+
2018
Huntingdon
PA
520
1805571
M
5
Un
-
+
2018
Huntingdon
PA
521
1805571
F
2
Semi
-
-
2018
Huntingdon
PA
522
1805571
F
5
Semi
-
+
2018
Huntingdon
PA
523
1805571
F
1
Fully
-
-
2018
Huntingdon
PA
524
1805574
F
5
Semi
-
-
2018
Huntingdon
PA
525
1805574
F
2
Fully
-
-
2018
Huntingdon
PA
526
51044
M
3
Un
+
+
2018
Clinton
PA
527
51044
M
5
Un
+
+
2018
Clinton
PA
528
51044
F
1
Un
-
-
2018
Clinton
PA
529
51044
F
1
Semi
-
-
2018
Clinton
PA
530
51044
F
3
Fully
-
+
2018
Clinton
PA
82
531
51178
N
1
Fully
-
-
2018
Clinton
PA
532
51178
F
4
Un
+
+
2018
Clinton
PA
533
51178
F
5
Un
-
+
2018
Clinton
PA
534
51178
F
5
Semi
-
-
2018
Clinton
PA
535
51178
F
1
Fully
-
-
2018
Clinton
PA
536
51036
M
4
Un
-
+
2018
Clinton
PA
537
51036
M
5
Un
-
-
2018
Clinton
PA
538
51036
F
1
Un
-
-
2018
Clinton
PA
539
51036
F
1
Semi
-
-
2018
Clinton
PA
540
51036
F
5
Semi
-
-
2018
Clinton
PA
541
51036
F
5
Semi
-
-
2018
Clinton
PA
542
51036
F
1
Fully
-
-
2018
Clinton
PA
543
1804200
M
1
Un
-
-
2018
Lycoming
PA
544
1804200
F
1
Semi
-
+
2018
Lycoming
PA
545
1804200
F
1
Semi
-
+
2018
Lycoming
PA
546
1804201
M
1
Un
-
-
2018
Lycoming
PA
547
1804201
M
1
Un
-
-
2018
Lycoming
PA
548
1804202
M
3
Un
-
+
2018
Lycoming
PA
549
1804202
F
1
Semi
-
+
2018
Lycoming
PA
550
1804202
F
1
Fully
-
-
2018
Lycoming
PA
551
1804204
F
1
Semi
+
-
2018
Lycoming
PA
552
1804203
M
5
Un
-
-
2018
Lycoming
PA
553
1804203
M
5
Un
-
-
2018
Lycoming
PA
554
1804203
M
5
Un
-
-
2018
Lycoming
PA
555
1804203
M
5
Un
-
-
2018
Lycoming
PA
556
1804203
F
5
Un
-
-
2018
Lycoming
PA
557
1804203
F
5
Un
-
+
2018
Lycoming
PA
558
1804203
F
5
Semi
-
-
2018
Lycoming
PA
559
1804203
F
5
Semi
-
-
2018
Lycoming
PA
560
1804203
M
2
Un
-
-
2018
Lycoming
PA
561
1804203
F
2
Un
-
-
2018
Lycoming
PA
562
1804207
F
1
Un
-
+
2018
Lycoming
PA
563
1804207
F
1
Semi
-
-
2018
Lycoming
PA
564
1804207
F
1
Fully
-
+
2018
Lycoming
PA
565
1804208
M
2
Un
-
+
2018
Lycoming
PA
566
1804208
M
5
Un
-
+
2018
Lycoming
PA
567
1804208
F
5
Un
-
+
2018
Lycoming
PA
568
1804208
F
5
Semi
-
+
2018
Lycoming
PA
83
569
1804208
F
4
Semi
-
+
2018
Lycoming
PA
570
1804208
F
1
Fully
-
-
2018
Lycoming
PA
571
1804209
M
2
Un
-
+
2018
Lycoming
PA
572
1804209
F
5
Un
-
+
2018
Lycoming
PA
573
1804209
F
2
Semi
-
+
2018
Lycoming
PA
574
1803379
M
1
Un
-
-
2018
Clearfield
PA
575
1803379
M
1
Un
-
-
2018
Clearfield
PA
576
1803379
F
1
Semi
-
-
2018
Clearfield
PA
577
1803380
M
1
Un
-
-
2018
Clearfield
PA
578
1803380
M
1
Un
-
-
2018
Clearfield
PA
579
1803380
M
1
Un
-
-
2018
Clearfield
PA
580
1803380
M
1
Un
-
-
2018
Clearfield
PA
581
1803378
F
1
Un
-
-
2018
Clearfield
PA
582
1803384
M
1
Un
-
-
2018
Clearfield
PA
583
1803384
F
1
Semi
-
-
2018
Clearfield
PA
584
1803388
M
5
Un
-
-
2018
Clearfield
PA
585
1803388
F
1
Semi
-
+
2018
Clearfield
PA
586
1803390
M
1
Un
+
-
2018
Clearfield
PA
587
1803390
M
1
Un
-
-
2018
Clearfield
PA
588
1803390
M
1
Un
-
-
2018
Clearfield
PA
589
1804278
M
1
Un
+
-
2018
Lycoming
PA
590
1804278
F
1
Fully
-
-
2018
Lycoming
PA
591
1804281
M
1
Un
-
+
2018
Lycoming
PA
592
1804281
F
1
Fully
-
+
2018
Lycoming
PA
593
1805549
M
1
Un
+
-
2018
Huntingdon
PA
594
1805549
F
1
Fully
-
-
2018
Huntingdon
PA
595
1810780
F
1
Semi
-
-
2018
Lycoming
PA
596
1810784
M
1
Un
+
-
2018
Lycoming
PA
597
1810787
M
1
Un
+
-
2018
Lycoming
PA
84
Appendix C: Statistical Raw Data
Powassan statistical data
Sex (male vs
female)
Adult
engorgement
Nymph
engorgement
Life stage
(adult vs
nymph)
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
-0.5594
0.836
1
-0.207
2.7060
6
-0.5783
0.920
3
-0.113
5.1210
7
-0.06677
0.976
1
-0.030
2.1948
5
0.6374
0.897
2
-0.129
3.4835
6
Borrelia burgdorferi statistical data
Sex (male vs
female)
Adult
engorgement
Nymph
engorgement
Life stage
(adult vs
nymph)
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
0.09929
0.945
1
0.069
1.4355
3
-0.4213
0.799
3
-0.254
1.6578
4
-0.6931
0.815
2
-0.234
1.6578
5
-0.4500
0.725
2
0.352
1.8119
5
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
0.06077
0.986
2
0.017
3.48817
6
-0.2834
0.906
1
-0.917
2.3999
5
-0.5254
0.852
1
-0.186
2.8238
6
Powassan State data
New Jersey
2015 / 2016 /
2017 / 2018
Pennsylvania
2017 / 2018
New Jersey
vs
Pennsylvania
85
B. burgdorferi State data
New Jersey
2015 / 2016 /
2017 / 2018
Pennsylvania
2017 / 2018
New Jersey
vs
Pennsylvania
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
-0.70299
0.720
3
-0.358
1.96366
4
-0.08109
0.955
1
-0.056
1.44226
3
-0.02498
0.986
1
-0.017
1.43616
3
Chi-Square tick collection data for difference between tick species collected from New
Jersey vs Pennsylvania
X2
Degrees of freedom
P-value
8
6
0.2381
Powassan county data to determine for significant difference between Sussex and Warren
County
Degrees
Fisher
Estimated p-value
of
Z value Std. error Scoring
freedom
iterations
Sussex vs
0.3791
0.917
1
0.105
3.6248
6
Warren
B. burgdorferi county data to determine for significant difference between Sussex and
Warren County
Degrees
Fisher
Estimated p-value
of
Z value Std. error Scoring
freedom
iterations
Sussex vs
0.1333
0.926
1
0.092
1.4428
3
Warren
86
BURGDORFERI) IN IXODES SCAPULARIS COLLECTED FROM NEW JERSEY
AND PENNSYLVANIA BLACK BEARS (URSUS AMERICANUS)
By
Kristine N. Bentkowski, B.S.
King’s College
A Thesis Submitted in Partial Fulfillment of
The Requirements for the Degree of
Master of Science in Biology
To the office of Graduate and Extended Studies of
East Stroudsburg University of Pennsylvania
May 10, 2019
SIGNATURE/APPROVAL PAGE
The signed approval page for this thesis was intentionally removed from the online copy by an
authorized administrator at Kemp Library.
The final approved signature page for this thesis is on file with the Office of Graduate and
Extended Studies. Please contact Theses@esu.edu with any questions.
ABSTRACT
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of
Master of Science in Biology to the office of Graduate and Extended Studies of East
Stroudsburg University of Pennsylvania
Student’s Name: Kristine N. Bentkowski
Title: Prevalence of Powassan Virus and Co-infection of Lyme Disease (Borrelia
burgdorferi) in Ixodes scapularis from New Jersey and Pennsylvania Black Bears (Ursus
americanus)
Date of Graduation: May 10, 2019
Thesis Chair: Joshua Loomis, Ph.D.
Thesis Member: Abdalla Aldras, Sc.D.
Thesis Member: Emily Rollinson, Ph.D.
Thesis Member: Nicole Chinnici, M.S.
Abstract
The blacklegged tick is the main vector for Lyme disease and Powassan virus
Lineage II (Deer Tick Virus) in the United States. The objective of this study was to identify
the prevalence of Powassan virus (DTV) and Lyme disease in adult and nymph blacklegged
ticks collected in New Jersey (2015-2018) and Pennsylvania (2017-2018). All ticks were
collected from lived trapped or hunter harvested black bears (Ursus americanus). A total of
2,713 ticks were collected, made up of four species. Only blacklegged ticks were analyzed in
this study. Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used
to amplify cDNA specific to the NS5 gene of POW Lineage II, and qPCR was used to
amplify the 16s-23s intergenic spacer region rDNA of Borrelia burgdorferi (Lyme disease).
A minimum infection rate (MIR) of 3.52% was determined for Powassan vitus and a MIR of
19.2% for Lyme disease. The findings in this study were similar to previous studies
conducted for Powassan and Lyme prevalence in Lyme endemic region.
Acknowledgments
I would like to acknowledge everyone that has helped me through my thesis.
From all my committee members, friends and family who were there to support,
encourage and guide me through this process.
I thank my graduate committee members Dr. Joshua Loomis, Dr. Emily
Rollinson, Dr. Abdalla Aldras and Nicole Chinnici for all their wisdom, help and
guidance through this process. Special thank you to Dr. Loomis for working through this
process together and figuring out everything that needed to be done to complete this
project and for being an amazing thesis chairperson. As well, thank you to Nicole for
encouraging me to apply to ESU for their graduate program and allowing to do my
research at the Northeast Wildlife DNA Laboratory.
Thank you to everyone at the NEWDL for their help and support throughout this
process. As well, to all the great friends who I have made who encouraged me to keep
going and helping get through the most stressful of times as we all worked towards out
Master’s degree. Especially Justin Clarke, Jon Adamski, Amanda Layden, Kacie Chern,
Eric, Sam and Ali Machrone who gave me a lot of support and created some great
friendships.
Lastly, I would like to thank my family and boyfriend. Thank you, mom, dad,
Ashley, Maggie and Matt, for all your encouragement and support you guys gave me
through all of this. I greatly appreciated it.
Table of Contents
List of Figures ............................................................................................................III
List of Tables ............................................................................................................ VI
Chapter I .................................................................................................................... 1
Introduction ......................................................................................................................1
Lyme Disease .......................................................................................................................................... 2
Immune Response.................................................................................................................................. 4
Lyme disease symptoms ........................................................................................................................ 6
Diagnosis and Treatment ....................................................................................................................... 7
Post Treatment Lyme Disease Syndrome .............................................................................................. 8
Powassan Virus .................................................................................................................................... 11
Vector ................................................................................................................................................... 13
Black Bears ........................................................................................................................................... 17
Study Objectives ................................................................................................................................... 19
Chapter II ................................................................................................................. 20
Materials and Methods .................................................................................................... 20
Study Area and Sample Collection ....................................................................................................... 20
Identification and Extraction ................................................................................................................ 23
Powassan virus (Lineage II) SYBR Assay Optimization ......................................................................... 25
RNA Analysis ......................................................................................................................................... 26
DNA analysis ......................................................................................................................................... 26
Statistical Analysis ................................................................................................................................ 28
Chapter III ................................................................................................................ 29
Results............................................................................................................................. 29
Tick Collections ..................................................................................................................................... 29
Optimization of Powassan Lineage II Assay ......................................................................................... 34
Disease and Statistical Analysis ............................................................................................................ 35
Powassan Virus .................................................................................................................................... 38
Borrelia burgdorferi.............................................................................................................................. 40
Co-infection .......................................................................................................................................... 42
County Data .......................................................................................................................................... 42
Powassan ......................................................................................................................................... 42
Borrelia burgdorferi......................................................................................................................... 44
Chapter IV ................................................................................................................ 46
Discussion........................................................................................................................ 46
Tick Collection Data .............................................................................................................................. 46
Powassan (DTV) Prevalence ................................................................................................................. 50
Borrelia burgdorferi Prevalence Data .................................................................................................. 53
Co-infection Prevalence Data............................................................................................................... 56
Conclusion ............................................................................................................................................ 57
Future Study ......................................................................................................................................... 58
Literature Cited ........................................................................................................ 60
I
Appendix A: Tick Collection Raw Data ............................................................................... 67
Appendix B: Powassan and Lyme Raw Data ...................................................................... 69
Appendix C: Statistical Raw Data ...................................................................................... 85
II
List of Figures
Figure
Page
Figure 1. Reported cases of Lyme disease in the United States (2017). Each blue dot
represents a confirmed case of Lyme disease. Endemic regions are illustrated within the
Northeast and Mid-west. Massachusetts surveillance method does not match the national
surveillance case definition set by the Council of State and Territorial Epidemiologist,
information on most Lyme disease cases are not sent to the CDC and are not represented
on this map. (Centers for Disease Control and Prevention).................................................4
Figure 2. Map of neuroinvaisve Powassan virus cases reported in each US state, 20082017. (Centers for Disease Control and Prevention) ........................................................12
Figure 3. Distribution of blacklegged ticks in the United States as of 2018 (Centers for
Disease Control and Prevention) .......................................................................................14
Figure 4. The two-year life cycle of the blacklegged tick. Broken down into when each
life stage is most prevalent and the main host each life stage feeds on. (Centers for
Disease and Control Prevention) .......................................................................................15
Figure 5. Sedated black bear cub, 2018 NJDFW research trapping (Photo credit Kristine
Bentkowski) .......................................................................................................................18
Figure 6. New Jersey counties where black bears were trapped or harvested, and ticks
were collected. Blue stripped counties had ticks analyzed for Powassan and Lyme. .......22
Figure 7. Pennsylvania counties where black bears were trapped or harvested, and ticks
were collected 2017-18. Blue counties ticks were collected in 2017 and pink counties
ticks were collected in 2018. ..............................................................................................23
III
Figure 8. Measurement of a female blacklegged tick using the scutal index (SI = b/a), a =
maximum width of scutum, b = length of tick from posterior to the basis capitulium
(Photo credit Kristine Bentkowski) ...................................................................................24
Figure 9. Examples of engorgement sizes of adult female blacklegged tick. Left to right:
Fully engorged, semi-engorged, semi-engorged, unengorged, unengorged (Photo credit
Kristine Bentkowski) .........................................................................................................25
Figure 10. Total number of ticks collected from 2015-18 in New Jersey by month .........31
Figure 11. Total adult I. scapularis collected in the month throughout the year from New
Jersey (2015-18).................................................................................................................32
Figure 12. Total number of ticks collected from 2017-18 in Pennsylvania by month ......33
Figure 13. Total number of adult I. scapularis collected in Pennsylvania in 2017-18 by
month .................................................................................................................................33
Figure 14. Total number of nymph and larval I. scapularis collected in Pennsylvania in
2017-18 by month ..............................................................................................................34
Figure 15. Plotted linear regression of the standard curve, using the log number of copies
of each dilution by the CT call from each triplicate ..........................................................35
Figure 16. Total number of I. scapularis female, male and nymph ticks analyzed (n=
1,277) from NJ (2015-18) and (PA 2017-18) ....................................................................38
Figure 17. Minimum infection rate (MIR) percent of adult I. scapularis with POW (DTV)
(n=1,213) from NJ (2015-18) and PA (2017-18)...............................................................39
IV
Figure 18. Minimum infection rate percent of nymph I. scapularis ticks with POW
(DTV) (n=64), from NJ and PA 2018 ................................................................................39
Figure 19. MIR (%) of adult I. scapularis ticks with B. burgdorferi (n=1,213) from NJ
(2015-18) and PA (2017-28) ..............................................................................................41
Figure 20. Minimum infection rate percent of nymph I. scapularis ticks with B.
burgdorferi (n=64), from NJ and PA 2018 ........................................................................41
V
List of Tables
Table
Page
Table 1. Average tick load of small to large animals identified by LoGiudice, Satefeld,
Scmidt & Keesing (2003) in Dutchess county, NY ...........................................................16
Table 2. Oligonucleotide primer used for PCR. SYBR green primer targeting the NS5
region of Powassan virus. Real-time primer targeting the 16S-23S intergenic spacer
region for Lyme (Borrelia species) and specific probe for Borrelia burgdorferi .............27
Table 3. Total number of blacklegged ticks analyzed from each county in New Jersey ...36
Table 4. Total number of blacklegged ticks analyzed from each county in Pennsylvania 37
Table 5. Total Powassan positive pools by year in each New Jersey county ....................43
Table 6. Total Powassan positive pools by year in each Pennsylvania county .................43
Table 7. Total B. burgdorferi positive tick pools by year in New Jersey counties ............44
Table 8. Total B. burgdorferi positive tick pools by year in Pennsylvania counties .........45
VI
Chapter I
Introduction
Tick-borne diseases affect thousands of people all over the globe every year.
Ticks have been vectors of disease for thousands of years and the spread of tick-borne
diseases has increased over the last decade, with over thirteen newly-recognized diseases
discovered over the last two decades48. The organisms that cause these diseases range
from bacterial to protozoan to viral, and each have their own unique transmission path
and can have various effects on those infected. The most common vector-borne disease in
the United States is Lyme disease, caused by the bacterium Borrelia burgdorferi. Lyme
disease is one of the top ten most recorded diseases in the United States but is not the
only tick-borne disease seen on the rise over the last decade33. Powassan virus is an
emerging tick-borne virus and is broken into two lineages, lineage I vectored by the
groundhog tick (Ixodes cookei) and lineage II (Deer Tick Virus) vectored by the
blacklegged tick (Ixodes scapularis)32. Powassan virus can be asymptomatic or can cause
encephalitis and severe neurological sequelae for those infected 36. The blacklegged tick is
the vector for many of these pathogens, including Lyme disease and Powassan virus, and
plays a large part in tick-borne disease transmission to humans. As the blacklegged tick
1
population continues to grow and expand, tick-borne diseases are becoming a
public health and safety crisis21,22. The increase in tick-borne diseases over the last
decade has sparked a nationwide concern for education on treatment and prevention.
Lyme Disease
Lyme disease, also known as Lyme borreliosis was first discovered in Lyme,
Connecticut in 1975 and since has had confirmed cases in North America, Europe and
Asia. Lyme disease is caused by several genospecies of the Borrelia burgdorferi sensu
lato group6. Of these, only two species of Borrelia bacteria, B. burgdorferi and Borrelia
mayonii, cause Lyme disease in the United States. Borrelia burgdorferi is the leading
cause of Lyme disease in the United States and Canada. In 2013, the Mayo Clinic
discovered B. mayonii, a new species of Borrelia that was causing Lyme disease like
symptoms in the upper Midwest37. Lyme disease is transmitted by the blacklegged tick
(Ixodes scapularis) in the Northeast and upper Midwest and by the western blacklegged
tick (I. pacificus) along the western coast of the U.S. The majority of Lyme disease cases
are reported during the late spring, summer and fall when blacklegged ticks are actively
seeking hosts.
Lyme disease is the fastest growing vector-borne disease in the United States. The
CDC estimates reports approximately 30,000 confirmed cases each year. With
accordance to the CDC and National Notifiable Diseases Surveillance System (NNDSS)
confirmed cases must present an erythema migran (EM) with a single primary lesion that
reaches greater than or equal to 5 cm in size across its largest diameter. The EM lesion is
accompanied by other acute symptoms, particularly fatigue, fever, headache, mildly stiff
2
neck, arthralgia, or myalgia. As well, a Physician must confirm laboratory evidence in the
form of a positive ELISA and western blot or a positive culture for B. burgdorferi. A
probable case is defined by the CDC when a Physician diagnoses a positive laboratory
test, but the patient lacks other evidence such as an erythema migran and no known
exposure in a high incidence state. In addition to strict diagnostic guidelines, studies have
shown only 10 percent of Lyme disease cases are reported. In 2008, Hinckley et, al.
surveyed 2.4 million specimens from laboratories throughout the U.S. Following
guidelines for testing Lyme disease recommended by the U.S. Public Health Service
Agencies and the Infectious Diseases Society of America, the estimated percentage of
true infection that year was upwards of 288,000 cases a year 33. Nelson et al. (2015),
evaluated the nationwide health insurance claims database from 2005-2010 identifying
patients with clinician diagnosed Lyme disease. Positive cases were determined using
ICD-9-CM codes for communicable diseases, along with a comprehensive analysis of the
positive predictive values (PPVs), and the case definition for Lyme disease by the CDC 57.
The ICD-9-CM codes are used by hospitals to assign diagnosis in patient charts. The
study determined based on clinical symptoms, there are approximately 329,000 cases of
Lyme disease each year1. In 2016, there were 36,429 confirmed and probable cases of
Lyme disease in the U.S. and 42,743 in 2017, an 8.5% increase4. Of these cases, 96% are
reported in 14 states located in the Northeast and upper Midwest35(Figure 1). The MidAtlantic states of Pennsylvania, New Jersey and New York have the highest number of
reported cases. In 2017, Pennsylvania reported 9,250 confirmed cases and New Jersey
reported 3,629 confirmed cases4.
3
Figure 1. Reported cases of Lyme disease in the United States (2017). Each blue dot
represents a confirmed case of Lyme disease. Endemic regions are illustrated within the
Northeast and Mid-west. Massachusetts surveillance method does not match the national
surveillance case definition set by the Council of State and Territorial Epidemiologist,
information on most Lyme disease cases are not sent to the CDC and are not represented
on this map. (Centers for Disease Control and Prevention)
Immune Response
Transmission time of Lyme disease from tick to host can be as short as 16 hours
or up to 48 hours15,44. Transmission of Lyme disease from vector to host causes the
bacterium to change its outer surface proteins to survive different environments. Within
the vector, B. burgdorferi resides and replicates in the midgut. During this time Outer
surface proteins are upregulated (OspA) allowing the bacterium to adhere to the tick’s
midgut until the tick feeds. As the tick attaches to a host and begins taking in a blood
meal, the OspA gene is downregulated and the OspC gene is expressed as a result of the
ticks midgut temperature and pH changes from the blood meal64,44.
4
The bacterium will than migrate from the tick’s midgut to the salivary glands; this
allows access for the bacterium to enter the new host. At the initial infection site B.
burgdorferi has several proteins that allow it to survive in the mammalian host and avoid
destruction. The OspC gene allows for the bacterium to survive the warmer temperatures
of the human body and basic pH level of the blood. The bacterium uses extra cellular
matrix (ECM) binding proteins DbpA and DbpB, to bind to decorin, and protein BBk32
to bind to fibronectin, as well as Bgp to bind to proteoglycans and P66 which further
binds to integrins64. These proteins may also play role in dissemination through
mammalian tissue and persistence in joints64. The OspC gene has been found to play a
possible role in dissemination by binding to human plasminogen44. These proteins at the
initial infection site are recognized by the innate immune system, recognized by
pathogen-associated molecular pattern (PAMP’s) and signal Toll-like receptors (TLR’s)
to release signals to the rest of the immune system. At the erythema migran lesion sites,
TLR2 has been found to play a specific role in releasing IFN, IL1 and IL6 cytotoxins52.
TLRs signal an influx of macrophages, dendritic cells and neutrophils to the initial
infection site. The immune system then produces pro-inflammatory cytokines such, TNF, IL-2, IL-6 and IFN’s. Production of cytokines recruit T-cells to the initial site of
infection which play an important role in activating complement and phagocytosis of the
bacterium during early localized infection. After several days of infection, the immune
system will begin to produce anti-inflammatory cytokine IL-10, this allows for surviving
bacteria to disseminate throughout the body66.
During early stage dissemination the surviving bacterium can travel through the
body hidden in tissue and being motile through viscous media found in the body. The
5
spirochete shape and flagella transverse the whole body protected under the outer
membrane, this helps the bacterium to penetrate host tissue and disseminate 64. As the
bacterium disseminates throughout the body the host immune system continues to
produce pro-inflammatory cytokines damaging tissue, joints, muscles and the heart due
to the inflammation caused by the host own immune system52.
Lyme disease symptoms
Lyme disease is a multisystem illness that can affect the skin, nervous system,
musculoskeletal system and the heart. Transmitted from a tick bite, symptoms may occur
3-30 days after exposure to the bacteria. There are three stages of symptomatology which
include: early localized, early disseminated and late disseminated Lyme disease.
In early localized (stage 1) Lyme disease, 60-90% of patients may develop a rash known
as an erythema migrans (EM)50. The rash tends to be localized in the area of the tick bite
and has the characteristic bulls-eye shape. Some patients may develop an uncharacteristic
rash that is patchy with no specific shape, and some patients may develop no rash at all
during their infection. Early Lyme disease may also include flu-like symptoms of fatigue,
malaise, fever, headache, arthralgias (joint pain), and myalgias (muscle pain)31. Lyme
disease symptoms for B. burgdorferi and B. mayonii infections are similar with additional
symptoms of B. mayonii including nausea, vomiting and diffuse rashes. Patients with B.
mayonii tend to have higher concentrations of bacteria circulating in the bloodstream 21.
If early localized Lyme disease goes untreated, patient symptoms may progress into the
early disseminated stage (stage 2). These symptoms can occur weeks or months after a
6
tick bite and symptoms may vary depending on the species of Borrelia causing the Lyme
disease infection. Early disseminated symptoms can include neck stiffness, facial palsy
(typically Bell’s palsy), lymphocytic meningitis, progressing into the loss of motor and
sensory function31. Lyme carditis may occur in this stage and cause an atrioventricular
block.
If Lyme disease is continued to be left untreated, it can persist into late
disseminated (stage 3) Lyme disease. This stage of Lyme disease can lead to severe
arthritis synovitis, severe neurological problems, such as memory loss and black outs,
and in rare cases cause encephalopathy31.
Diagnosis and Treatment
In endemic regions, clinicians use characteristic signs of Lyme disease described
and presented by patients for diagnosis. These characteristic signs include the patient
reporting a known tick bite, developing an EM rash, or developing other symptoms
common to early-localized Lyme disease. Not all patients develop an EM rash or
remember a tick bite. These patients will present symptoms similar to other diseases such
as fibromyalgia, multiple sclerosis and the flu. These patients may not receive the correct
treatment resulting in symptoms developing into early and late disseminated Lyme
disease30.
Current testing recommended by the CDC is a two-tier serological test. The first
step uses an ELISA (enzyme-linked immunosorbent assay) to detect IgG and IgM
antibodies against B. burgdorferi, if positive, the second test is an immunoblot (Western
7
blot) to measure IgG and IgM30,44. During early Lyme disease serological testing can be
inaccurate due to low antibody production and sensitivity from the serological test. If a
patient continues to have symptoms after a negative ELISA, they can be retested 2-4
weeks after the initial exposure. IgM can be detected 2-4 weeks after initial infection and
reaches peak antibody production at six weeks before the titer drops 66. Studies are being
conducting to develop new methods for diagnosing Lyme disease with higher sensitivity
and accuracy44.
Lyme disease can be treated with antibiotics. Doxycycline is the most commonly
used antibiotic to treat Lyme disease; however, other antibiotics such as amoxicillin, or
cefuroxime axetil can be used. Antibiotics are prescribed orally one to two times a day
for 14-30 days47.
Post Treatment Lyme Disease Syndrome
In most Lyme disease cases, patients clear the infection and no longer have
symptoms following a course of oral antibiotics. However, 10 to 20% of treated patients
continue to have symptoms for months to years following completion of their treatment 46.
This condition was previously referred to as Chronic Lyme Disease (CLD), and more
recently referred to as Post Treatment Lyme Disease Syndrome (PTLDS) or Post Lyme
disease Syndrome (PLDS)23. PTLDS received a case definition from the Infectious
Disease Society of America (IDSA) stating that the individual must have a documented
case of Lyme disease who has completed treatment but continues to show a relapse of
symptoms including fatigue, musculoskeletal pain, and complaints of cognitive
difficulties for a minimum of 6 months from treatment completion55.
8
The cause of PTLDS is unknown but there are many studies with potential
hypotheses as to the cause55. These theories include, the body is having an autoimmune
response as a result of Lyme disease, the antibiotic course fails to clear the Lyme disease
infection, the bacterium has the ability to change form and create biofilms during
environmental stress and a secondary infection by a different pathogen with similar
symptoms to Lyme disease55.
The first theory focuses on the idea that PTLDS is a delayed autoimmune
response to Lyme disease. Singh & Girschick (2004) reviewed the effects of T-cells on
inflammation in the joints during Lyme disease. They found elevated levels of Tlymphocytes in synovial fluid and peripheral blood in adults who were showing
symptoms of PTLDS58. Maccallini et al. (2018) evaluated the role of B cells and the
similarity between human enolase and Borrelia enolase. They found that human and
Borrelia enolase share a conformational B cell epitope which can cause a release of
autoantibodies against enolase. These antibodies have been seen in other autoimmune
diseases that affect the brain41. Further studies testing this theory must be conducted
using several case studies with known Lyme disease patients and those who are suspected
to have PTLDS41.
Lyme disease is typically treated with a course of oral antibiotics between 14-28
days and is supposed to clear the infection. Cameron, Johnson & Maloney (2016)
reviewed several studies conducted to determine the effectiveness of clearing Lyme
disease after a course of antibiotics and found that those treated during early localized
and early disseminated failed to bring 16% to 48% of the patients back to their pre-Lyme
health status. A observation trial found that 33% of patients treated with a three week
9
course of doxycycline continued to have symptoms three to six months post-treatment12.
A study conducted by Logigian, Kaplan & Steere (1990) observed patients with late
disseminated Lyme disease and found that 63% of patients treated with intravenous (IV)
ceftriaxone for 14 days showed improvement, 15% showed no health improvement and
22% showed initial improvement and relapsed with symptoms six months after
treatment39. Failure to clear the infection with an oral or IV course of antibiotics can lead
to these symptoms and be a possible cause for PTLDS. Patients were not tested for other
tick-borne diseases, lasting symptoms may have been caused by a TBD that could not be
treated with antibiotics.
Recent studies have found that the Lyme bacterium has the ability to change
under environmental stresses (pH, temperature, immune attack, nutrient starvation) and
form biofilms to protect itself. It was determined that B. burgdorferi has several forms,
spirochete (stationary phase and log phase spirochete), round body and an aggregated
microcolony that has the ability to form biofilms 17,28. A study conducted by Feng,
Tingting & Zhang (2018) determined that when studied in vitro B. burgdorferi can persist
in variant forms and protect itself from antibiotics and, in vivo mice, the microcolony
form and stationary phase caused more server inflammation in joints that log phase.
Antibiotics were able to destroy log phase spirochete and some round body forms of the
bacterium but failed to completely destroy stationary spirochete phase and the biofilm
aggregates17,28. These forms can prevent the destruction of all B. burgdorferi bacterium in
the body, leaving persistent bacteria behind and may play a role in PTLDS.
The last hypothesis states that PTLDS may be caused by a secondary infection
masked by Lyme disease. One of the main co-infections that has been hypothesized to
10
cause PTLDS is Powassan virus. Powassan virus 63 has similar symptoms to those present
in PTLDS such as fever, fatigue, myalgia, dizziness, confusion, memory loss and in
severe cases, encephalitis and death63. Deer tick virus is not a well-known or studied
virus but has been increasing in the U.S. over the last decade and can be hard to diagnosis
and detect29. Frost et al. (2017) tested 41 patients with known Lyme disease for Powassan
virus and identified 10 (4.1%) of those patients were also positive for Powassan virus.
Furthermore, Thomm et al. (2018) tested 106 patients for Powassan virus which have
been diagnosed with at least one tick-borne disease and identified 10 (9.4%) of these
patients were also positive for Powassan virus 63.
Powassan Virus
Powassan virus (POW) is an emerging tick-borne virus that is on the rise in North
America. It was first discovered in Powassan, Ontario in 1958 after a young boy died of
encephalitis49. POW has since been found in the Great Lake Region and along the
Northeast in the U.S., up into Canada and in the Primorsky region of Russia51. It is a
ssRNA Flavivirus, part of Flavividae family19. It is part of the tick-borne encephalitis
complex (TBC-E), and transmitted by the bite of an infected tick51,63. There are two
lineages of POW; lineage I was first discovered in the 1958 Powassan, Canada case and,
lineage II was discovered in 1996 and referred to as Deer Tick Virus (DTV). The two
lineages are associated with having different vertebrate reservoirs and vectors 62. Lineage
I transmission cycle is maintained by the woodchuck tick (Ixodes cookei) and medium
sized mammals such as the woodchuck (Marmota monax) whereas lineage II
transmission cycle is maintained by the blacklegged tick (Ixodes scapularis) and the
11
white-footed mouse (Peromyscus leucopus)51. Although they are two distinct lineages,
phylogenetic studies have found them to have 84% nucleotide similarity and an amino
acid similarity of 93%5.
POW lineage I is more aggressive with a higher possibility to be fatal than DTV.
DTV is less aggressive20 but studies over the last decade have found that DTV can cause
fatal encephalitis or lasting neurological effects similar to lineage I26,61,63. The CDC has
reported an increase in neuroinvasive POW cases from 6 in 2015 to 33 in 2017 60.
Neuroinvasive cases have been confirmed within 11 states from 2008 to 2017 (Figure 2).
Since 2008, NJ has had 5 cases and PA has had 7. In 2017, PA and NJ both had 4 cases
of confirmed POW, an increase from 0 diagnosed in 2016 in both states 60.
Figure 2. Map of neuroinvaisve Powassan virus cases reported in each US state, 20082017. (Centers for Disease Control and Prevention)
POW lineage II is typically asymptomatic, however it can cause life-threating
conditions such as encephalitis and meningitis to those infected 49. Symptoms occur
12
between 1-5 weeks after initial exposure by a tick bite53. They can range from headaches,
drowsiness, nausea and disorientation during early infection and can progress into
encephalitis, meningoencephalitis and coma during later stages of infection32. The
mortality rate can be as high as 15% for patients infected, and 50% of the surviving
patients are diagnosed with neurological sequelae53. Patients can be tested using their
CSF or serum targeting POW IgM antibodies29,53. Current serological testing consist of
using IgM ELISA, IgM immunofluorescence antibody assay (IFA), IgG ELISA and
conformation testing with a > 90% or > 50% plaque neutralization test (PRNT90 or
PRNT50) with a >4-fold increase in antibody titers from acute- and convalescent-phase
sera29,63. Based on the sensitivity of current testing protocols, there may be more
confirmed cases of POW then what is being reported 29,53. Confirming lineage I vs lineage
II POW requires using neutralization assays such as qPCR and sequencing following a
positive serological test49. Transmission of POW from vector to host is as fast as 15
minutes following attachment as the virus resides within the salivary glands of the tick18 .
Vector
There are hundreds of ticks worldwide which fall into one of three
families: Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae (ticks of South
Africa)27. The largest family Ixodidae, play a role in vectoring and transmitting a
majority of tick-borne diseases globally. Ixodes scapularis (blacklegged tick or deer tick)
is a medically-important tick as it contributes to many tick borne diseases in North
America38. The blacklegged tick is distributed up into southeastern Canada down the
13
northeast coast and as far west as Texas, Oklahoma and parts of North and South Dakota
(Figure 3).
The blacklegged tick is a 3-host tick with three life stages: larval, nymph and
adult, that lives a two-year life cycle, molting between each life stage38 (Figure 4). Larval
ticks hatch in May and are active until August, feeding on small rodents such as the
white-footed mouse (Peromyscus leucopus) and birds. Once a larval tick has fed to
engorgement, it will drop off its host and molt into a nymph and enter diapause until the
late spring and summer24. The nymph will quest for its second host which can be small to
medium-sized animals such as rodents, lagomorphs, ungulates, cats, dogs and humans.
Following engorgement, the nymph will molt into an adult. Adult females will use larger
animals as a host before copulating with adult males and laying their eggs in the early
spring38, laying up to 3,000 eggs.
Figure 3. Distribution of blacklegged ticks in the United States as of 2018 (Centers for
Disease Control and Prevention)
14
Figure 4. The two-year life cycle of the blacklegged tick. Broken down into when each
life stage is most prevalent and the main host each life stage feeds on. (Centers for
Disease and Control Prevention)
Blacklegged ticks acquire pathogens transovarially or transstadially depending on
the pathogen. Transovarial pathogens are able to be passed on from an infected mother
tick to the eggs. It is unknown if POW is transovarially passed down to the next
generation of an infected tick. A transstadial pathogen is picked up by a vector when
feeding on an infected reservoir. The bacterium that causes Lyme disease is a transstadial
pathogen that will continue to live within the tick once acquired from a reservoir40. A
reservoir host is able to survive living with a pathogen, without presenting symptoms or
infection.
15
The white footed-mouse (Peromyscus leucopus) is the reservoir for several tickborne pathogens that are vectored by the blacklegged tick. The most notable pathogen is
B. burgdorferi (Lyme disease), in addition to Anaplasma phagocytophilum
(Anaplasmosis), Babesia microti (Babesiosis), Borrelia miyamotoi and, DTV (Powassan
virus lineage II)16. Other rodents, such as chipmunks, squirrels, shrews and woodchucks
are competent reservoirs for tick-borne pathogens by the blacklegged tick10. Although
only a few animals are reservoir host, other large animals play a role in the distribution
and spread of infected ticks. Studies enumerating the number of ticks on black bears have
identified on average 400 ticks3. A study conducted by LoGiudice, Ostfeld, Schmidt &
Keesing (2003) identified the host diversity and community composition of ticks in
Dutchess County, New York seen in Table 1. 40. The average tick load on an animal can
play a large role in pathogen and tick distribution depending on the animal. The whitefooted mouse is a key reservoir host for many tick-borne pathogens and a high tick load
can increase infected ticks in an area. While, other larger animals such as the white-tailed
deer and black bear are not known reservoir host but can play crucial roles in carrying
large tick loads and distributing them into and out of urban and rural areas. This
mechanism of transport can result in introducing new tick species and diseases to an area
inhabited by humans.
Table 1. Average tick load of small to large animals identified by LoGiudice, Satefeld,
Scmidt & Keesing (2003) in Dutchess County, NY
Animal
White-footed mouse (Peromyscus leucopus)
Eastern chipmunk (Tamias striatus)
White-tailed deer (Odocoileus virginianus)
16
Average tick load
27.8
36.0
239
Short-tailed shrew (Blarina brevicauda)
Grey Squirrel (Sciurus carolinensis)
62.9
142
Black Bears
The American black bear (Ursus americanus) is medium-sized and historically
found in the United States, Canada and Mexico. Black bears have great mobility, are
generalist and have an omnivorous diet. This allows black bears to live in a wide range of
habitats, from swamps to semi-desserts and dense forests. Black bear males (called boars)
can weigh between 150 to 600lbs and females (called sows) can weigh from 150 to
400lbs8. Female black bears will typically have a home range between 2.5-10 square
miles, while males can have a much larger home range between 10-59 square miles9. In
the Northeast, they typically are black in color with a brown muzzle and can have a white
blaze on their chest. Some bears are an atypical cinnamon color. Black bears are strong
swimmers and good climbers due to their five toes and long curved claws 45. In the wild,
black bears can live up to 25 years. Although bears prefer to eat wild foods such as
acorns, skunk cabbage, and blueberries they will also eat from human garbage and bird
feeders. Merkle et al.(2013) surveyed black bears from 2009 and 2010 in Missoula,
Montana where bears were 80% more likely to choose urban grounds for food than in the
wild42. Black bears will do a wide range of damage to homes, sheds that have food stored
in them, destroy garbage cans and bird feeders.
Black bears typically begin to mate around the age of three, but can start as early
as two years old. Mating occurs late May through July. Females may enter their dens as
early as the end of October and males may enter their dens as late as mid-December.
During January, sows will give birth in their dens and can have a litter of one to five
17
cubs. Bears will begin to exit their dens in April, with the cubs following the sow for
about a year to year and half before they go off on their own8,45.
Figure 5. Sedated black bear cub, 2018 NJDFW research trapping (Photo credit Kristine
Bentkowski)
Black bear populations in NJ, PA and NY have been increasing over the last
decade. The New Jersey Division of Fish and Wildlife (NJDFW) has seen an expansion
of the black bear population from the northeast region of the state in 1995 to sightings in
every county to date45. Today in northwestern NJ, there are as many as three bears per
square mile45. In PA, the Pennsylvania Game Commission (PAGC) has also seen an
increase in the black bear population over the last decade. As a result of increased
populations, both states have had significant increases in nuisance bear reports. Nuisance
bears defined by the PAGC and NJDFW are black bears that are entering residential
areas, destroying farmland or homeowner property. To control and monitor black bear
18
populations, the PAGC and NJDFW conduct annual research trappings, and an annual
black bear harvest to control overpopulation (Figure 5). With the increase of interactions
between black bears and humans, there is an increasing public health concern for
zoonotic diseases. Very little research has been conducted to determine if black bears are
reservoirs to some tick-borne diseases, however, these mammals play an important role in
tick dispersal carrying ticks into residential areas.
Study Objectives
Monitoring the prevalence of Lyme disease and Powassan virus are important to
public health and safety. This will be the first study conducted in Pennsylvania and New
Jersey to determine the prevalence of Powassan virus and co-infection prevalence with
Lyme disease. The goal of this study was to determine the prevalence of Lyme disease
and Powassan virus in blacklegged ticks collected from black bears in New Jersey and
Pennsylvania from 2015-2018. The objectives were to:
1.
Determine the prevalence of Powassan virus (DTV) in blacklegged ticks from
bears in NJ and PA from 2015-208
2.
Determine the prevalence of Lyme disease in blacklegged ticks collected from
black bears in NJ and PA from 2015-2018
3.
Evaluate the co-infection rate of Powassan virus and Lyme disease in NJ and PA
19
Chapter II
Materials and Methods
Study Area and Sample Collection
From 2015-2018, ticks were collected from black bears (Ursus americanus) with
assistance from the New Jersey Division of Fish and Wildlife (NJDFW). Ticks were
collected from Hunterdon, Morris, Passaic, Sussex, and Warren Counties in New Jersey
(Figure 6). They were collected three to four times throughout the year during research
trapping and at the black bear hunt check stations. Biannual research trapping occurred
May through June and again in the fall from August through September. Research
trapping sites were selected based on bear activity, proximity to food sources such as
cornfields, acorn mass, and reported bear sightings. Black bears were captured using
Aldrich foot snares and culvert traps. Trained personnel used a mixture of ketamine and
xylazine (ZooPharm Inc, Windsor, CO) to anesthetize captured animals. Morphological
measurements were collected, and each animal was tagged, tattooed with corresponding
tag number, and sexed. To determine age, the premolar of yearling and adult bears was
removed. Ticks were also collected from hunter-harvested bears during NJDFWs hunting
season. Segment A of the bear hunt occurred for one week in October, and segment B
20
occurred for one week in December. Harvested black bears were brought into check
stations, where morphological measurements were collected, and tick searches were
completed.
During the annual research trapping and black bear harvest, a 5 to 10-minute tick
check focusing on the ears, around the eyes, neck, under the armpits and around the groin
region was conducted. Ticks were collected with forceps and placed into 2mL screw top
tubes labeled with the bear’s ID number, date and location of capture. Ticks were stored
in a cooler on ice until being brought back to the Northeast Wildlife DNA Lab
(NEWDL), where they were stored at -20˚C.
21
Figure 6. New Jersey counties where black bears were trapped or harvested, and ticks
were collected. Ticks collected from blue stripped counties were analyzed for Powassan
and B. burgdorferi.
Ticks were collected from black bears in Pennsylvania between 2017-2018 with
the assistance of the Pennsylvania Game Commission (PAGC) and collaborators at Penn
State University. In 2017, ticks were collected from Monroe and Pike Counties, and in
2018 from Centre, Clearfield, Clinton, Huntingdon, Lycoming, Potter, and Tioga
Counties (Figure 7). The PAGC collected ticks from nuisance and vehicle-strike bears
between September and October of 2017. In 2018 ticks were collected throughout the
year from June to December during the annual research trapping in the Fall and Spring,
the bear hunt in October and December and bears caught throughout the time period with
suspicion of mange infection from central Pennsylvania. Tick check methods were
22
designed by Hannah Greenberg from Pennsylvania State University from an ongoing
study to determine the abundance and distribution of ticks on American black bears in
Pennsylvania. Tick checks were conducted over 16 designated body regions on the black
bear with 4" X 4" standardized tick square. There was no time constraint on tick checks.
Ticks were placed in 2mL screw top tubes filled with 70% ethanol and had the black
bear’s age, sex, and date.
Figure 7. Pennsylvania counties where black bears were trapped or harvested, and ticks
were collected 2017-18. Blue counties ticks were collected in 2017 and pink counties
ticks were collected in 2018.
Identification and Extraction
All ticks were identified to species and life stage following Ward’s Guide to
North American Ticks (Ward’s, Rochester, NY). This was done by examining their
festoons, geographic location and scutum (shield) pattern, as each tick has their own
unique pattern or color. The body of the I. scapularis begins to engorge and stretch as it
feeds, while the hard scutum continues to keep its size and shape. A scutal index can be
used to measure the engorgement level of the tick and estimate duration of attachment.
The engorgement level of I. scapularis females and nymphs were determined using the
23
scutal index (SI = b/a) measuring the body length from the posterior edge to the basis
capitulum and the maximum width of the scutum (Figure 8). Based on the SI, estimated
engorgement hours were determined, and ticks were then labeled as unengorged (≤ 14
hours), semi-engorged (≤ 15-92 hours) or fully engorged (≥ 93 hours) (Figure 9).
A tick pool consisted of ticks of the same sex, life stage, engorgement level,
individual black bear and county. Bears that had more than five ticks of the same
engorgement, sex and life stage were pooled together but if the bear had less than five
ticks each tick was analyzed individually. For example, a bear with three ticks collected
from it had each tick analyzed alone, while a bear with five females of the same
engorgement had one pool containing all five ticks. Pools typically ranged from 1-5 ticks.
Ticks in the same pool were all placed into one tube for extraction of RNA and DNA and
analyzed as one sample.
Figure 8. Measurement of a female blacklegged tick using the scutal index (SI = b/a), a =
maximum width of scutum, b = length of tick from posterior to the basis capitulium
24
Figure 9. Examples of engorgement sizes of adult female blacklegged tick. Left to right:
Fully engorged, semi-engorged, semi-engorged, unengorged, unengorged (Photo credit
Kristine Bentkowski)
Powassan virus (Lineage II) SYBR Assay Optimization
To optimize a reverse transcriptase real-time PCR (RT- PCR) assay for Powassan
virus Lineage II, a standard curve analysis was performed. Using a synthetic positive,
serial 1:10 dilutions were created ranging from 10 -1 to 10-10. Each dilution was performed
in triplicate to validate accuracy and a negative control of nuclease free water was used to
confirm absence of contamination. A synthetic positive sample was created by
GENEWIZ (South Plainfield, NJ) with the NS5 target primers. PCR was performed in
25L reactions. Each reaction contained 0.2M of forward primer
5’gaagctgggtgagtttggag 3’ and 0.2M reverse primer 5’cctgagcaaccaaccaagat 3’ targeting
a 318 base pair region of the NS5 gene (Knox et al. 2017). The PCR protocol followed
manufacture guidelines with the modification of using 0.25L of SYBR enzyme mix
(Thermofisher, Waltham, MA). The 25L RT-PCR reaction consisted of 1X SYBR
Green Master Mix (Thermofisher, Waltham, MA), 1.0L premixed forward and reverse
primers, 6.25L Qiagen nuclease free water, 0.25L SYBR enzyme mix, and 5L of
synthetic positive. The standard curve was run on an Applied Biosystems StepOnePlus TM
25
PCR system. Thermal cycling conditions for RT-PCR was performed at 50C for 20
mins, 95C for 5 minutes, followed by 45 cycles of 94C for 10 seconds, 55C for 5
seconds and 60C for 25 seconds. A standard curve was created using the log dilution of
copies (ng) by the CT (cycle threshold) value. Each CT value was determined by how
many cycles it took for the fluorescent signal of the sample to cross the threshold.
RNA Analysis
RNA was extracted from tick pools following the Qiagen viral RNA extraction
protocol (Qiagen, Germantown, MD). The protocol was modified to include an overnight
incubation at room temperature. A blank was included in each analysis extraction to
confirm absence of contamination during the extraction process.
Samples were analyzed for Powassan virus using the optimized Powassan virus
Lineage II SYBR green assay. Specific primers were used to target the NS5 region of the
Powassan virus genome (Table 2). Each analysis was run with a positive to validate the
assay and negative to confirm the absence of contamination. All RT-PCR was conducted
on an Applied Biosystems StepOnePlusTM PCR system. Positive samples were identified
with CT values of 30-44 and a threshold of 1.725.
DNA analysis
DNA was extracted from tick pools along with RNA following the Qiagen viral
RNA extraction protocol (Qiagen, Germantown, MD) and modifications. A blank was
included in the extraction process to confirm the absence of contamination during
extraction. To identify the presence of Borrelia burgdorferi, DNA was amplified using a
26
TaqMan real-time PCR protocol. A specific primer and probe targeting the 16s-23s
intergenic spacer region of Borrelia burgdorferi was used (Table 2). A 25µL TaqMan
real-time PCR (qPCR) reaction containing 1X TaqMan Master Mix (Thermofisher,
Waltham, MA) was used for qPCR. The reaction was made up of 12.5L TaqMan master
mix, 5L Qiagen nuclease free water, 4.48L of premixed forward and reverse primer,
0.56L B. burgdorferi probe, and 2.5L of sample DNA. Thermal cycler conditions for
qPCR were performed at 50C for 2 minutes, an enzyme activation of 95C for 10
minutes, followed by 50 cycles at 95C for 15 seconds and 60C for 1 minute. Positive
samples were determined with CT values of 30-38 at a threshold of 0.047. A positive
control was run with each analysis to validate the assay and a negative control to confirm
the absence of contamination.
Table 2. Oligonucleotide primer used for PCR. SYBR green primer targeting the NS5
region of Powassan virus. Real-time primer targeting the 16S-23S intergenic spacer
region for Lyme (Borrelia species) and specific probe for Borrelia burgdorferi
Target
Organism
Powassan
virus (DTV)
Powassan
Virus
(DTV)
Gene
Target
Sequence (5’ → 3’)
Amplicon Size
(bp)
F 5’-AACATGATGGGAAAGAGAGAG-3’
NS5
318bp
R 5’ -CAGATCCTTCGGTACATGGAA-3’
Borrelia
species
16S-23S
intergenic
spacer
Borrelia
burgdorferi
Probe
F 5’-GCTGTAAACGATGCACACTTGGT-3’
R 5’-GGCGGCACACTTAACACGTTAG-3’
6FAM-TTCGGTACTAACTTTTAGTTAAQSY
27
69bp
Statistical Analysis
The minimum infection rate (MIR) was determined by dividing the total number
of positive individuals and pools by the total number of ticks analyzed. This value
assumes only one tick per pool was infected; therefore, it is a conservative estimate of
prevalence. The minimum infection rate calculated from the tested ticks was used as an
estimate of the minimum prevalence of infected ticks in New Jersey and Pennsylvania.
Prevalence rates were determined by life stage for adults and nymphs.
Statistical analysis was conducted using the statistical computing program
R34,56,67. A Chi-square test was used to determine if there was significance between the
proportion of tick species collected in New Jersey and Pennsylvania. A generalized linear
model (GLM) was used to test for the effect of engorgement status (unengorged, semiengorged or fully engorged) and life stage (nymph or adult) on the minimum infection
rate. All factors were treated as fixed. A GLM was also used to test for the effect of the
state (New Jersey/Pennsylvania) and year (2015-18 in New Jersey, 2017-28 in
Pennsylvania) on infection rate. Lastly, a GLM was used to test for effect of New Jersey
counties on infection rate. For all statistical analyses, the criterion for significance was
set to = 0.05.
28
Chapter III
Results
Tick Collections
Appendix A presents the total number of ticks collected from 2015-2018 by
collection season (Fall harvest and Fall or Spring research trapping), year, state, species
and life stage. Overall, four species of ticks were collected: Ixodes scapularis, Ixodes
cookei, Amblyomma americanum and Dermacentor variabilis. Larval, nymph and, adult
life stages were collected of I. scapularis were collected. All other tick species were
collected in their adult life stage. The most commonly collected tick was Ixodes
scapularis (n=2,119) (78.1%), followed by Dermacentor variabilis (n = 590) (21.7%),
Amblyomma americanum (n=2) (0.07%) and Ixodes cookei (n=2) (0.07%). The average
tick load per black was 6.33. The total number of ticks collected from New Jersey
between 2015-18 was 2,133 and in Pennsylvania from 2017-18 was 580. There was no
significant difference between New Jersey and Pennsylvania in the relative abundance of
tick species collected (chi-squared test; X2= 8, p = 0.2381, df = 6).
29
In New Jersey, four different tick species were collected throughout the year, with
the majority of collected ticks being adult I. scapularis in October and D. variabilis in
30
June. Overall the majority of ticks were collected in October when adult I.
scapularis are most active, then in June when I. scapularis nymphs and adult D.
variabilis are most active (Figure 10). Fewer ticks were collected in August, most likely
due to the hot, dry weather unfit for tick activity. Fig. 10 presents the total number of all
ticks collected in the month of October between 2015-18, in June 2015-18 and August
2015-18. There were 1,554 I. scapularis collected between 2015-18 in New Jersey with
98% being adults, 1.6% being nymphs and 0.1% being larval. The majority of adults
were collected each year in October (Figure 11).
1600
Total Ticks Collected
1400
1200
1000
800
600
400
200
0
October
June
August
Month
Figure 10. Total number of ticks collected from 2015-18 in New Jersey by month
31
Total I. scapularis Collected
600
500
400
300
200
100
0
October October June 2016 August October June 2018 August
2015
2016
2017
2017
2018
October
2018
Month and Year
Figure 11. Total adult I. scapularis collected in the month throughout the year from New
Jersey (2015-18)
Three tick species collected in Pennsylvania between 2017-18. The majority of
ticks in Pennsylvania were collected in May, June, and November, active months for
nymphs in the Spring and Summer, and adults in the Spring and Fall (Figure 12). The
majority of ticks collected were I. scapularis, with 90.6% consisting of adults, 5.3%
nymphs and 4.1% larval. Adult I. scapularis were collected through the majority of
months but were most prevalent in May, June and November during periods that I.
scapularis adults are known to be most active (Figure 13). Nymph I. scapularis were
collected in five months out of the year, with May, June and August having the highest
collection rate (Figure 14). Larval I. scapularis were collected four months out of the
year with August being the peak month for larval collection (Figure 14).
32
Total number of ticks collected
200
180
160
140
120
100
80
60
40
20
0
Month
Figure 12. Total number of ticks collected from 2017-18 in Pennsylvania by month
Total I. scapularis collected
160
140
120
100
80
60
40
20
0
Month anf Year
Figure 13. Total number of adult I. scapularis collected in Pennsylvania in 2017-18 by
month
33
Total Number of larval and nymph
collected
30
25
20
15
Nymph
Larval
10
5
0
March
May
June
July
August
Month
Figure 14. Total number of nymph and larval I. scapularis collected in Pennsylvania in
2017-18 by month
Optimization of Powassan Lineage II Assay
In order to standardize the Powassan virus Lineage II RT-PCR assay a standard
curve was created using a synthetic positive created by GENEWIZ (South Plainfield, NJ).
The synthetic positive control was created from the targeted NS5 primers used in the
assay. Using 10 fold serial dilutions ranging from 10-1 to 10-10 with a starting
concentration of 1.6711 copies/L were used and performed in triplicate to validate
accuracy. The 10-7 dilution was the last dilution to have a CT call for all three samples.
CT values from dilutions 10 -1 to 10-7 were inserted into Microsoft Excel and a linear
regression was created using the log of number of copies per each dilution. The slope
formula was determined to be y = -5.036x + 74.524, with an R2 of 0.9853 (Figure 15).
The CT cutoff value was determined to be 44.7 by plugging in the highest triplicate value
34
from the 10-7 dilution into the slope formula. A threshold generated by the Applied
Biosystems StepOnePlus standard curve was 1.725 for Powassan virus (DTV)
50
45
y = -5.036x + 74.524
R² = 0.9853
40
35
CT
30
25
20
15
10
5
0
5
6
7
8
9
10
11
12
13
Log of copy number
Figure 15. Plotted linear regression of the standard curve, using the log number of copies
of each dilution by the CT call from each triplicate
Disease and Statistical Analysis
Ixodes scapularis were selected for analysis from three counites in New Jersey
and nine counties in Pennsylvania because they are the known vectors of Powassan virus
Lineage II. Samples that were selected to be analyzed were separated by state, county and
year and each individual was accounted for (Table 3,Table 4). Counites in New Jersey
were picked according to which had the highest yield of I. scapularis collected.
Hunterdon County was also included as it has had a known human case of Powassan
virus. All I. scapularis nymph and adult ticks from Pennsylvania were analyzed due to
having only two years of ticks collected. A total of 1,277 blacklegged ticks were selected
35
to be analyzed for Powassan virus Lineage II and Lyme disease (Borrelia burgdorferi)
using the polymerase chain reaction (PCR). A total 595 samples consisting of 344
individuals and 251 pools were analyzed (Appendix B). Of these, 831 (64.9%) were
female, 384 (30.0%) were male and 64 (5.1%) were nymphs (Figure 16).
Table 3. Total number of blacklegged ticks analyzed from each county
in New Jersey
YEAR
Hunterdon
Sussex
Warren
Total
2015
26
23
21
90
2016
0
184
47
231
2017
0
109
91
200
2018
0
108
117
225
36
37
32
0
2017
2018
0
3
10
0
22
0
304
0
23
0
111
0
5
0
23
0
498
35
YEAR Monroe Pike Centre Clearfield Clinton Huntingdon Lycoming Potter Toga Total
Table 4. Total number of blacklegged ticks analyzed from each county in Pennsylvania
300
Number of Ticks
250
200
Female
150
Male
Nymph
100
50
0
NJ 2015 NJ 2016 NJ 2017 NJ 2018 PA 2017 PA 2018
State and Year
Figure 16. Total number of I. scapularis female, male and nymph ticks analyzed (n=
1,277) from NJ (2015-18) and (PA 2017-18)
Powassan Virus
The overall minimum infection (MIR) rate was determined for positive tick pools
for Powassan virus as 3.52%. The overall minimum infection rate was determined for
adult blacklegged ticks positive for Powassan virus as 3.54%. The MIR of adults I.
scapularis infected in New Jersey ranged from 0-3.0% between 2015-18, with 2017
having the highest MIR with 3.0%. The MIR in northeast Pennsylvania counties was
5.7%, compared to 4.0% in central Pennsylvania counties (Figure 17). The minimum
infection rate was determined for nymph blacklegged ticks positive for Powassan virus in
New Jersey 0%, and Pennsylvania 3.92% for 2018 (Figure 18).
38
6.0%
MIR % Positive
5.0%
4.0%
3.0%
2.0%
1.0%
0.0%
NJ 2015
NJ 2016
NJ 2017
NJ 2018
PA 2017
PA 2018
State and Year
Figure 17. Minimum infection rate (MIR) percent of adult I. scapularis with POW (DTV)
(n=1,213) from NJ (2015-18) and PA (2017-18)
5%
4%
MIR % Positive
4%
3%
3%
2%
2%
1%
1%
0%
NJ 2018
PA 2018
State and Year
Figure 18. Minimum infection rate percent of nymph I. scapularis ticks with POW
(DTV) (n=64), from NJ and PA 2018
39
Ixodes scapularis nymphs and adults did not differ in Powassan infection
prevalence (GLM df = 2, p = 0.897). Powassan prevalence also did not vary with
engorgement state in either adults (df = 3, p = 0.910) or nymphs (df = 1, p = 0.976).
There was no significant difference between males and females in Powassan infection (df
= 1, p = 0.836). There was no significant difference between New Jersey and
Pennsylvania overall in Powassan infection (df = 1, p = 0.852). Appendix C consist of
full statistical tables.
Borrelia burgdorferi
The overall minimum infection rate was determined as 19.2% for ticks positive
for B. burgdorferi. The overall minimum infection rate was determined as 19.8%. for
adult blacklegged ticks positive for B. burgdorferi. The MIR of adults I. scapularis
infected in New Jersey ranged from 17.7-23.0% between 2015-18, increasing each year
(Figure 19). The MIR in northeast Pennsylvania counties was 28.5%, compared to 18.1%
in central Pennsylvania counties. The minimum infection rate was determined for nymph
blacklegged ticks positive for B. burgdorferi in New Jersey 0%, and Pennsylvania 7.84 %
for 2018 (Figure 20).
40
30.0%
MIR % Positive
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
NJ 2015
NJ 2016
NJ 2017
NJ 2018
PA 2017
PA 2018
State and Year
Figure 19. MIR (%) of adult I. scapularis ticks with B. burgdorferi (n=1,213) from NJ
(2015-18) and PA (2017-28)
9%
8%
MIR% Positive
7%
6%
5%
4%
3%
2%
1%
0%
NJ 2018
PA 2018
State and Year
Figure 20. Minimum infection rate percent of nymph I. scapularis ticks with B.
burgdorferi (n=64), from NJ and PA 2018
41
I. scapularis nymphs and adults did not differ in Borrelia burgdorferi infection
prevalence (GLM df = 2, p = 0.725). B. burgdorferi prevalence also did not vary with
engorgement state in either adults (df = 3, p = 0.799) or nymphs (df = 2, p = 0.815).
There was no significant difference between males and females in B. burgdorferi
infection (df = 1, p = 0.945). There was no significant difference between New Jersey
and Pennsylvania in B. burgdorferi infection (df = 1, p = 0.986).
Co-infection
Overall of the 45 positive Powassan ticks, 25 of them were co-infected with B.
burgdorferi (55.5%). Of these, 23 were adults (53.4%) and the two Powassan positive
nymph pools were both co-infected with B. burgdorferi (100%). New Jersey had 11 coinfected tick pools and Pennsylvania had 14. There were 12 co-infected female pools, 11
co-infected male pools and 2 co-infected nymph pools. The majority of co-infected ticks
came from central Pennsylvania in 2018, compared to New Jersey 2015-18 and
Pennsylvania 2017.
County Data
Powassan
Positive pool samples of Powassan virus in New Jersey counties were consistent
over the three years 2016-18. Sussex County had the most positive tick pools compared
to Warren or Hunterdon Counties (Table 5). No counites in 2015 had a positive pool for
42
Powassan virus. Northeast Pennsylvania presented a lower positive pool sample of
Powassan virus than central Pennsylvania (Table 6). Clinton County in central
Pennsylvania had the highest amount of positive pools of Powassan virus throughout
New Jersey or Pennsylvania with 16 positive tick pools.
New Jersey did not differ in Powassan infection prevalence between 2015- 2018
(GLM df = 2, p = 0.986). Pennsylvania did not differ in Powassan infection prevalence
between 2017 and 2018 (df = 1, p = 0.906). There was no significant difference between
Sussex and Warren county (df = 1, p = 0.917), Hunterdon county was excluded due to no
positive ticks. No statistical test was run for Pennsylvania counties due to uneven
distribution of ticks collected between counites.
Table 5. Total Powassan positive pools by year in each New Jersey county
COUNTY
HUNTERDON
SUSSEX
WARREN
TOTAL
2015
0
0
0
0
2016
0
4
2
6
2017
0
5
1
6
2018
0
4
1
5
Table 6. Total Powassan positive pools by year in each Pennsylvania county
COUNTY
PIKE
MONROE
CENTRE
CLEARFIELD
CLINTON
HUNTINGDON
LYCOMING
POTTER
2017
0
2
0
0
0
0
0
0
2018
0
0
0
2
16
1
5
0
43
TIOGA
TOTAL
0
2
2
26
Borrelia burgdorferi
The amount of positive tick pools overall and in Sussex County fluctuated over
the years. In contrast, Warren had a continuous increase of positive tick pools between
2015-18 (Table 7). Northeast Pennsylvania had fewer positive pools than central
Pennsylvania for B. burgdorferi (Table 8). Overall, Clinton County had the most positive
tick pools in Pennsylvania and New Jersey for B. burgdorferi.
New Jersey did not differ in B. burgdorferi infection prevalence between 2015
and 2018 (GLM df = 3, p = 0.885). Pennsylvania did not have a significant difference in
B. burgdorferi infection prevalence between 2017 and 2018 (df = 1, p = 0.955). There
was no significant difference between counties in New Jersey 2015-18 (d=3, p = 0. 720).
Statistical analysis were not conducted for Pennsylvania counties due to uneven
distribution of tick collection.
Table 7. Total B. burgdorferi positive tick pools by year in New Jersey counties
COUNTY
HUNTERDON
SUSSEX
WARREN
TOTAL
2015
4
6
6
16
2016
0
36
9
45
2017
0
27
14
41
44
2018
0
25
22
47
Table 8. Total B. burgdorferi positive tick pools by year in Pennsylvania counties
PIKE
MONROE
CENTRE
CLEARFIELD
CLINTON
HUNTINGDON
LYCOMING
POTTER
TIOGA
TOTAL
2017
0
10
0
0
0
0
0
0
0
10
2018
0
0
0
2
55
3
19
1
6
86
45
Chapter IV
Discussion
Powassan virus is an emerging tick-borne virus that has the potential to be
debilitating and deadly to those infected. Lyme disease is one of the top vector-borne
diseases in the United States and has been increasing in the amount of cases reported
every year1. Conducting prevalence studies is an important tool to understanding tickborne diseases in areas with high human and tick interactions. This is the first study
identifying the prevalence of Powassan virus (DTV) and Lyme disease (Borrelia
burgdorferi) co-infection in Ixodes scapularis collected from black bears in New Jersey
and Pennsylvania.
Tick Collection Data
Tick distribution in the United States has been changing over the last decade.
Ticks not established in regions are becoming established and populations of ticks in
established regions are on the rise59. In 2014-2018, the distribution of I. scapularis has
increased from the Northeast and Great Lake regions down south further into Texas and
46
west into the eastern part of Nebraska and Kansas. Eisen, Eisen & Beard (2017)
examined the county-scale distribution of I. scapularis and noted that it had been
documented in 1,420 of the 3,100 continental US counties as of 2016. This represented
an increase of 44.7% when compared to the previous county-scale distribution map
created in 199823. Other tick species such as Amblyomma americanum (Lone star tick)
have been increasing in geographic distribution. The Lone star ticks distribution has
increased from established areas in southern states to newly established areas as far north
as New York and Maine25.
Several factors play a key role in the increased distribution of tick populations in
the United States, with the two main hypotheses focused around climate change and
habitat fragmentation. To survive, the majority of ticks need moderately warm
temperatures. In fact, I. scapularis cannot survive temperatures over 40C or under
-10C59. Additionally, ticks need a moderately humid climate to survive, as too not dry
out or become overly saturated24. Climate change in the U.S. has caused warmer winters
and wetter summers. This has allowed ticks to have a higher survival rate over the winter
and summer24. In Warren County, NJ the average temperature in June 2015 was 24.6C
and in June 2018 increased to 24.8C. June of 2015 in Warren county had a high average
precipitation of 7.73 inches compared to June 2018 which had an average precipitation of
3.43 inches. In Monroe County the average temperature in June 2015 was 25.0C and in
June of 2018 it was 26.0C with an average precipitation of 10.95 inches in 2015 and
3.62 inches in 2018. In Clinton County the average temperature was 25.6C in 2015 and
25.2C in 2018 with an above average precipitation in 2015 of 8.36 inches and an
47
average precipitation of 5.28 inches in 2018. Between 2015 and 2018 in these three
counties temperatures tended to increase and had lower precipitation each year. The high
precipitation in 2015 may of caused the tick population that year to become overly
saturated with water, while in 2018 the precipitation was close to the known average for
each county and could of created a more stable humid for ticks to survive in 14.
Changes due to human growth have not just impacted the climate but wildlife
habitat too. Human growth and expansion have increased forest fragmentation and
changed the micro-habitat of wildlife. Allan, Keesing & Ostfeld (2003) conducted a study
to determine if forest fragmentation increased the white-footed mouse (Peromyscus
leucopus) population and if it affected the infection rate of B. burgdorferi in larval and
nymph I. scapularis. They concluded that in highly fragmented areas, the white-footed
mouse population increased, possibly due to low predator abundance and low
competition of resources. As the white-footed mouse is the main reservoir for many tickborne pathogens, they noted larval and nymph infection rates of B. burgdorferi increased
in highly fragmented areas2. Furthermore, forest fragmentations create edge habitats that
are suitable for high populations of white-tailed deer (Odocoileus virginianus), a
favorable host for I. scapularis adults to feed and reproduce on11. Forest fragmentations
have increased the risk of human-tick interactions as many of these forest fragmentations
border homes11 and increased populations of ticks are present in them.
In this study, Lone star ticks were collected from black bears in 2018 in northern
New Jersey and northeast and central Pennsylvania. Lone star ticks were not found on
black bears in previous years in either states. Previous studies conducted by Zolink et al.
(2015) and Chern, Bird & Frey (2016) collected ticks from black bears in New Jersey and
48
found the majority of ticks to be adult D. variabilis and adult and nymph I.
scapularis13,68. Although, nymph and larval I. scapularis were only found in 2017 and
2018 on black bears during this study. The collection of larval and nymph I. scapularis
from black bears could be due to an increase in the nymph and larval populations in 2017
and 2018 and choosing a larger mammal to feed on due to accessibility of feeding space.
Ticks used in this study from 2015 and 2016 were collected by previous students and all
nymphs collected those years may have been used for other studies. Lastly, another
reason could be due to a more thorough search on the black bears by collectors, looking
for all life stages of ticks, and not just the large adults easily visible to the eye. Black
bears were combed over in 10-15-minute increments, a short period of time to search for
ticks. The average bear had 6.7 ticks collected from it, with the low being 0 and the
highest being 44 ticks. Al-Warid et al. (2017) determined an average tick community
composition of 400 ticks on black bears in Missouri. Al-Warid did not state if there was a
time restraint for each tick search. A longer search duration may yield a higher average of
ticks on black bears in New Jersey and Pennsylvania and may more accurately describe
the community composition of ticks on these bears.
Black bears may play an important role in the dilution of tick-borne diseases in an
area. Black bears, like white-tailed deer, are not known reservoirs of many tick-borne
pathogens but can host many ticks on their bodies. Huang et al. (2019) conducted a study
on Block Island, RI and determined when nymphs and larval I. scapularis fed on whitetailed deer, the infection rate of B. burgdorferi in I. scapularis decreased the next year.
Black bears may also help to decrease the infection rate of I. scapularis if larval and
nymphs begin to feed on them as their first and second meals. Black bear populations in
49
the New York, New Jersey and Pennsylvania are on the rise and if they are becoming a
primary host for larval, nymph and adult I. scapularis the infection rate of tick-borne
disease may decrease in high-density black bear areas. It is unknown if black bears are
reservoirs for Powassan virus and is unclear at this time if they play a role the
transmission cycle.
Powassan (DTV) Prevalence
Few prevalence studies for Powassan virus lineage I and/or lineage II have been
conducted over the last decade. A study conducted by Brackney et al. (2008) determined
that 1.3% of I. scapularis adults tested were DTV positive in Wisconsin. Another study
conducted in 2011-12 by Knox et al (2017) analyzed four quadrants of Wisconsin and
determined a MIR range for DTV between the four quadrants to be 1.56-4.62%.
Anderson and Armstrong (2012) analyzed adult and nymph I. scapularis in Connecticut
and found 0.8% to 1.6% DTV-positive tick pools in Bridgeport and 0.4% to 3.9% DTVpositive pools in North Branford. A study conducted by Dupuis II et al. (2013) analyzed
adult and nymph I. scapularis collected from several counties in Hudson Valley, NY
between 2007 and 2012 and determined a Maximum Likelihood Estimate (MLE) range to
be 0.2-6.0% for adults. Results from this study were similar to those found in previous
studies, with a minimum infection rate (MIR) prevalence ranging from 0.0-3.0% in New
Jersey (2015-18) and 4.0-5.7%% in Pennsylvania (2017-18). As the MIR was calculated
to determine the prevalence of tick pools in New Jersey and Pennsylvania, the true
prevalence rate of each state may be higher, as it was assumed only one tick was positive
for Powassan virus in a pool and not multiple ticks in the pool being positive.
50
The prevalence of Powassan virus (DTV) in Warren and Sussex County, New
Jersey appear to be stable, as each year the amount of positive pools either increased by
one or decreased by one. It is unclear what the true prevalence of Powassan virus (DTV)
is in Hunterdon County, as only one year had tick pool samples analyzed and zero were
positive. The stability of Powassan virus (DTV) in Pennsylvania could not be determined
by this study for reported counties as each county was only analyzed for one year.
Although it was not found to be statistically significant Pennsylvania did have a higher
prevalence rate than New Jersey in 2018 with 2.2% and 4.0% in Pennsylvania. These
results could possibly be biased due to the sample size difference between the two states
in 2018, with 102 more sample pools analyzed in Pennsylvania than New Jersey. Other
biases may be seen in the results from Pennsylvania 2017 due to a small sample size of
only 26 tick pools. An uneven number of ticks were collected from counties in
Pennsylvania, creating an uneven distribution of tick pools per county. This could create
biased results for counties with small tick pools such as Potter (5 ticks pools) and Centre
(4 tick pools) compared to Clinton which had 203 tick pools. Although several of the
counties had small sample sizes, Powassan positive tick pools came from eight of the
twelve counties analyzed. This indicates a risk in several counties in New Jersey and
Pennsylvania for humans to come into contact with Powassan infected ticks.
All positive pools for Powassan in New Jersey came from ticks collected in
October, while the majority of positive ticks in Pennsylvania were collected in May and
June. One possible explanation for this difference is that the population of the whitefooted mouse, the primary reservoir of Powassan virus (DTV), may be different in New
Jersey and Pennsylvania. If there are more white-footed mice in one area than another
51
ticks have a greater opportunity to feed on these mice as their first or second meal. This
could cause some ticks to be infected earlier in the year and others to be infected later in
the year. The majority of ticks analyzed were adults and 43 of the 45 positive tick pools
were composed of adults. In New Jersey, adult I. scapularis ticks are most active in the
Fall and as noted the majority of Powassan positive ticks were collected in October. The
Fall months, notably October, in New Jersey are potential high-risk months for Powassan
virus infection from I. scapularis. In Pennsylvania, ticks had the highest rates of
Powassan infection in May and June. Although adults are not typically the most active in
the Spring and Summer, they can be found questing for a host and will attach to a host if
they find one. Nymphs are most active during this season and with two positive nymph
pools, there is a risk that Powassan infection is present in these months in Pennsylvania.
A concern with nymph ticks being positive for infection is the fact they are very small,
and the majority of humans will not know to check or may miss them during a tick check,
giving these ticks ample time to feed and transmit tick-borne diseases. As Powassan virus
can transmit within 15 minutes adults and nymphs are both likely to be able to transmit
the virus even if the tick is found within a short amount of time of attaching and feeding
before being removed.
There was no significant effect of gender, life stage, or engorgement on Powassan
infection of I. scapularis. This indicates there is no difference in the chance of
contracting Powassan virus from a I. scapularis depending on its sex, life stage or
engorgement level. There is also no significant difference in Powassan infection between
2015-18 in New Jersey or 2017-18 in Pennsylvania. Lastly, there was no significant
difference between the overall positive pools in New Jersey and Pennsylvania.
52
Powassan virus (DTV) cases have been increasing over the last decade with 6 human
cases reported in the US in 2015, 21 reported in 2016, and 33 reported in 2018.
Prevalence research has been sparse for Powassan virus over the last decade, although it
is on the rise. Most cases of Powassan virus have occurred in the Northeast and Great
Lakes regions of the U.S., which are in Lyme endemic regions 63. It is important for states
with confirmed Powassan virus (DTV) cases to monitor the tick population to better
understand the prevalence rate in high-risk areas for infection.
It is unclear how Powassan virus interacts with other tick-borne diseases, such as
Lyme disease, or why some individuals are asymptomatic to the virus and others develop
severe symptoms. This study did find I. scapularis adults and nymphs capable of being
co-infected with Powassan virus and B. burgdorferi. Further research needs to be
conducted on Powassan virus to better understand prevalence, transmission effects in
presence of other tick-borne diseases and its ability to cause disease in those infected.
Borrelia burgdorferi Prevalence Data
Previous studies have been conducted on the prevalence of B. burgdorferi in I.
scapularis ticks in Lyme disease endemic regions of the U.S.. Courtney et al. (2003)
conducted a prevalence study in Northwestern and Southeastern Pennsylvania and
determined a prevalence of 61.6% and 13.1%, respectively. A study conducted by Steiner
et al. (2008) collected adult I. scapularis from Indiana, Maine, Pennsylvania and
Wisconsin and found prevalence rates that ranged between to 35% and 70%. In central
New Jersey, Schulze et al. (2005) analyzed adult I. scapularis and determined an
infection prevalence for B. burgdorferi to be 50.3%. Prusinski et al. (2014) collected I.
53
scapularis adults and nymphs from eight New York Counties from 2003 to 2006 and
determined an overall prevalence rate of B. burgdorferi 14.4% in adults and 45.7% in
nymphs.
The current study determined a MIR prevalence range of 17.7-22.6% in New
Jersey 2015-18 for B. burgdorferi. The MIR prevalence in Pennsylvania in 2017 and
2018 was 28.5% and 18.9%. These findings are comparable to other previous studies
conducted for B. burgdorferi prevalence in Lyme endemic regions. In a thesis study
conducted by Bird (2014) found a MIR of 0% of I. scapularis larval pools positive and
41% adult I. scapularis pools positive for B. burgdorferi7. The 9.6% difference between
2017 and 2018 could be caused by the large sample size difference between 2017 (26 tick
pools) and 2018 (203 tick pools). Additionally, different regions of Pennsylvania may
have different prevalence rates of Lyme disease, as seen with 2017 ticks collected from
the Northeast region of Pennsylvania and 2018 ticks collected from the Central region of
Pennsylvania.
Overall, out of 245 positive pools 98.3% were adults and 1.63% were nymphs.
Similar to Powassan, the majority of positive B. burgdorferi tick pools in New Jersey
were ticks collected in October. These may be due to the increased number of adult I.
scapularis out questing during this time of the year. Female adult I. scapularis accounted
for 71.8% of positive ticks pool, males made up 26.5% of the positive pools and nymphs
made up 1.63% positive pools. The Fall is a high-risk season for Lyme disease with large
populations adults questing, especially females in New Jersey. Pennsylvania had the
majority of B. burgdorferi positive tick pools from ticks collected in May (23 positive
tick pools), June (22 positive tick pools) and November (17 positive tick pools). All other
54
positive tick pools were from ticks collected in March, July, August, and December. Risk
for Lyme disease in Pennsylvania is high throughout the Spring, Summer and Fall due to
the high percentage of nymphs questing in the Spring and Summer and the adults
questing in the Fall. Human contact with ticks increases in the Spring, Summer, and Fall
as the warmer weather increases outdoor activities such as hiking and camping increase.
There was no significant difference between life stage, or engorgement and B.
burgdorferi positive ticks. There is no difference in chance of contracting B. burgdorferi
from a I. scapularis depending on its life stage or engorgement level. There was no
significant difference between 2015-18 positive tick pools in New Jersey or 2017-18 in
Pennsylvania. There was no significant difference between the amount of overall positive
tick pools between New Jersey and Pennsylvania.
As nymph ticks play a crucial role in causing Lyme disease infections in humans,
this study is not consistent with other studies with regards to prevalence rates in nymph I.
scapularis ticks. This can be due to the small sample size of nymphs. Also, the MIR was
calculated for prevalence and these numbers may be underestimating the true percentage
of positives ticks, as it was assuming only one out of the number of ticks in the pool was
positive for adult and nymph pools analyzed.
The CDC reported 12,801 confirmed cases of Lyme disease in 1997. Just20 years
later, the CDC reported 29,513 confirmed cases and 13,230 probable cases. The rise of
Lyme disease in the Northeastern and Midwest United States is a cause for alarm and a
need for continuous prevalence and surveillance studies to monitor high risk.
55
Co-infection Prevalence Data
I. scapularis is the vector to many different pathogens and is able to harbor more
than one pathogen at once. Many of these pathogens such as B. burgdorferi and
Powassan virus (DTV) cause infection in humans. Studies that have examined the ability
of I. scapularis to have co-infections and found them to be able to harbor up to four or
more pathogens65. Studies have focused on the infection rate and co-infection rate of B.
burgdorferi with the other tick-borne pathogens that include Anaplasma
phagocytophilum, Babesia microti, Powassan virus (DTV), and Borrelia miyamotoi65.
Many of these pathogens share the reservoir host of small mammals, specifically the
white-footed mouse (Peromyscus leucopus)16. Larval and nymphs can become infected
when they feed off of the reservoir host during their first or second meal. The larval and
nymph ticks will continue to carry these pathogens between molting to the next life stage
and can transmit the pathogen(s) to its next host during feeding.
Tokarz et al. (2010) conducted a study in New York where 70% of I. scapularis
had one pathogen and 30% had a polymicrobial infection. Specifically, they found that
24% were co-infected with B. burgdorferi/A. phagocytophilum, 31% were co-infected
with B. burgdorferi/Babesia microti, and five ticks were Powassan virus (DTV) positive,
with two being co-infected with Powassan (DTV)/B. burgdorferi (40%). Knox et al.
(2017) analyzed adult I. scapularis in Wisconsin and found that eight were positive for
Powassan virus (DTV) and four of the eight were co-infected Powassan (DTV)/B.
burgdorferi (50%).
This current study had similar co-infection rates to Knox et al. (2017) and Tokarz
et al. (2010) in adult I. scapularis, with 53.4% Powassan virus (DTV) positive adults
56
being co-infected with B. burgdorferi. Frost et al. (2015) tested patients with one known
tick-borne disease for Powassan and 17.1% of patients showed serological evidence that
they were infected with both B. burgdorferi and Powassan virus. Three patients (7.3%)
were laboratory confirmed (PCR) to have a Powassan and B. burgdorferi co-infection.
Co-infections increase the difficulty for accurately diagnosing and treating tickborne diseases43. Pathogens can range from bacterial to protozoan to viral and need
different treatment methods. Symptoms of one tick-borne disease can be masked by
another when a co-infection occurs. This allows one pathogen to be treated but the other
to continue causing symptoms and illness. Some studies, such as those conducted by
Thomm et al (2018), have suggested Post Treatment Lyme Disease Syndrome (PTLDS)
may be caused by an undiagnosed co-infection. Thomm et al (2018) has worked to
develop and validate a serological test panel for the detection of Powassan virus and has
suggested those who have been treated for Lyme disease but have persistent symptoms
consistent with PTLDS should be tested for Powassan virus due to a possible coinfection. With co-infections as high as 28% in I. scapularis ticks in Lyme disease
endemic regions in the U.S., surveillance studies are important to monitor co-infection
rates in these areas.
Conclusion
The overall increase in tick populations and distribution can have severe impacts
on human infection and tick-borne diseases. Ticks established in new areas increase the
risk of tick-borne disease brought into areas that they were not present in before. The
increased distribution of I. scapularis may be a possible cause of the increase in Lyme
57
disease infections in the U.S. and may lead to an increase of other tick-borne diseases
such as Powassan virus (DTV). Lone star ticks are known to carry the tick-borne
pathogens Ehrlichia chaffeensis, E. ewingii, Borrelia lonestari (STARI), and tularemia.
Human and pet populations in New Jersey and Pennsylvania can be at risk of being bitten
by Lone star ticks and becoming infected with these diseases, as they are now known to
inhabit these areas.
Tick surveillance studies such as this one help to determine the prevalence rate
and high-risk areas for tick-borne diseases. Pennsylvania and New Jersey are the top two
states for Lyme disease cases in the US 4. This is the first study to determine the MIR of
Powassan virus in New Jersey and Pennsylvania and found the MIR to be similar to rates
determined in previous studies. This indicates that these states are at risk for human tickborne infections and should be continued to be monitored for human, pet and wildlife
health. Tick surveillance is important to continue to monitor populations of new and
established ticks and diseases for the safety and health of those living in tick populated
areas.
Future Study
As this was the first study in New Jersey or Pennsylvania to survey the prevalence
of Powassan virus (DTV) and co-infection with Lyme disease (B. burgdorferi), there is
ample opportunity to continue monitoring the prevalence rates in these states. With
Powassan virus, as an emerging tick-borne virus capable of causing deadly disease in
humans, it is important to monitor the prevalence rate of it to determine if risk to humans
increases. This allows for physicians to be aware should a patient exhibit symptoms of it.
58
Additionally, only three of the twenty-one counties in New Jersey had ticks analyzed for
Powassan virus (DTV), leaving a need for other counties in New Jersey to be analyzed in
future work. Pennsylvania had nine out of sixty-seven counites analyzed with uneven tick
samples from each county, future studies can conduct more thorough analysis of counties.
Blacks bears in this study had several different ticks’ species and life stages
attached to them. Future studies can conduct a more thorough search on black bears to
better determine what the tick community composition consists of on black bears in New
Jersey and Pennsylvania. Another study can be done using serological test (ELISA, IFA
or PRNT50 or 90) with blood to determine if black bears have the capability to be reservoirs
of Powassan virus, Lineage I or Lineage II, as they were found to be host to I. scapularis
and I. cookei.
There has not been a study conducted to determine the prevalence rate of infected
white-footed mice with Powassan virus (DTV) in Pennsylvania or New Jersey. As the
reservoir host to many tick-borne pathogens, observing the prevalence of these pathogens
in these mice can help to determine the prevalence rate of DTV and the chances of ticks
obtaining the infection when feeding on wild mice.
59
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Appendix A: Tick Collection Raw Data
Tick Collection New Jersey 2015
County
Ixodes scapularis
Adult
Hunterdon
25
Morris
Passaic
Sussex
Warren
Total
6
0
23
51
105
Tick Collection New Jersey 2016
Ixodes scapularis
Dermacentor variabilis
County
Hunterdon
Morris
Passaic
Sussex
Warren
Total
County
Morris
Passaic
Sussex
Warren
Total
Adult
0
28
10
309
54
401
Adult
0
0
14
48
145
207
Tick Collection New Jersey 2017
Ixodes scapularis
Dermacentor
variabilis
Adult
Nymph
Larval
Adult
54
0
0
0
20
0
0
0
262
0
0
1
93
5
1
72
429
5
1
73
67
Ixodes
cookie
Adult
0
0
0
1
1
County
Morris
Passaic
Sussex
Warren
Total
Tick Collection New Jersey 2018
Ixodes scapularis
Dermacentor Ixodes Amblyomma
variabilis
cookei americanum
Adult Nymph Larval
Adult
Adult
Adult
19
0
0
1
0
0
12
1
0
0
0
0
199
6
0
3
0
0
149
9
1
121
1
1
379
16
1
125
1
1
Tick Collection Pennsylvania 2017
County
Ixodes scapularis
Adult
Lackawanna
1
Monroe
35
Pike
3
Total
39
County
Centre
Clearfield
Clinton
Huntingdon
Lycoming
Potter
Tioga
Total
Tick Collection Pennsylvania 2018
Ixodes scapularis
Dermacentor
variabilis
Adult
Nymph
Larval
Adult
1
9
0
0
19
3
1
0
276
28
22
1
23
0
0
0
102
9
0
13
3
2
0
0
23
0
0
0
447
51
23
14
68
Amblyomma
americanum
Adult
0
0
1
0
0
0
0
1
Appendix B: Powassan and Lyme Raw Data
ID
Bear ID
Sex
Pooled
Ticks
Engorgement
POW
Lyme
Year
County
State
1
86
F
1
Semi
-
-
2017
Sussex
NJ
2
86
M
1
Un
-
-
2017
Sussex
NJ
3
86
F
1
Un
-
-
2017
Sussex
NJ
4
92
F
1
Un
-
-
2017
Sussex
NJ
5
111
F
1
Un
-
+
2017
Sussex
NJ
6
111
M
1
Un
-
-
2017
Sussex
NJ
7
291
F
1
Un
-
-
2017
Sussex
NJ
8
453
F
1
Semi
-
-
2017
Sussex
NJ
9
453
F
1
Semi
-
-
2017
Sussex
NJ
10
453
F
1
Fully
-
-
2017
Sussex
NJ
11
10
F
1
Semi
-
-
2017
Sussex
NJ
12
10
F
5
Semi
-
+
2017
Sussex
NJ
13
32
F
1
Semi
-
+
2017
Sussex
NJ
14
32
F
5
Semi
-
-
2017
Sussex
NJ
15
605
F
5
Semi
-
+
2017
Sussex
NJ
16
318
F
1
Fully
-
-
2017
Sussex
NJ
17
318
F
1
Semi
-
-
2017
Sussex
NJ
18
318
F
1
Un
-
+
2017
Sussex
NJ
19
106
M
1
Un
-
-
2017
Sussex
NJ
20
106
M
1
Un
-
-
2017
Sussex
NJ
21
103
F
1
Semi
-
+
2017
Sussex
NJ
22
69
F
1
Un
-
+
2017
Warren
NJ
23
69
F
1
Un
-
+
2017
Warren
NJ
24
69
M
1
Un
-
-
2017
Warren
NJ
25
41
F
1
Semi
-
+
2017
Warren
NJ
26
41
F
1
Semi
-
-
2017
Warren
NJ
27
41
F
1
Semi
-
+
2017
Warren
NJ
28
41
F
1
Semi
-
+
2017
Warren
NJ
29
41
M
1
Un
-
-
2017
Warren
NJ
30
296
F
5
Semi
-
-
2017
Warren
NJ
31
9273
F
1
Semi
-
-
2018
Warren
NJ
32
9273
F
1
Un
-
+
2018
Warren
NJ
33
9273
M
1
Un
-
-
2018
Warren
NJ
34
10202
F
1
Semi
-
+
2018
Warren
NJ
69
35
10202
F
1
Semi
-
+
2018
Warren
NJ
36
10202
F
1
Un
-
-
2018
Warren
NJ
37
10202
F
1
Un
-
-
2018
Warren
NJ
38
10202
M
2
Un
-
+
2018
Warren
NJ
39
9473
F
4
Semi
-
+
2018
Warren
NJ
40
9473
F
1
Fully
-
-
2018
Warren
NJ
41
9473
M
1
Un
-
-
2018
Warren
NJ
42
9194
F
1
Fully
-
-
2018
Sussex
NJ
43
9194
N
1
Fully
-
-
2018
Sussex
NJ
44
10041
M
1
Un
-
-
2018
Sussex
NJ
45
10041
N
1
Semi
-
-
2018
Sussex
NJ
46
10041
F
1
Semi
-
-
2018
Sussex
NJ
47
6573
M
1
Un
-
-
2018
Warren
NJ
48
6573
F
5
Un
-
+
2018
Warren
NJ
49
10003
M
1
Un
-
+
2018
Sussex
NJ
50
10003
F
1
Semi
-
-
2018
Sussex
NJ
51
10003
F
1
Semi
-
-
2018
Sussex
NJ
52
10003
F
1
Semi
-
-
2018
Sussex
NJ
53
10004
F
1
Semi
-
-
2018
Sussex
NJ
54
9149
N
1
Un
-
-
2018
Sussex
NJ
55
9887
F
1
Semi
-
-
2018
Sussex
NJ
56
9198
M
1
Un
-
+
2018
Sussex
NJ
57
9198
F
1
Semi
-
-
2018
Sussex
NJ
58
9848
F
1
Fully
-
-
2018
Sussex
NJ
59
9848
M
1
Un
-
-
2018
Sussex
NJ
60
10204
F
1
Un
-
+
2018
Warren
NJ
61
10204
F
1
Semi
-
+
2018
Warren
NJ
62
10204
F
1
Semi
-
+
2018
Warren
NJ
63
9776
F
1
Fully
-
-
2018
Sussex
NJ
64
9848
M
4
Un
-
+
2018
Sussex
NJ
66
19
F
1
Un
-
-
2018
Sussex
NJ
67
19
F
1
Un
-
-
2018
Sussex
NJ
68
7900
M
5
Un
-
-
2018
Warren
NJ
69
7900
F
3
Un
-
-
2018
Warren
NJ
70
7900
F
1
Semi
-
-
2018
Warren
NJ
71
7900
N
1
Fully
-
-
2018
Warren
NJ
72
9848
N
1
Fully
-
-
2018
Warren
NJ
73
9848
F
1
Un
-
-
2018
Warren
NJ
70
74
9848
F
3
Semi
-
-
2018
Warren
NJ
75
9848
F
4
Semi
-
-
2018
Warren
NJ
76
8814
F
1
Fully
-
-
2018
Warren
NJ
77
8844
M
5
Un
-
+
2018
Warren
NJ
78
8844
F
4
Semi
-
-
2018
Warren
NJ
79
8844
F
4
Semi
-
-
2018
Warren
NJ
80
8844
F
2
Fully
-
-
2018
Warren
NJ
81
8844
N
3
Fully
-
-
2018
Warren
NJ
82
295
M
3
Un
-
+
2017
Warren
NJ
83
632
F
1
Fully
-
-
2015
Warren
NJ
84
632
F
1
Fully
-
+
2015
Warren
NJ
85
631
F
1
Semi
-
-
2015
Warren
NJ
86
631
F
1
Semi
-
-
2015
Warren
NJ
87
631
F
1
Fully
-
+
2015
Warren
NJ
88
631
M
1
Un
-
-
2015
Warren
NJ
89
633
F
3
Semi
-
-
2015
Hunterdon
NJ
90
633
F
4
Semi
-
+
2015
Hunterdon
NJ
91
633
F
1
Fully
-
-
2015
Hunterdon
NJ
92
633
M
1
Un
-
-
2015
Hunterdon
NJ
93
630
F
1
Semi
-
+
2015
Warren
NJ
94
616
F
1
Semi
-
-
2015
Hunterdon
NJ
95
616
F
1
Semi
-
-
2015
Hunterdon
NJ
96
628
F
1
Semi
-
-
2015
Warren
NJ
97
628
F
1
Semi
-
-
2015
Warren
NJ
98
628
F
1
Fully
-
-
2015
Warren
NJ
99
628
F
1
Un
-
-
2015
Warren
NJ
100
628
M
1
Un
-
-
2015
Warren
NJ
101
627
M
1
Un
-
-
2015
Warren
NJ
102
627
F
1
Semi
-
-
2015
Warren
NJ
103
627
F
1
Semi
-
-
2015
Warren
NJ
104
625
F
1
Fully
-
-
2015
Warren
NJ
105
625
F
5
Semi
-
+
2015
Warren
NJ
106
625
M
3
Un
-
+
2015
Warren
NJ
107
624
F
1
Semi
-
-
2015
Warren
NJ
108
624
F
1
Un
-
-
2015
Warren
NJ
109
623
F
1
Semi
-
-
2015
Warren
NJ
110
623
F
1
Semi
-
-
2015
Warren
NJ
111
623
F
1
Un
-
-
2015
Warren
NJ
71
112
622
F
4
Fully
-
-
2015
Warren
NJ
113
622
F
3
Semi
-
-
2015
Warren
NJ
114
622
M
1
Un
-
-
2015
Warren
NJ
115
619
F
2
Semi
-
+
2015
Hunterdon
NJ
116
619
F
3
Fully
-
-
2015
Hunterdon
NJ
117
619
M
3
Un
-
-
2015
Hunterdon
NJ
118
618
F
1
Semi
-
+
2015
Hunterdon
NJ
119
618
F
1
Fully
-
+
2015
Hunterdon
NJ
120
618
M
1
Un
-
-
2015
Hunterdon
NJ
121
617
F
1
Fully
-
-
2015
Hunterdon
NJ
122
617
F
1
Fully
-
-
2015
Hunterdon
NJ
123
617
F
1
Fully
-
-
2015
Hunterdon
NJ
124
617
M
1
Un
-
-
2015
Hunterdon
NJ
125
21
F
1
Fully
-
-
2015
Warren
NJ
126
20
F
1
Fully
-
-
2015
Warren
NJ
127
15
F
1
Semi
-
+
2015
Warren
NJ
128
15
F
1
Semi
-
-
2015
Warren
NJ
129
41
F
1
Fully
-
-
2015
Sussex
NJ
130
39
F
1
Semi
-
-
2015
Sussex
NJ
131
39
F
1
Semi
-
-
2015
Sussex
NJ
132
35
F
1
Fully
-
-
2015
Sussex
NJ
133
34
F
1
Fully
-
+
2015
Sussex
NJ
134
34
M
1
Un
-
-
2015
Sussex
NJ
135
31
F
1
Fully
-
-
2015
Sussex
NJ
136
31
M
1
Un
-
-
2015
Sussex
NJ
137
30
F
1
Fully
-
+
2015
Sussex
NJ
138
30
M
1
Un
-
-
2015
Sussex
NJ
139
28
F
3
Fully
-
+
2015
Sussex
NJ
140
28
F
1
Semi
-
-
2015
Sussex
NJ
141
28
M
1
Un
-
+
2015
Sussex
NJ
142
22
F
1
Un
-
+
2015
Sussex
NJ
143
22
F
1
Fully
-
-
2015
Sussex
NJ
144
18
F
1
Semi
-
-
2015
Sussex
NJ
145
18
M
1
Un
-
-
2015
Sussex
NJ
146
13
F
1
Semi
-
-
2015
Sussex
NJ
147
13
M
1
Un
-
-
2015
Sussex
NJ
148
12
F
1
Fully
-
+
2015
Sussex
NJ
149
11
F
1
Semi
-
-
2015
Sussex
NJ
72
150
2
M
3
Un
-
-
2016
Sussex
NJ
151
2
F
4
Semi
-
+
2016
Sussex
NJ
152
2
F
4
Semi
-
-
2016
Sussex
NJ
153
2
F
1
Fully
-
+
2016
Sussex
NJ
154
3
M
4
Un
-
-
2016
Sussex
NJ
155
3
F
1
Un
-
-
2016
Sussex
NJ
156
3
F
5
Semi
-
-
2016
Sussex
NJ
157
3
F
4
Semi
-
+
2016
Sussex
NJ
158
3
F
2
Fully
-
+
2016
Sussex
NJ
159
4
F
1
Semi
-
-
2016
Sussex
NJ
160
5
M
1
Un
-
-
2016
Sussex
NJ
161
5
M
1
Un
-
-
2016
Sussex
NJ
162
5
F
1
Semi
-
+
2016
Sussex
NJ
163
5
F
1
Semi
-
-
2016
Sussex
NJ
164
6
M
1
Un
-
-
2016
Sussex
NJ
165
6
F
1
Semi
-
-
2016
Sussex
NJ
166
6
F
1
Semi
-
-
2016
Sussex
NJ
167
6
F
1
Semi
-
+
2016
Sussex
NJ
168
8
F
1
Semi
+
+
2016
Sussex
NJ
169
8
F
1
Semi
-
+
2016
Sussex
NJ
170
8
F
1
Semi
-
-
2016
Sussex
NJ
171
8
F
1
Semi
-
-
2016
Sussex
NJ
172
9
M
1
Un
-
-
2016
Sussex
NJ
173
9
F
1
Semi
-
-
2016
Sussex
NJ
174
10
M
4
Un
-
+
2016
Sussex
NJ
175
10
F
2
Un
-
+
2016
Sussex
NJ
176
10
F
4
Semi
-
+
2016
Sussex
NJ
177
10
F
4
Semi
-
+
2016
Sussex
NJ
178
11
M
5
Un
-
-
2016
Sussex
NJ
179
11
F
5
Un
-
+
2016
Sussex
NJ
180
11
F
5
Semi
-
-
2016
Sussex
NJ
181
11
F
1
Fully
-
-
2016
Sussex
NJ
182
13
M
3
Un
-
+
2016
Sussex
NJ
183
13
F
5
Semi
-
+
2016
Sussex
NJ
184
13
F
5
Semi
-
-
2016
Sussex
NJ
185
13
F
3
Fully
-
-
2016
Sussex
NJ
186
14
M
2
Un
-
-
2016
Sussex
NJ
187
14
F
3
Semi
-
+
2016
Sussex
NJ
73
188
15
M
1
Un
+
-
2016
Sussex
NJ
189
15
F
3
Un
-
+
2016
Sussex
NJ
190
15
F
2
Semi
-
+
2016
Sussex
NJ
191
17
M
3
Un
-
+
2016
Sussex
NJ
192
17
F
5
Semi
-
-
2016
Sussex
NJ
193
20
M
7
Un
-
+
2016
Sussex
NJ
194
20
F
1
Un
-
-
2016
Sussex
NJ
195
20
F
5
Semi
-
+
2016
Sussex
NJ
196
20
F
5
Semi
-
+
2016
Sussex
NJ
197
20
F
5
Semi
-
+
2016
Sussex
NJ
198
22
M
2
Un
-
-
2016
Sussex
NJ
199
22
F
4
Semi
-
-
2016
Sussex
NJ
200
16
M
2
Un
-
-
2016
Warren
NJ
201
16
F
2
Un
+
+
2016
Warren
NJ
202
16
F
5
Semi
-
+
2016
Warren
NJ
203
30
M
3
Un
-
-
2016
Warren
NJ
204
30
F
3
Un
-
+
2016
Warren
NJ
205
30
F
4
Semi
-
+
2016
Warren
NJ
206
154
F
1
Fully
-
+
2016
Warren
NJ
207
162
M
2
Un
-
+
2016
Warren
NJ
208
162
F
4
Semi
-
+
2016
Warren
NJ
209
167
M
2
Un
-
-
2016
Warren
NJ
210
167
F
1
Un
-
-
2016
Warren
NJ
211
167
F
3
Semi
-
+
2016
Warren
NJ
212
169
M
1
Un
+
-
2016
Warren
NJ
213
169
F
3
Semi
-
-
2016
Warren
NJ
214
169
F
2
Fully
-
-
2016
Warren
NJ
215
8819
M
1
Un
-
-
2016
Warren
NJ
216
8819
F
1
Semi
-
-
2016
Warren
NJ
217
8819
F
1
Fully
-
-
2016
Warren
NJ
218
8819
F
1
Fully
-
-
2016
Warren
NJ
219
9209
F
1
Semi
-
-
2016
Warren
NJ
220
9553
M
1
Un
-
-
2016
Warren
NJ
221
9553
M
1
Un
-
-
2016
Warren
NJ
222
9553
F
1
Fully
-
-
2016
Warren
NJ
223
9553
F
1
Fully
-
+
2016
Warren
NJ
224
28
M
3
Un
-
-
2016
Sussex
NJ
225
28
F
1
Un
-
-
2016
Sussex
NJ
74
226
28
F
5
Semi
-
+
2016
Sussex
NJ
227
29
M
3
Un
-
+
2016
Sussex
NJ
228
29
F
3
Un
-
+
2016
Sussex
NJ
229
31
M
1
Un
+
+
2016
Sussex
NJ
230
31
F
1
Un
-
+
2016
Sussex
NJ
231
31
F
4
Semi
-
+
2016
Sussex
NJ
232
144
M
3
Un
+
+
2016
Sussex
NJ
233
144
F
5
Semi
-
-
2016
Sussex
NJ
234
145
M
1
Un
-
-
2016
Sussex
NJ
235
145
M
1
Un
-
-
2016
Sussex
NJ
236
145
F
1
Un
-
-
2016
Sussex
NJ
237
145
F
1
Semi
-
+
2016
Sussex
NJ
238
145
F
1
Semi
-
-
2016
Sussex
NJ
239
147
F
1
Semi
-
-
2016
Sussex
NJ
240
147
F
1
Semi
-
+
2016
Sussex
NJ
241
148
M
1
Un
-
-
2016
Sussex
NJ
242
148
F
1
Un
-
+
2016
Sussex
NJ
243
148
F
3
Semi
-
-
2016
Sussex
NJ
244
149
M
1
Un
-
-
2016
Sussex
NJ
245
149
F
1
Un
-
-
2016
Sussex
NJ
246
149
F
1
Semi
-
-
2016
Sussex
NJ
247
249
F
1
Semi
-
+
2016
Sussex
NJ
248
379
F
1
Semi
-
+
2016
Sussex
NJ
249
379
F
1
Semi
-
+
2016
Sussex
NJ
250
91
M
4
Un
-
-
2017
Warren
NJ
251
91
F
1
Un
-
-
2017
Warren
NJ
252
91
F
5
Semi
-
+
2017
Warren
NJ
253
91
F
5
Semi
-
+
2017
Warren
NJ
254
91
F
3
Semi
-
-
2017
Warren
NJ
255
105
M
2
Un
-
-
2017
Warren
NJ
256
105
F
2
Semi
-
+
2017
Warren
NJ
257
105
F
1
Fully
-
-
2017
Warren
NJ
259
110
M
7
Un
-
+
2017
Warren
NJ
260
110
F
2
Un
+
+
2017
Warren
NJ
261
110
F
3
Semi
-
+
2017
Warren
NJ
262
6
M
5
Un
-
-
2017
Warren
NJ
263
6
F
1
Un
-
-
2017
Warren
NJ
264
6
F
5
Semi
-
+
2017
Warren
NJ
75
265
6
F
5
Semi
-
+
2017
Warren
NJ
266
6
F
5
Semi
-
-
2017
Warren
NJ
267
6
F
5
Semi
-
-
2017
Warren
NJ
268
6
F
5
Semi
-
-
2017
Warren
NJ
269
6
F
5
Semi
-
-
2017
Warren
NJ
270
90
M
1
Un
-
-
2017
Sussex
NJ
271
90
F
2
Un
-
+
2017
Sussex
NJ
272
90
F
2
Semi
-
+
2017
Sussex
NJ
273
93
M
4
Un
-
+
2017
Sussex
NJ
274
93
F
1
Un
+
-
2017
Sussex
NJ
275
93
F
1
Semi
-
+
2017
Sussex
NJ
276
94
M
1
Un
-
+
2017
Sussex
NJ
277
94
F
1
Semi
-
+
2017
Sussex
NJ
278
94
F
1
Fully
-
+
2017
Sussex
NJ
279
30
M
1
Un
-
-
2017
Warren
NJ
280
30
F
1
Semi
-
-
2017
Warren
NJ
281
30
F
1
Semi
-
-
2017
Warren
NJ
282
30
F
1
Semi
-
-
2017
Warren
NJ
283
101
M
1
Un
-
-
2017
Sussex
NJ
284
101
F
1
Semi
-
-
2017
Sussex
NJ
285
101
F
1
Semi
-
-
2017
Sussex
NJ
286
104
F
1
Un
-
-
2017
Sussex
NJ
287
9
M
1
Un
-
+
2017
Sussex
NJ
288
9
F
6
Semi
-
-
2017
Sussex
NJ
289
9
F
1
Fully
-
+
2017
Sussex
NJ
290
55
F
1
Semi
-
-
2017
Sussex
NJ
291
55
F
1
Semi
-
+
2017
Sussex
NJ
292
55
F
1
Semi
-
-
2017
Sussex
NJ
293
55
F
1
Semi
-
+
2017
Sussex
NJ
294
64
M
1
Un
+
-
2017
Sussex
NJ
295
64
F
2
Un
-
-
2017
Sussex
NJ
296
64
F
2
Semi
-
-
2017
Sussex
NJ
297
67
M
2
Un
-
+
2017
Sussex
NJ
298
67
F
2
Un
-
+
2017
Sussex
NJ
299
67
F
2
Semi
-
-
2017
Sussex
NJ
300
68
M
1
Un
-
+
2017
Sussex
NJ
301
252
M
1
Un
+
-
2017
Sussex
NJ
302
252
F
1
Semi
-
-
2017
Sussex
NJ
76
303
252
F
1
Semi
-
-
2017
Sussex
NJ
304
302
M
1
Un
-
-
2017
Sussex
NJ
305
302
F
1
Un
-
-
2017
Sussex
NJ
306
302
F
1
Un
-
-
2017
Sussex
NJ
307
302
F
1
Fully
-
-
2017
Sussex
NJ
308
306
F
1
Un
+
+
2017
Sussex
NJ
309
306
F
1
Un
-
-
2017
Sussex
NJ
310
608
M
4
Un
-
-
2017
Sussex
NJ
311
608
F
1
Un
+
-
2017
Sussex
NJ
312
608
F
4
Semi
-
+
2017
Sussex
NJ
313
608
F
5
Fully
-
+
2017
Sussex
NJ
314
608
F
5
Fully
-
-
2017
Sussex
NJ
315
299
F
1
Un
-
+
2017
Sussex
NJ
316
299
F
1
Fully
-
+
2017
Sussex
NJ
317
299
F
1
Un
-
+
2017
Sussex
NJ
318
304
F
1
Un
-
-
2017
Sussex
NJ
319
304
F
1
Un
-
+
2017
Sussex
NJ
320
14
M
1
Un
+
-
2017
Monroe
PA
321
38
F
1
Semi
-
-
2017
Monroe
PA
322
37
M
1
Un
-
+
2017
Monroe
PA
323
37
M
1
Un
-
+
2017
Monroe
PA
324
37
F
1
Semi
-
-
2017
Monroe
PA
325
37
F
1
Semi
-
-
2017
Monroe
PA
326
37
F
1
Semi
-
+
2017
Monroe
PA
327
44
F
1
Un
+
+
2017
Monroe
PA
328
44
F
1
Semi
-
-
2017
Monroe
PA
329
44
F
1
Semi
-
-
2017
Monroe
PA
330
44
M
1
Un
-
-
2017
Monroe
PA
331
55
F
1
Fully
-
-
2017
Pike
PA
332
55
M
1
Un
-
-
2017
Pike
PA
333
43
F
1
Fully
-
-
2017
Pike
PA
334
1723665
M
2
Un
-
-
2017
Monroe
PA
335
1723665
F
2
Semi
-
+
2017
Monroe
PA
336
1723665
F
1
Fully
-
-
2017
Monroe
PA
337
1723667
M
1
Un
-
+
2017
Monroe
PA
338
1723667
F
4
Semi
-
+
2017
Monroe
PA
339
1723667
F
1
Fully
-
-
2017
Monroe
PA
340
1723668
M
2
Un
-
-
2017
Monroe
PA
77
341
1723668
F
2
Un
-
+
2017
Monroe
PA
342
172366
F
1
Semi
-
-
2017
Monroe
PA
343
172366
M
2
Un
-
-
2017
Monroe
PA
344
172366
F
1
Un
-
+
2017
Monroe
PA
345
172366
F
2
Semi
-
+
2017
Monroe
PA
346
51100
F
1
Un
-
-
2018
Clinton
PA
347
51100
N
1
Fully
-
-
2018
Clinton
PA
348
52030
F
1
Un
-
-
2018
Lycoming
PA
349
35998
M
5
Un
-
+
2018
Clinton
PA
350
35998
M
5
Un
-
+
2018
Clinton
PA
351
35998
F
1
Un
-
+
2018
Clinton
PA
352
35998
F
2
Semi
-
-
2018
Clinton
PA
353
35998
F
2
Fully
-
-
2018
Clinton
PA
354
27518
F
1
Un
-
-
2018
Clinton
PA
355
27518
F
1
Un
+
-
2018
Clinton
PA
356
27518
F
1
Semi
-
-
2018
Clinton
PA
357
51039
M
5
Un
-
+
2018
Clinton
PA
358
51039
M
5
Un
-
+
2018
Clinton
PA
359
51039
M
3
Un
-
+
2018
Clinton
PA
360
51039
F
5
Semi
-
+
2018
Clinton
PA
361
51039
F
5
Semi
-
+
2018
Clinton
PA
362
51039
F
5
Semi
-
+
2018
Clinton
PA
363
51039
F
1
Fully
-
+
2018
Clinton
PA
364
48076
N
1
Fully
-
-
2018
Lycoming
PA
365
48076
F
1
Semi
+
-
2018
Lycoming
PA
366
48076
F
1
Semi
-
-
2018
Lycoming
PA
367
51660
F
1
Un
-
+
2018
Potter
PA
368
51049
M
1
Un
-
-
2018
Clinton
PA
369
51049
F
1
Un
+
-
2018
Clinton
PA
370
51049
F
1
Semi
-
+
2018
Clinton
PA
371
52028
F
1
Un
-
+
2018
Clinton
PA
372
52028
F
4
Semi
-
+
2018
Clinton
PA
373
52028
F
1
Fully
-
+
2018
Clinton
PA
374
51032
M
5
Un
-
+
2018
Clinton
PA
375
51032
M
5
Un
+
+
2018
Clinton
PA
376
51032
M
2
Un
-
+
2018
Clinton
PA
377
51032
F
3
Un
+
+
2018
Clinton
PA
378
51032
F
3
Semi
-
+
2018
Clinton
PA
78
379
51032
F
2
Fully
-
+
2018
Clinton
PA
380
27516
M
5
Un
-
+
2018
Clinton
PA
381
27516
M
5
Un
+
+
2018
Clinton
PA
382
27516
M
1
Un
-
-
2018
Clinton
PA
383
27516
F
1
Un
-
-
2018
Clinton
PA
384
27516
F
5
Semi
-
+
2018
Clinton
PA
385
27516
F
5
Semi
-
+
2018
Clinton
PA
386
27516
F
1
Fully
-
+
2018
Clinton
PA
387
27516
F
1
Fully
-
-
2018
Clinton
PA
388
27516
F
1
Fully
-
-
2018
Clinton
PA
389
27516
N
1
Fully
-
-
2018
Clinton
PA
390
41630
M
5
Un
-
+
2018
Tioga
PA
391
41630
M
1
Un
+
+
2018
Tioga
PA
392
41630
F
5
Un
+
+
2018
Tioga
PA
393
41630
F
2
Semi
-
+
2018
Tioga
PA
394
41638
M
4
Un
-
+
2018
Tioga
PA
395
41638
F
5
Semi
-
+
2018
Tioga
PA
396
41638
F
1
Fully
-
-
2018
Tioga
PA
397
23095
F
1
Un
-
-
2018
Lycoming
PA
398
23095
N
1
Fully
-
+
2018
Lycoming
PA
399
51047
M
1
Un
-
-
2018
Clinton
PA
400
51047
F
1
Semi
+
-
2018
Clinton
PA
401
33138
M
2
Un
-
+
2018
Clinton
PA
402
33138
F
2
Un
-
-
2018
Clinton
PA
403
33138
F
1
Fully
-
-
2018
Clinton
PA
404
51942
M
1
Un
-
-
2018
Clinton
PA
405
51942
F
1
Un
-
+
2018
Clinton
PA
406
41688
N
1
Fully
-
-
2018
Potter
PA
407
33142
M
3
Un
-
+
2018
Clinton
PA
408
33142
F
3
Semi
-
-
2018
Clinton
PA
409
33142
F
1
Fully
-
-
2018
Clinton
PA
410
35414
M
2
Un
-
-
2018
Clinton
PA
411
35414
F
1
Un
-
+
2018
Clinton
PA
412
35414
F
2
Semi
-
+
2018
Clinton
PA
413
51636
F
1
Fully
-
+
2018
Clinton
PA
414
51172
F
1
Semi
-
+
2018
Clinton
PA
415
51172
F
1
Un
-
+
2018
Clinton
PA
416
51172
F
1
Un
-
-
2018
Clinton
PA
79
417
51172
F
1
Fully
-
-
2018
Clinton
PA
418
51949
M
4
Un
+
+
2018
Clinton
PA
419
51949
F
2
Un
-
+
2018
Clinton
PA
420
1
M
3
Un
+
+
2018
Warren
NJ
421
1
F
2
Semi
-
-
2018
Warren
NJ
422
2
M
1
Un
-
+
2018
Warren
NJ
423
2
F
6
Semi
-
+
2018
Warren
NJ
424
2
F
3
Un
-
+
2018
Warren
NJ
425
2
F
1
Fully
-
+
2018
Warren
NJ
426
2
N
2
Fully
-
-
2018
Warren
NJ
427
3
M
1
Un
-
-
2018
Warren
NJ
428
3
F
5
Semi
-
-
2018
Warren
NJ
429
3
F
5
Semi
-
+
2018
Warren
NJ
430
4
M
1
Un
-
-
2018
Sussex
NJ
431
4
F
2
Un
-
-
2018
Sussex
NJ
432
4
F
1
Semi
-
+
2018
Sussex
NJ
433
4
F
1
Fully
-
-
2018
Sussex
NJ
434
44
M
3
Un
-
+
2018
Sussex
NJ
435
44
F
2
Un
+
+
2018
Sussex
NJ
436
44
F
3
Semi
-
+
2018
Sussex
NJ
437
6
F
3
Un
+
+
2018
Sussex
NJ
438
6
F
5
Semi
-
-
2018
Sussex
NJ
439
7
M
5
Un
-
+
2018
Warren
NJ
440
7
F
2
Un
-
+
2018
Warren
NJ
441
7
F
2
Semi
-
+
2018
Warren
NJ
442
7
N
1
Fully
-
-
2018
Warren
NJ
443
8
M
1
Un
+
+
2018
Sussex
NJ
444
8
F
1
Un
-
+
2018
Sussex
NJ
445
8
F
3
Semi
-
+
2018
Sussex
NJ
446
8
N
1
Semi
-
-
2018
Sussex
NJ
447
9
M
1
Un
-
-
2018
Warren
NJ
448
9
F
3
Un
-
+
2018
Warren
NJ
449
9
F
6
Semi
-
+
2018
Warren
NJ
450
9
F
5
Semi
-
+
2018
Warren
NJ
451
45
M
5
Un
-
-
2018
Sussex
NJ
452
45
F
5
Un
-
+
2018
Sussex
NJ
453
45
F
2
Un
-
-
2018
Sussex
NJ
454
45
F
1
Semi
-
+
2018
Sussex
NJ
80
455
45
F
2
Fully
-
+
2018
Sussex
NJ
456
56
F
1
Fully
-
-
2018
Warren
NJ
457
15
M
5
Un
-
+
2018
Sussex
NJ
458
15
F
6
Semi
-
+
2018
Sussex
NJ
459
15
F
6
Semi
-
+
2018
Sussex
NJ
460
14
M
4
Un
-
+
2018
Sussex
NJ
461
14
F
4
Un
-
+
2018
Sussex
NJ
462
14
F
4
Semi
-
+
2018
Sussex
NJ
463
13
M
1
Un
-
+
2018
Sussex
NJ
464
13
F
4
Semi
-
+
2018
Sussex
NJ
465
13
F
4
Semi
-
+
2018
Sussex
NJ
466
13
F
2
Fully
+
+
2018
Sussex
NJ
467
44
F
1
Fully
-
+
2018
Sussex
NJ
468
56
N
1
Un
-
-
2018
Sussex
NJ
469
33168
F
1
Un
-
-
2018
Clinton
PA
470
33168
F
1
Semi
-
-
2018
Clinton
PA
471
51357
F
1
Semi
-
-
2018
Potter
PA
472
51357
F
1
Fully
-
-
2018
Potter
PA
473
51357
N
1
Semi
-
-
2018
Potter
PA
474
35676
M
3
Un
-
+
2018
Clinton
PA
475
35676
F
4
Un
-
+
2018
Clinton
PA
476
35676
F
2
Semi
-
+
2018
Clinton
PA
477
35676
F
3
Fully
-
-
2018
Clinton
PA
478
51370
N
3
Semi
-
+
2018
Clinton
PA
479
52026
N
2
Un
-
-
2018
Centre
PA
480
52026
N
5
Semi
-
-
2018
Centre
PA
481
52026
N
2
Semi
-
-
2018
Centre
PA
482
52026
F
1
Fully
-
-
2018
Centre
PA
483
35918
N
1
Un
-
-
2018
Clinton
PA
484
35918
N
1
Semi
-
-
2018
Clinton
PA
485
35918
N
1
Fully
-
-
2018
Clinton
PA
486
35918
N
1
Fully
-
-
2018
Clinton
PA
487
41479
N
2
Un
-
-
2018
Lycoming
PA
488
41479
N
5
Semi
-
-
2018
Lycoming
PA
489
41479
F
2
Semi
-
+
2018
Lycoming
PA
490
51486
M
5
Un
-
+
2018
Clinton
PA
491
51486
M
4
Un
-
+
2018
Clinton
PA
492
51486
F
1
Un
-
-
2018
Clinton
PA
81
493
51486
F
3
Semi
-
-
2018
Clinton
PA
494
51486
F
2
Fully
-
-
2018
Clinton
PA
495
51366
N
3
Un
+
+
2018
Clearfield
PA
496
36361
N
2
Un
-
-
2018
Clinton
PA
497
35017
N
5
Un
-
-
2018
Clinton
PA
498
35017
N
5
Semi
+
+
2018
Clinton
PA
499
35017
N
6
Fully
-
-
2018
Clinton
PA
500
35017
M
1
Un
+
-
2018
Clinton
PA
501
35017
F
2
Un
-
-
2018
Clinton
PA
502
51234
M
1
Un
-
-
2018
Clinton
PA
503
51234
M
5
Un
+
+
2018
Clinton
PA
504
51234
F
4
Un
-
+
2018
Clinton
PA
505
51234
F
2
Semi
-
+
2018
Clinton
PA
506
51234
F
1
Fully
-
-
2018
Clinton
PA
507
51034
M
4
Un
-
+
2018
Clinton
PA
508
51034
F
4
Un
-
+
2018
Clinton
PA
509
51034
F
5
Semi
-
-
2018
Clinton
PA
510
51034
F
5
Semi
-
-
2018
Clinton
PA
511
51034
F
2
Fully
+
-
2018
Clinton
PA
512
35017
M
5
Un
+
-
2018
Clinton
PA
513
35017
M
5
Un
-
-
2018
Clinton
PA
514
35017
M
5
Un
-
+
2018
Clinton
PA
515
35017
F
5
Un
+
+
2018
Clinton
PA
516
35017
F
2
Semi
-
-
2018
Clinton
PA
517
35017
F
4
Semi
-
-
2018
Clinton
PA
518
35017
F
1
Fully
-
-
2018
Clinton
PA
519
1805571
M
1
Un
-
+
2018
Huntingdon
PA
520
1805571
M
5
Un
-
+
2018
Huntingdon
PA
521
1805571
F
2
Semi
-
-
2018
Huntingdon
PA
522
1805571
F
5
Semi
-
+
2018
Huntingdon
PA
523
1805571
F
1
Fully
-
-
2018
Huntingdon
PA
524
1805574
F
5
Semi
-
-
2018
Huntingdon
PA
525
1805574
F
2
Fully
-
-
2018
Huntingdon
PA
526
51044
M
3
Un
+
+
2018
Clinton
PA
527
51044
M
5
Un
+
+
2018
Clinton
PA
528
51044
F
1
Un
-
-
2018
Clinton
PA
529
51044
F
1
Semi
-
-
2018
Clinton
PA
530
51044
F
3
Fully
-
+
2018
Clinton
PA
82
531
51178
N
1
Fully
-
-
2018
Clinton
PA
532
51178
F
4
Un
+
+
2018
Clinton
PA
533
51178
F
5
Un
-
+
2018
Clinton
PA
534
51178
F
5
Semi
-
-
2018
Clinton
PA
535
51178
F
1
Fully
-
-
2018
Clinton
PA
536
51036
M
4
Un
-
+
2018
Clinton
PA
537
51036
M
5
Un
-
-
2018
Clinton
PA
538
51036
F
1
Un
-
-
2018
Clinton
PA
539
51036
F
1
Semi
-
-
2018
Clinton
PA
540
51036
F
5
Semi
-
-
2018
Clinton
PA
541
51036
F
5
Semi
-
-
2018
Clinton
PA
542
51036
F
1
Fully
-
-
2018
Clinton
PA
543
1804200
M
1
Un
-
-
2018
Lycoming
PA
544
1804200
F
1
Semi
-
+
2018
Lycoming
PA
545
1804200
F
1
Semi
-
+
2018
Lycoming
PA
546
1804201
M
1
Un
-
-
2018
Lycoming
PA
547
1804201
M
1
Un
-
-
2018
Lycoming
PA
548
1804202
M
3
Un
-
+
2018
Lycoming
PA
549
1804202
F
1
Semi
-
+
2018
Lycoming
PA
550
1804202
F
1
Fully
-
-
2018
Lycoming
PA
551
1804204
F
1
Semi
+
-
2018
Lycoming
PA
552
1804203
M
5
Un
-
-
2018
Lycoming
PA
553
1804203
M
5
Un
-
-
2018
Lycoming
PA
554
1804203
M
5
Un
-
-
2018
Lycoming
PA
555
1804203
M
5
Un
-
-
2018
Lycoming
PA
556
1804203
F
5
Un
-
-
2018
Lycoming
PA
557
1804203
F
5
Un
-
+
2018
Lycoming
PA
558
1804203
F
5
Semi
-
-
2018
Lycoming
PA
559
1804203
F
5
Semi
-
-
2018
Lycoming
PA
560
1804203
M
2
Un
-
-
2018
Lycoming
PA
561
1804203
F
2
Un
-
-
2018
Lycoming
PA
562
1804207
F
1
Un
-
+
2018
Lycoming
PA
563
1804207
F
1
Semi
-
-
2018
Lycoming
PA
564
1804207
F
1
Fully
-
+
2018
Lycoming
PA
565
1804208
M
2
Un
-
+
2018
Lycoming
PA
566
1804208
M
5
Un
-
+
2018
Lycoming
PA
567
1804208
F
5
Un
-
+
2018
Lycoming
PA
568
1804208
F
5
Semi
-
+
2018
Lycoming
PA
83
569
1804208
F
4
Semi
-
+
2018
Lycoming
PA
570
1804208
F
1
Fully
-
-
2018
Lycoming
PA
571
1804209
M
2
Un
-
+
2018
Lycoming
PA
572
1804209
F
5
Un
-
+
2018
Lycoming
PA
573
1804209
F
2
Semi
-
+
2018
Lycoming
PA
574
1803379
M
1
Un
-
-
2018
Clearfield
PA
575
1803379
M
1
Un
-
-
2018
Clearfield
PA
576
1803379
F
1
Semi
-
-
2018
Clearfield
PA
577
1803380
M
1
Un
-
-
2018
Clearfield
PA
578
1803380
M
1
Un
-
-
2018
Clearfield
PA
579
1803380
M
1
Un
-
-
2018
Clearfield
PA
580
1803380
M
1
Un
-
-
2018
Clearfield
PA
581
1803378
F
1
Un
-
-
2018
Clearfield
PA
582
1803384
M
1
Un
-
-
2018
Clearfield
PA
583
1803384
F
1
Semi
-
-
2018
Clearfield
PA
584
1803388
M
5
Un
-
-
2018
Clearfield
PA
585
1803388
F
1
Semi
-
+
2018
Clearfield
PA
586
1803390
M
1
Un
+
-
2018
Clearfield
PA
587
1803390
M
1
Un
-
-
2018
Clearfield
PA
588
1803390
M
1
Un
-
-
2018
Clearfield
PA
589
1804278
M
1
Un
+
-
2018
Lycoming
PA
590
1804278
F
1
Fully
-
-
2018
Lycoming
PA
591
1804281
M
1
Un
-
+
2018
Lycoming
PA
592
1804281
F
1
Fully
-
+
2018
Lycoming
PA
593
1805549
M
1
Un
+
-
2018
Huntingdon
PA
594
1805549
F
1
Fully
-
-
2018
Huntingdon
PA
595
1810780
F
1
Semi
-
-
2018
Lycoming
PA
596
1810784
M
1
Un
+
-
2018
Lycoming
PA
597
1810787
M
1
Un
+
-
2018
Lycoming
PA
84
Appendix C: Statistical Raw Data
Powassan statistical data
Sex (male vs
female)
Adult
engorgement
Nymph
engorgement
Life stage
(adult vs
nymph)
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
-0.5594
0.836
1
-0.207
2.7060
6
-0.5783
0.920
3
-0.113
5.1210
7
-0.06677
0.976
1
-0.030
2.1948
5
0.6374
0.897
2
-0.129
3.4835
6
Borrelia burgdorferi statistical data
Sex (male vs
female)
Adult
engorgement
Nymph
engorgement
Life stage
(adult vs
nymph)
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
0.09929
0.945
1
0.069
1.4355
3
-0.4213
0.799
3
-0.254
1.6578
4
-0.6931
0.815
2
-0.234
1.6578
5
-0.4500
0.725
2
0.352
1.8119
5
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
0.06077
0.986
2
0.017
3.48817
6
-0.2834
0.906
1
-0.917
2.3999
5
-0.5254
0.852
1
-0.186
2.8238
6
Powassan State data
New Jersey
2015 / 2016 /
2017 / 2018
Pennsylvania
2017 / 2018
New Jersey
vs
Pennsylvania
85
B. burgdorferi State data
New Jersey
2015 / 2016 /
2017 / 2018
Pennsylvania
2017 / 2018
New Jersey
vs
Pennsylvania
Estimated
p- value
Degrees
of
freedom
Z value
Std. error
Fisher
Scoring
iterations
-0.70299
0.720
3
-0.358
1.96366
4
-0.08109
0.955
1
-0.056
1.44226
3
-0.02498
0.986
1
-0.017
1.43616
3
Chi-Square tick collection data for difference between tick species collected from New
Jersey vs Pennsylvania
X2
Degrees of freedom
P-value
8
6
0.2381
Powassan county data to determine for significant difference between Sussex and Warren
County
Degrees
Fisher
Estimated p-value
of
Z value Std. error Scoring
freedom
iterations
Sussex vs
0.3791
0.917
1
0.105
3.6248
6
Warren
B. burgdorferi county data to determine for significant difference between Sussex and
Warren County
Degrees
Fisher
Estimated p-value
of
Z value Std. error Scoring
freedom
iterations
Sussex vs
0.1333
0.926
1
0.092
1.4428
3
Warren
86