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Spotted Fever Group Rickettsiae in Southern Indiana Ticks
A THESIS SUBMITTED TO THE HONORS COLLEGE
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
for the degree
BACHELOR OF SCIENCE
by
KATRINA N. MESINA
tat /?a~~
Advisor
BALL STATE UNIVERSITY
MUNCIE, INDIANA
JULY, 2001
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THESIS ABSTRACT
, /'t1'17
THESIS:
Spotted Fever Group Rickettsiae in Southern Indiana Ticks
Student:
Katrina N. Mesina
Degree:
Bachelor of Science
College:
Ball State University
Date:
July 2001
Pages:
37
This study was designed to determine the presence and identity of spotted fever
group (SFG) rickettsiae in adult Dennacentor variabilis ticks from southern Indiana.
Ticks were collected from 13 sites in 8 counties and tested for presence of SFG
rickettsiae using the polymerase chain reaction (PCR) technique and agarose gel
electrophoresis.
D. variabilis ticks were collected in May 2000, separated by site and county,
pooled (3 ticks per pool), and stored. DNA extractions were performed, followed by
PCR and agarose gel electrophoresis.
Results from this study indicate a presence of SFG rickettsiae in ticks collected at
6 of 13 sites in 4 southern Indiana counties. Further research needs to be conducted to
determine the identity of these SFG rickettsiae.
ACKNOWLEDGEMENTS
Special thanks to Fresia Steiner, M.S. for assisting with this research project and
Dr. Robert Pinger for making this project possible. Thanks to Dr. Lorenza Beati for
providing positive controls. This research was supported by grants from the Indiana State
Department of Health and Ball State University.
SPOTTED FEVER GROUP RICKETTSIAE IN
SOUTHERN INDIANA TICKS
A THESIS
SUBMITTED TO THE HONORS COLLEGE
For the degree
BACHELOR OF SCIENCE
by
KATRINA N. MESINA
ADVISOR: ROBERT R. PINGER
BALL STATE UNIVERSITY
MUNCIE, INDIANA
JULY, 2001
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TABLE OF CONTENTS
LIST OF ILLUSTRATIONS
IV
Chapter
I. INTRODUCTION
1
REVIEW OF LITERATURE
4
Characteristics of Rickettsiae
Structure and Composition
Growth and Development
Epidemiology
Rocky Mountain Spotted Fever
Other Spotted Fever Group Rickettsiae
PCR Analysis
Gel Electrophoresis
4
6
8
III.
METHODS AND MATERIALS
Tick Collection
Template Preparation
Oligonucleotide Primers
PCR Amplifications
18
18
18
19
21
IV.
RESULTS
Extraction of DNA
PCR Amplification of DNA with Citrate Synthase Primers
DNA Amplification with R. rickettsii 190-kDa Antigen Primers
23
23
23
25
V.
DISCUSSION
29
II.
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REFERENCES CITED
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10
10
12
15
16
32
iii
LIST OF ILLUSTRATIONS
TABLES
1.
2.
3.
Prominent Rickettsial Species and Their Characteristics
Oligonucleotide Primers
Summary of Surveyed Indiana Counties
5
21
25
FIGURES
1.
2.
3.
4.
5.
Example of high molecular DNA in a 0.7% agarose gel
electrophoresis
Amplification of the 381 bp product of the citrate synthase gene
from tick pools with primers RpCS.877p and RpCS.1258n
County location and numbers of collected adult D. variabilis tick
pools in Indiana during May 2000
County location and numbers of adult D. variabilis tick pools
positive for the genus Rickettsia in Indiana
County location and numbers of adult D. variabilis tick pools
positive for SFG rickettsiae
IV
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23
24
26
27
28
CHAPTER I
INTRODUCTION
Rickettsiae are bacterial, obligate, intracellular parasites ranging from harmless
endosymbionts to agents of some of the most devastating diseases known. Many of these
diseases are characterized by a rash which occurs over most of the body, which is why
they are termed spotted fevers (Sonenshine 1993). Rickettsiae are primarily associated
with arthropod vectors in which they exist commensally and typically only accidentally
infect humans (Hackstadt 1996).
Rickettsiae are divided into three groups: the typhus group, the scrub typhUS
group, and the spotted fever group (La Scola & Raoult 1997). Spotted fever group (SFG)
rickettsiae include both pathogenic and nonpathogenic bacteria. In most cases, the
passage or development of tick-borne rickettsiae in a vertebrate host is not essential for
survival of the bacteria because rickettsiae are maintained through transstadial and
transovarial transmission within the arthropod vector. Most tick-borne rickettsiae
typically have no significant damaging effects on the arthropod host itself (Macaluso et
al. 2001). Exceptions exist in which the bacteria are capable of manipulating cellular
functions of the arthropod host or even result in mortality of the host (Werren 1997). For
example, Rickettsia prowazekii has a lethal effect on its arthropod vector, the human
body louse (Azad 1988), as does Rickettsia rickettsii on its tick vectors, Dennacentor
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andersoni and Dennacentor variabilis (Niebylski et al. 1999).
2
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The association between rickettsiae and arthropods is the result of an evolutionary
relationship in which highly adapted rickettsiae co-exist within their arthropod host. One
unique aspect of this relationship is the higher occurrence of nonpathogenic rickettsiae
reported in ticks, compared with the causative agent of Rocky Mountain spotted fever
(RMSF), Rickettsia rickettsii, in the United States. This relationship, or competition, of
rickettsiae within the arthropod host is of interest from an evolutionary standpoint, as
well as from a pathogen control aspect as well. Presently, the mechanisms of
interspecific competition between rickettsiae within the tick vector are unclear (Macaluso
et al. 2001).
In Indiana, D. variabilis ticks are responsible for carrying R. rickettsii as well as
other bacteria of the spotted fever group. Since 1970, 225 cases of RMSF have been
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confirmed in Indiana. Although cases have been reported from 60 different counties (out
of 92), the majority of cases have been reported from southern counties. Vanderburgh
County has reported the highest number of cases for an individual county, a total of 46
cases in the past 31 years. D. variabilis, the primary reservoir for R. ricketts ii, as well as
other SFG rickettsiae, has been recorded from all 92 counties in Indiana (RR Pinger,
unpublished data).
Previous studies conducted at the Public Health Entomology Laboratory (PHEL)
at Ball State University on dog serum found that R. montana, R. rhipicephali, and R.
rickettsii are the predominant species of SFG rickettsiae in Indiana (J.M. Stauffer,
unpublished data). Approximately 1% - 3% of D. variabilis that PHEL receives every
year are Gimenez +, but IFA negative for R. rickettsii in the hemolymph test. To
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determine which particular SFG rickettsiae were responsible for these results, a study was
3
designed to identify the rickettsiae infecting D. variabilis ticks from southern Indiana.
This study was designed to determine the presence and identity of these
pathogenic and nonpathogenic SFG rickettsiae by molecular biology-based techniques
such as the polymerase chain reaction (peR) and agarose gel electrophoresis.
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4
CHAPTER II
REVIEW OF LITERATURE
Characteristics of Rickettsiae
Rickettsiae are obligate intracellular bacteria that are transmitted to mammals by a
number of arthropod vectors including mites, lice, fleas, and ticks. Rickettsiae include
the genera Rickettsia, Ehrlichia, Orientia, and Coxiella (Hackstadt 1996).
Rickettsiae are small bacilli that have evolved in such close association with
athropod hosts that they are adapted to survive within the host cells. They represent a
rather diverse collection of bacteria, making a listing of characteristics that apply to the
entire group difficult. Some common characteristics are their epidemiology, their
obligate intracellular lifestyle, and the laboratory technology required to work with them.
Rickettsiae cannot be cultivated in the laboratory on agar plates or in broth, but only in
viable eukaryotic host cells (Walker 1998).
These arthropod-borne rickettsiae are all members of the genus Rickettsia, named
in honor of Howard T. Ricketts. Ricketts discovered these fastidious bacteria in the
tissues and eggs of ticks and also proved their transmission to humans through biting
ticks. The rickettsiae responsible for the spotted fevers multiply in tick body tissues, are
transmitted to their offspring, and are injected into vertebrates when the ticks feed. In
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terms of their ecology, the spotted fever rickettsiae are zoonotic (Sonenshine 1993).
5
Zoonotic diseases overflow or bridge from the enzootic cycle to infect and cause illness
in humans. The numerous tick-transmitted pathogens causing important infections in
humans are considered to be zoonotic because humans are typically incidental hosts for
tick vectors. The emergence of a zoonosis requires unusual circumstances such as high
vector and reservoir density, promoting overflow, or the introduction of an aggressive
bridge vector that bites humans (Telford et al. 1997).
The genus Rickettsia is divided into three groups of antigenic ally related
microorganisms: the spotted-fever group, the typhus group, and the distantly related
scrub typhus group (La Scola & Raoult 1997). Table 1 represents the prominent species
in each group, the disease commonly associated with each species, geographical
distribution, and primary arthropod vectors.
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TABLE 1
PROMINENT RICKETTSIAL SPECIES
AND THEIR CHARACTERISTICS
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Organism
Spotted fever group
Rickettsia rickettsii
Rickettsia conorii
Disease
Distribution
Vector
Rocky Mountain Spotted Fever
Mediterranean Spotted Fever
Ticks
Ticks
Rickettsia akari
Rickettsia australis
Rickettsia sibirica
Rickettsia montana
Rickettsia parkeri
Rickettsia rhipicephali
Typhus group
Rickettsia prowazekii
Rickettsia typhi
Rickettsia canada
Scrub typhus group
Rickettsia tsutsugamushi
Rickettsialpox
Queensland Tick Typhus
North Asian Tick Typhus
(Avirulent)
(Avirulent)
(A virulent)
North & South America
Southern Europe,
Mideast, Africa
Worldwide
Australia
Asia
North America
North America
North America
Epidemic Typhus
Murine Typhus
(Avirulent)
Worldwide
Worldwide
North America
Lice
Fleas
Ticks
Scrub Typhus
Southeast Asia
Mites
Ticks
Ticks
Ticks
Ticks
Ticks
Chiggers
6
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Structure and Composition
The rickettsiae comprise a group of Gram-negative, obligate intracellular,
pleomorphic rod or coccoid-shaped bacteria. The genome size is small but comparable to
that of other free-living bacteria and more than twice as large as some mycoplasma
(Sonenshine 1993). Using pulsed-field gel electrophoresis (PFGE), researchers have
shown previously that the genome size of SFG rickettsiae is approximately 1200-1300
kilobase pairs (Roux & Raoult 1993; Roux & Raoult 2000).
Like all bacteria, rickettsiae contain both RNA and DNA. The three-layered cell
wall evident in very high magnification electron micrographs is typical of gram-negative
bacteria, with an inner and outer membrane separated by a peptidoglycan layer
(Hackstadt 1996).
The cell wall proteins of rickettsiae display several interesting features. Rickettsia
rickettsii, the causative agent of Rocky Mountain spotted fever, possesses two large
immunodominant surface protein antigens (Anacker et al. 1984; Anacker et al. 1985;
Anacker et al. 1986; Williams et al. 1986; Anacker et al. 1987). These proteins have
been termed rOmpA and rOmpB for rickettsial outer membrane proteins A and B.
rOmpA from R. rickettsii had previously been known as the 155-kDa, 170-kDa, and 190kDa antigen. Similarly, rOmpB from R. rickettsii had been termed the 120-kDa, 133kDa, and 135-kDa antigen and refered to as SPA in the typhus-group rickettsia. Not only
did confusion result from the varying names in nomenclature, but the actual size of both
proteins differs between species (Anacker et al. 1987; Gilmore & Hackstadt 1991). Both
rOmpA and rOmpB show heterogeneity between and within species. Monoclonal
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7
antibodies have defined genus, group, species, and subspecies reactivities of each of these
proteins (Anacker et al. 1987).
The 190-kDa rOmpA protein of R. rickettsii contains one of the largest tandem
repeat regions known among prokaryotes both in number and in size of repeat units
(Austin & Winkler 1988). Although these repeat regions are very similar, they can be
separated into different types: type I and type II. The type II regions can also be
subclassified into type IIa and type lIb. The rOmpA protein appears to be present
primarily in the spotted fever group (Gilmore 1993).
Southern blot analysis and polymerase chain reaction (PCR) techniques have
indicated polymorphisms in the repeat regions and that they vary in size or number
between rickettsiae (Gilmore & Hackstadt 1991). Comparisons of the DNA sequences of
the repeat regions for R. rickettsii, R. conorii, and R. akari indicate that the number of
repeat units differs between the three species (Gilmore 1993).
The rOmpB protein is the most abundant surface protein on rickettsiae and has
stimulated interest because of its location, strong immunogenicity, and reactivity with
monoclonal antibodies that protect mice against lethal rickettsial challenge (Anacker et
al. 1984; Anacker et al. 1986; Williams et al. 1986; Anacker et al. 1987). Ultrastructural
analyses of the rickettsial outer membrane indicate a regularly arrayed surface structure
comprised of a paracrystalline surface array or S-layer (Palmer et al. 1974; Palmer et al.
1974; Sleytr 1983). rOmpB has been found to have a structural role in the S-layer due to
its abundance, its amino acid composition, and the release of the rOmpB homolog from
typhus-group rickettsiae by hypotonic shock (Ching et al. 1990; Hackstadt 1996).
8
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Surrounding the cell wall is a coat of slime layer. In thin sections of infected host
cells this layer appears as a translucent zone surrounding each organism (Austin &
Winkler 1988). The slime layer is the likely site for the major group antigens and serves
to attach the rickettsiae to potential host cells. The slime layer is also thought to be the
site for a phenomenon known as "reactiviation" in which rickettsiae surviving in hungry,
unfed ticks are able to revert to the virulent state. When ticks infected with R. rickettsii
are starved, the slime layer deteriorates, but is restored upon feeding. These observations
suggest that the slime layer is important in the disease process (Austin & Winkler 1988;
Sonenshine 1993).
Growth and Development
Rickettsiae may survive outside of the eUkaryotic cell for varying periods, but
they can only multiply within living cells. Typically, rickettsiae are grown in chick
embryo yolk sacs or in vitro cell cultures. Once within the host cell, they multiply by
binary fission. They grow prominently in the cytoplasm and occasionally appear within
the nucleus (Sonenshine 1993).
Rickettsiae are well adapted to intracellular life, competing efficiently with the
host cell for essential nutrients. Inhabitation of the cytoplasm of eukaryotic cells places
rickettsiae in an environment extremely rich in biosynthetic precursors (Hackstadt 1996).
Tick-borne rickettsiae can either multiply freely within the cytoplasm of host cells or
occur only as a colony within an intracytoplasmic membrane-bound inclusion (Gothe &
Kreier 1977; Kocan & Bezzuidenhout 1987).
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9
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The mode of entry of rickettsiae into the host cell is not fully understood .
Although some rickettsiae are able to invade and grow in a wide variety of cell types,
others are highly selective and parasitize only a few specific cell types. The selective
rickettsiae are believed to adhere to specific ligands on specific host cells and are
internalized by phagocytosis. Once within the cell, rickettsiae may escape or remain in
the phagosome and may reside in the cytosol (Sonenshine 1993). Other rickettsiae may
invade the nucleus or invade the rough endoplasmic reticulum. R. sibirica infecting fat
body cells of the tick vector Dennacentor reticulatus are primarily found surrounded by
host cell membranes, isolating the rickettsiae from the cell cytoplasm which is regarded
as a protection against lysis by cytoplasmic enzymes (Gosteva et al. 1991). According to
Austin and Winkler (1988), rapid multiplication of highly virulent organisms leads to cell
lysis and release of the rickettsiae in a single "burst". However, this mode of
multiplication which is common for the typhus organism, R. prowazekii, is not associated
with R. rickettsii. R. rickettsii divide only a few times before invading other cells.
Whether rickettsiae can also escape by exocytosis from living cells is uncertain
(Sonenshine 1993).
In some studies, rickettsiae were found to traverse the cell membrane many times
and did not always induce a cytopathic effect. Rickettsiae are believed to injure the cells
directly during the entry phase, probably due to phospholipase injury to the plasma
membrane (Walker 1998). In the tick vectors, rickettsiae can invade virtually all cells
and tissues following their multiplication in the midgut and penetration through the gut
wall. Intense multiplication within these tissues (salivary glands, hemocytes, etc.)
commonly takes place during feeding (Burgdorfer 1988a).
10
Epidemiology
The geographic distribution of rickettsial disease prevalence is detennined by the
ecology of the infected arthropod (Walker 1998). In all cases except epidemic typhus,
humans are accidental hosts. The prevalence of the rickettsiae in the vector population
and the interaction of the arthropod vectors with humans are indicative of the
epidemiology of rickettsial diseases (Hackstadt 1996).
SFG rickettsiae, such as R. rickettsii, R. conorii, R. australis, and R. sibirica, vary
in the severity of their associated diseases while several species of these bacteria are
nonpathogenic for humans (Hackstadt 1996). The belief that every rickettsial species
may have pathogenic potential, provided that its arthropod vector is capable of biting
humans, has stimulated interest in the identification and diagnosis of old and new
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rickettsial diseases (La Scola & Raoult 1997).
Rocky Mountain Spotted Fever
The most severe and intensively studied disease caused by SFG rickettsiae is
Rocky Mountain spotted fever (Hackstadt 1996). Also known as tick-borne typhus fever,
RMSF can have mortality rates ranging from 20-25% in untreated cases. RMSF is the
most common fatal human tick-borne disease in the United States, with a minimal
average of 351 confinned human cases (minimum casel fatality ratio =4.0%) occurring
annually and undoubtedly many more going unreported (Dalton et al. 1995). The
incidence of disease is a reflection of the geographic distribution of infected Dermacentor
variabilis ticks in the midwestern and eastern United States and D. andersoni in the
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Rocky Mountain states (Walker 1998; Burgdorfer 1975; Burgdorfer 1988; McDade &
Newhouse 1986).
RMSF is a severe, self-limiting disease characterized by fever, headache, and a
generalized rash. Following a brief incubation period of about 7 days, onset is abrupt
with a sudden sharp rise in fever (40°C or higher), severe frontal headache, nausea,
prostration, and is often accompanied by chills as well as aches in the muscles and joints
(Walker 1998). Within 5 days, nearly 90% of all affected persons will experience a rash,
which classically starts on the wrists, ankles, hands, and feet, and can spread to toward
the trunk. Particularly in dark-skinned patients, cases of spotless fever are encountered.
Other symptoms may include vomiting, abdominal pain, and occasional diarrhea and
cough. Without prompt antibiotic therapy, death can occur. Currently, death is reported
in 1% to 5% of treated patients and approximately 20% of untreated patients (NagyAgren & Blevins 2001).
R. rickettsii is the prototype of the spotted fever group and fits all of the
morphologic and physiologic criteria. Individual rickettsiae range in size from 0.2 to 0.4
urn wide by 1.0 to 1.8 urn wide. They can multiply in the cytoplasm and in the nuclei of
host cells. SFG rickettsiae grow readily in tick tissues, chick embryo tissues, mammalian
cells, and in vitro cell cultures. Rickettsial isolates from different regions of the world
vary greatly in virulence (Walker 1998).
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Other Spotted Fever Group Rickettsiae
The advent of new culture techniques, such as the shell vial-centrifugation
technique, and the detection of rickettsial DNA has dramatically increased the number of
rickettsial species detected in recent years (Zhang et al. 2000). Before 1984, only six
SFG rickettsiae were recognized in the world (Raoult & Roux 1997): Rocky Mountain
spotted fever caused by R. rickettsii, Mediterranean spotted fever caused by R. conorii,
Siberian tick typhus caused by R. sibirica, Queensland tick typhus caused by R. australis,
rickettsialpox caused by R. akari, and Israeli spotted fever caused by Israeli tick typhus
rickettsia (Zhang et al. 2000).
Nonpathogenic rickettsiae resembling R. rickettsii occur in many tick vectors
implicated in transmission of RMSF, complicating attempts to understand the ecology of
R. rickettsii in nature. Early researchers, including Ricketts, recognized the existence of
these nonpathogenic rickettsiae. R. montana, isolated from D. variabilis and D.
andersoni in eastern Montana, is a nonpathogenic rickettsia which is unable to induce
toxin-neutralizing antibodies protective against R. rickettsii and is antigenically distinct
from that species. R. rhipicephali, isolated from the brown dog tick, Rhipicephalus
sanguineus, is also nonpathogenic (Burgdorfter 1988b). These two species of rickettsia
are morphologically indistinguishable from each other (Sonenshine 1993).
A nonpathogenic rickettsia, identified only as WB-8-2, has been isolated from
lone star ticks, Amblyomma americanum (Goddard 1987). Another nonpathogenic SFG
rickettsia, R. belli, was isolated from D. variabilis collected in Arkansas but discovered to
be widely distributed throughout the United States (Sonenshine 1993).
13
R. montana, R. rhipicephaU, and R. belli can be differentiated from R. rickettsii by
microimmunofluorescence (micro-IF). The use of the micro-IF test has made it possible
to discriminate most species of rickettsiae from vertebrates and ticks. The use of nucleic
acid probes and the polymerase chain reaction (peR) facilitates the identification of these
species even further (Sonenshine 1993).
In recent years, two additional rickettsial agents have been isolated in central and
eastern Europe. While testing for the occurrence of Coxiella burnetti in D. marginatus
ticks collected in central Slovakia, two strains of a SFG rickettsia were isolated and found
to have a DNA base similar to that of R. sibirica and R. conorii. However, results
obtained from the complement fixation test confirmed that these strains had a unique
antigenic composition and did not cross-react with either R. sibirica, R. conorii, or R.
rickettsii. These strains were considered to be a new species of rickettsia, subsequently
named Rickettsia slovaca. Experiments in the laboratory produced mild scrotal reactions
and fever in guinea-pigs. Strains isolated from Germany, Hungary, and Armenia suggest
that the distribution of this agent may be widespread (Rehacek et al. 1990).
In 1978, a different rickettsial organism, Rickettsia helvetica, was isolated from
Ixodes ricinus in Switzerland. Using direct FA, serotyping tests, and protein analysis, R.
helvetica was found to be a member of the spotted fever group but distinct from all other
species tested. Although chick embryos are killed by the organism, laboratory animals
did not develop a clinical response (Sonenshine 1993).
Another SFG rickettsia, known as R. aeschlimannii, was isolated from Hyalomma
marginatum marginatum ticks collected in Morocco in 1997. In Morocco, H.
marginatum marginatum is one of the most widely distributed tick species, representing
14
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up to 42% of the tick burden of cattle (Bailly-Choumara et al 1989; Ouhelli et al. 1985).
This tick species usually bites birds in its juvenile stage, but under particular
circumstances it may also infest humans (Hoogstraal 1956). The spotted fever group
rickettsial strain MC 16T was isolated from Moroccan H. marginatum marginatum and the
name was changed to R. aeschlimannii, in honor of Andre Aeschlimann, a Swiss
zoologist and parasitologist (Beati, et al. 1997).
During recent years, new rickettsioses have been reported. These new emerging
diseases include Japanese spotted fever due to R. japonica, first reported in 1984 (Mahara
1984); Flinders Island spotted fever caused by R. honei, described in 1991 (Stewart
1991); Astrakhan fever caused by Astrakhan fever rickettsia, reported in 1991
(Tarasevich et al. 1991); African tick bite fever caused by R. africae, reported in 1992
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(Kellyet al. 1992); the pseudotyphus of California due to R.jelis, described in a patient
in 1994 (Higgins et al. 1996); and two new spotted fevers caused by "R.
mongolotimonae," reported in 1996 (Raoult et al. 1996), and the other due to R. slovaca,
reported in 1997 (Raoult et al. 1997; La Scola & Raoult 1997; Zhang et aI2000).
Infections due to several spotted fever group rickettsiae, at present classified as
nonhuman pathogens, are suspected to increase the size of this group of new emerging
diseases. Isolation of etiologic agents also allows for the recognition of rickettsial
diseases in regions where they were not previously identified, as demonstrated by the
isolation of R. akari in Croatia, an area where Mediterranean spotted fever is endemic
(Radulovic et al. 1996; La Scola & Raoult 1997). The development ofPCR
amplification-based approaches and techniques for the analysis of amplified fragments,
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15
_
especially automatic DNA sequencing, allows convenient and rapid identification of
rickettsiae, even in nonreference laboratories (La Scola & Raoult 1997).
peR Analysis
Recombinant DNA techniques were developed in the early 1970s, and in
subsequent years they revolutionized the way molecular biologists and geneticists
conduct research (Klug & Cummings 1997). In 1986, a new technique called the
polymerase chain reaction (PCR) was developed by scientists at the former Cetus
Corporation, which has greatly extended the power of recombinant DNA research. The
PCR technique is an in vitro reaction which produces enzymatic synthesis of millions of
copies of a specific DNA segment from a complex template without the use of cloning
(Erlich 1995; Klug & Cummings 1997). The reaction is based on the amplification of a
DNA segment using DNA polymerase and oligonucleotide primers that hybridize to
opposite strands of the sequence to be amplified. There are three basic steps that
constitute a PCR cycle, and the amount of amplified DNA produced is limited
theoretically only by the number of times the steps are repeated (Klug & Cummings
1997).
In the first step, the DNA to be amplified is denatured into single strands. This
DNA does not have to be purified or cloned, and it can come from a variety of sources,
including genomic DNA, forensic samples, samples stored as part of medical records,
single hairs, mummified remains, and fossils. The double-stranded DNA is denatured by
heating until it dissociates into single strands.
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16
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After denaturation of DNA, each primer hybridizes to one of the two separated
strands such that extension from each 3' -hydroxy end is directed toward each other
(Erlich 1995). These primers are synthetic oligonucleotides that anneal to sequences
flanking the segment to be amplified. Each primer has a sequence complimentary to only
one of the strands of DNA.
In the third step, a heat-resistant DNA polymerase (Taq polymerase) is added to
the reaction mixture. The annealed primers are then extended in the 5' -3' direction, using
the single-strand DNA bound to the primer as a template. The product is a doublestranded DNA molecule with the primers incorporated into the final product (Klug &
Cummings 1997).
These three steps-denaturation of the double-stranded product, annealing of
primers, and extension of polymerase-constitute a single PCR cycle. Each step of the
cycle is generally performed at a different temperature. Repeated cycles of PCR result in
the exponential accumulation of a discrete fragment. The process is automated and uses
a machine called a thermocycler, which can be programmed to carry out a predetermined
number of cycles, producing large amounts of amplified DNA segment in a few hours
(Klug & Cummings 1997).
Gel Electrophoresis
After the PCR method has been applied, products are visualized by using a gel
electrophoresis. Electrophoresis is a technique that measures the rate of migration of
charged molecules in a liquid-containing medium when an electrical field is applied to
-
the liquid. Negatively charged molecules, or anions, will migrate towards the positive
17
_
electrode (anode) and positively charged molecules, or cations, will migrate towards the
negative electrode (cathode) (Klug & Cummings 1997). The DNA migrates through the
gel in response to the field, and when this motion occurs, smaller particles travel faster
and move further than larger ones (Meyers 1995).
Electrophoresis operates using a buffer system~ a medium (paper, cellulose
acetate, starch gel, agarose gel, or polyacrylamide gel)~ and a source of direct current.
Samples are inserted, and current is passed through the system for an appropriate time.
Following migration of molecules, the gel or paper may be treated with selective stains to
reveal the location of the separated components (Klug & Cummings 1997).
Although gels can be run on a variety of mediums, acrylamide and agarose gels
are most commonly used by researchers. Both are porous gels, acting as molecular sieves
(the higher the percentage of acrylamide or agarose, the smaller the pores), and
theoretically, either could be used to separate DNA, RNA, or protein. However,
acrylamide, at low percentages, is very floppy and can be difficult to handle. It is usually
used only at high percentages to analyze proteins and small oligonucleotides. Low
percentage agarose gels are relatively rigid and easy to handle, and are used to separate
large molecules such as DNA and RNA, and very large proteins and protein complexes
(Barker 1998).
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18
CHAPfERIII
METHODS AND MATERIALS
Tick Collection
During May 2000, D. variabilis adult ticks were collected from 13 sites in 8
Indiana counties. These included Harrison, Jefferson, Lawrence, Perry, Pike, Spencer,
Warrick, and Washington counties.
Ticks were collected from vegetation by dragging a 1-m 2 white corduroy cloth
over the ground, particularly at the margins of wooded areas. In the laboratory ticks were
sorted by collection site. Adult ticks (males and females) were pooled, 3 ticks per pool.
Pools were placed in heat sealable plastic bags, labeled according to county and site, and
stored at -70 D C until DNA extractions were performed
Template Preparation
DNA was extracted from pools of ticks using the modified cetyltrimethylammonium bromide (CTAB) procedure previously described (Burket et al. 1998). Total
nucleic acids were isolated from all pools of ticks using this method. Ticks were
homogenized in 0.4 ml high salt buffer (1.4 M NaCI, 2% [wtvol] CTAB, 20mM Na
EDTA, IOOmM Tris-HCI [pH 8.0], and a 2-mercaptoethanol to 0.2% [vol:vol] [added just
-
prior to use]) in the plastic bag using a hammer (Johnson et al. 1992). The homogenate
19
-
was removed from the bag, placed into a 1.5-ml microcentrifuge tube, and incubated at
65°C for 35 minutes. For the phenol extraction, 200 III each of phenol and chloroform
were added and the solution was mixed. The phases were separated by centrifugation at
14,000 x g, and the aqueous phase was removed and stored on ice. Since much of the
DNA segregated into the organic phase under the high-salt conditions, the organic phase
was re-extracted with buffer lacking the CTAB detergent, separated by centrifugation
(14,000 x g), and the second aqueous phase was removed. After combining the aqueous
phases, an equal volume of chloroform: isoamyl alcohol (24:1, vol:vol) was added and
the mixture vortexed. The phases were separated by centrifugation (14,000 x g) and the
aqueous one was removed to a clean tube. Nucleic acid was precipitated with
isopropanol (2/3 vol), stored at -20°C for a minimum of 1 hour, and pelleted by
centrifugation (14,000 x g). Pellets were washed with 80% EtOH, centrifuged
(14,000 X g), and allowed to air dry. Finally, pellets were suspended in 50 III of TE
(Burket et al 1998).
Oligonucleotide Primers
Oligonucleotide primers used for priming PCRs are shown in Table 2 (Life
Technologies Inc., Gaithersburg, MD). Generic rickettsial oligonucleotide primers for
PCR were derived from two areas of the citrate synthase gene which have significant
homologies for both R. prowazekii and Escherichia coli analogous sequences (Ner et al.
1983; Wood et al. 1987). The exact nucleotide sequences of both primers were derived
from the nucleotide sequence of the R. prowazekii gene. Sequences conserved between
-
20
-
diverse bacterial genera are believed to also be conserved within either of the genera from
which the sequences were initially derived.
Oligonucleotide primers for PCR amplification of regions of the 190-kDa SFG
antigen gene were derived from the R. rickettsii sequence without specific prior
information regarding possible genus or species genetic conservation (Anderson et al.
1990).
Primer pairs were referred to by the initials of the genus and species from which
the nucleotide sequence was derived, combined with a simple alphanumeric identifier of
the gene amplified. This was followed by the position of the first nucleotide of the
"positive" strand of the primer and then the position of the last nucleotide of the
"negative" strand of the corresponding primer from the complementary strand of DNA.
This system allowed for primer pair designation that also displayed the position and
extent of the PCR-amplified product from the organism from which the original sequence
was obtained. For example, RpCS.877p and RpCS.1258n refer to the R. prowazekii
citrate synthase primer pair for a region between nucleotides 877 and 1258. Likewise,
Rr190.70p and Rr190.602n refer to primers derived from the R. rickettsii 190-kDa
antigen gene sequence and which prime an amplified product between nucleotides 70 and
602 of the homologous DNA sequence (Regnery et al. 1991). These primers are
associated with rickettsial outer membrane protein A (rOmpA), which is present
primarily in the spotted fever group (Gilmore 1993).
-
21
-
TABLE 2
OLIGONUCLEOTIDE PRIMERS
Nucleotide sequence
(5'-3')
Produce
size (bp)
Primer
Species
Gene
RpCS.877p
R. prowazekii
Citrate Synthase
GGGGGCCTGCTCACGGCGG
381
RpCS.1258n
R. prowazekii
Citrate Synthase
ATTGCAAAAAGTACAGTGAACA
381
Rr190.7Op
R. rickettsii
190-kDa antigen
ATGGCGAATATTTCTCCAAAA
532
Rr190.602n
R. rickettsii
190-kDa antigen
AGTGCAGCATTCGCTCCCCCT
532
peR Amplifications
PCR amplifications were performed in a Perkin Elmer 2400 thermal cycler using
a total reaction volume of 50 III in a MicroAmp reaction tube (Perkin-Elmer Cetus, Foster
City, CA). Reactions were assembled in a manner to minimize contamination and carryover of previously amplified products. Thus, reaction assembly was performed in a
laminar flow hood and disposable gloves were worn. Materials such as pipettemen and
tips were UV-irradiated prior to use. For each reaction, both a positive (R. slovaca,
kindly provided by Lorenza Beati, Yale University) and a negative control (water blank)
were included.
To screen nucleic acid isolated from tick pools and ensure equal distribution of
reagents, a master mix was prepared which, following the addition of nucleic acid,
contained IX thermophilic buffer, 2.0 mM MgCI ,dNTPs (150 uM each dNTP), 1.5 u
Taq DNA polymerase (Promega, Madison, WI), and 2 pmolllli each of primers
RpCS.877p and RpCS.1258n. These primers amplify a 381 base pair product of the
-
22
-
Rickettsia citrate synthase gene (Regnery et al. 1991). Template nucleic acid was added
to aliquots of the master mix.
Polymerase chain reactions were subjected to 35 cycles of denaturing at 95°C for
15 s, primer annealing at 50°C for 20 s, and primer extension at 72°C for 45 s, and a final
extension at 72°C for 5 min. Aliquots of the amplification products (10 j.ll each of each
50 ul reaction) were analyzed by 1.5% agarose gel electrophoresis and the image was
captured on a Gel Print 2000 Imaging System (Biophotonics, Ann Arbor, MI). Pools
containing an amplified band of 381 bp were scored as positive.
A second PCR was conducted on those pools that tested positive for the Rickettsia
citrate synthase gene sequence. Conditions were the same as described above, with the
exception of primers. Primers used in this PCR were Rr190.70p and Rr190.602n, which
-
amplify a 532 bp product of the 190-kDa SFG antigen gene. Results were visualized in
2% agarose gel electrophoresis and pools containing an amplified band of 532 bp were
scored as positive.
-
23
.-
CHAPTER IV
RESULTS
Extraction of DNA
Figure 1 shows the extraction of high molecular weight DNA isolated by the
CTAB method, as visualized in a 0.7% agarose gel electrophoresis.
-
1
2
3
4
5
6
M
-23,130bp
Fig. 1. Example of high molecular DNA in a 0.7% agarose gel electrophoresis. Lanes
1-6, tick and bacterial DNA extracted from D. variabilis; lane M, molecular marker. If the
bacteria would have been infecting the ticks, its DNA would have moved with the tick DNA.
.-
24
.-
PCR Amplification of DNA with Citrate Synthase Primers
PCR was performed on 343 pools of adult D. variabilis ticks using the primers
RpCS.877p and RpCS.1258n for the citrate synthase gene. Successful amplification with
these primers generated a 381 base pair product.
DNA extracted from 28 out of 343 pools of ticks was clearly positive for the
citrate synthase gene product following PCR.
1
2
3
4
5
678
9
10
11 12
M
381
Fig. 2. Amplification of the 381 bp product of the citrate synthase gene from tick
pools with primers RpCS.877p and RpCS.1258n. Example of adult D. variabilis tick
pool clearly positive (lane 8) following PCR and gel electrophoresis. Lane 1, no DNA
control; lane 2, positive control (R. slovaca); lanes 3-12 contain PCR products from tick
pools; lane M, molecular marker.
.-
25
DNA Amplification with R. rickettsii 190-kDa Antigen Primers
A second PCR was conducted on the 28 D. variabilis tick pools positive for the
R. prowazekii citrate synthase gene using primers Rr190.7p and RrI90.602n. Successful
amplification with these primers amplified the 190-kDa antigen gene sequence and
generated a 532 base pair product. Eight out of 28 tick pools were positive for the 190kDa antigen gene product following PCR.
TABLE 3
SUMMARY OF SURVEYED INDIANA COUNTIES
-
No. pools positive
for genus Rickettsia"
No. pools positive
for SFG rickettsiaeb
6
57
13
19
40
7
2
3
7
8
31
52
98
1
5
2
3
2
0
0
1
1
0
2
4
7
0
1
0
0
1
0
0
343
28
8
County/Site no.
No. pools
Harrison/l
Harrison/2
Jefferson
Lawrencell
Lawrence/2
Perry
Pike
Spencer
Warrickll
Warrickl2
Washington/ 1
Washington/2
Washington/3
Total
"PCR using primers RpCS.877p and RpCS.1258n
bpCR using primers Rrl90.7Op and Rrl90.602n
1
()
0
1
1
3
26
laGrange
Elchart
Steuben
OeKaIb
Noble
Monhol
Kosciusko
Alon
F..on
ttm.g.
ton
Wo..
Adams
cnnt
Howord
a.aekford
Jay
Clrton
TIptOn
Machon
Delaware
Rand~
Montgomery
Hamilon
Boone
Herwy
Wayne
Hendric:b
Nnam
Marion
Hancoct<
Ruoh
unon
Shelby
Jollnson
Franlclrl
Oec:atur
Brown
-.
Bartholomew
Fig. 3. County location and numbers of collected adult D. variabilis tick pools
in Indiana during May 2000.
27
laGrange
steuben
OeKaIb
Noble
Alon
Huntitgton
Web
TIPPecanoe
Howard
Blackford
Adams
Joy
WaITOfl
Clinton
Tipton
Madison
Delaware
Randolph
Mortgomery
Hamilon
Boone
"-
Nnam
--
Henry
Wayne
Marton
....ncock
Rush
Union
Shelly
Johnson
Franklin
Decat"
Brown
-
Bartholomew
Fig. 4. County location and numbers of adult D. variabilis tick pools positive for the
genus Rickettsia in Indiana.
28
laGrnge
StelJ)en
DeKl1b
Hunting-
ton
Weh
Howard
Blackford
Adams
Jay
warren
CInIon
Tipton
Machon
Dellware
Randolph
Montgome<y
Boone
-
Nnam
-
Ha""on
Herwy
Wayne
Ma_
Hancock
Rush
Shelly
Jolmon
Fnlnldin
DecatlM"
Brown
Bartholomew
Fig. 5. County location and numbers of adult D. variabilis tick pools positive for SFG
rickettsiae in Indiana.
29
CHAPTER V
DISCUSSION
Results from peR and agarose gel electrophoresis indicate that SFG rickettsiae
infest adult D. variabilis ticks from southern Indiana.
peR with citrate synthase primers amplified SFG rickettsiae plus other members
from the genus Rickettsia. Out of the 343 tick pools collected, 28 were found to be
positive for the citrate synthase gene, implying that these bacteria were members of the
_
Rickettsia genus. These positive samples were from the following 6 southern Indiana
counties: Harrison, Jefferson, Lawrence, Spencer, Warrick, and Washington (Fig. 5).
Future studies will determine the species of bacteria that were present in the 20 tick pools
that were positive for the genus Rickettsia but not for SFG rickettsiae.
A second peR conducted on the 28 tick pools positve for the Rickettsia genus
amplified only SFG rickettsiae. Out of these 28 tick pools, 8 were found to be positive
for SFG rickettsiae. These positive samples were from the following 4 counties:
Harrison, Lawrence, Spencer, and Washington Previous studies suggest that R. montana,
R. rhipicelphali, and R. rickettsii are the predominant species of SFG rickettsiae in
Indiana (J.M. Stauffer, unpublished data). Future research using additional peR and a
technique referred to as restriction fragment length polymorphism (RFLP) will
-
30
distinguish between these positive samples and determine which particular species of
SFG rickettsiae were present in the survey.
Although these species of SFG rickettsiae have yet to be distinguished, there is
expectation of a greater number of nonpathogenic bacteria than pathogenic bacteria. This
belief is based on previous findings of the lethal effects of pathogens on their vectors.
For example, the agents of Rocky Mountain spotted fever (R. rickettsii), epidemic typhus
(R. prowazekii), bubonic plague (Yersinia pestis), leishmaniasis (Leishmania major and
L. in!antum), onchocerciasis (Onchocera spp.), filariasis (Brugia spp. and Dirofilaria
immitis), malaria (Plasmodiu spp.), mosquito-borne encephalitides, and African swine
fever have been reported to reduce the survival, fecundity, or fitness of their vectors
(Niebylski et al. 1999). Less clear is effect of the agents of tularemia (Francisella
_
tularensis) and Lyme disease (Borrelia burgdorferi) on their tick vectors, although
pathological effects have been reported (Balashov 1972; Burgdorfer et al. 1989;
Neibylski et al. 1999). Future studies on the transmission of arthropod-borne microorganisms are needed to determine their potential effects on vector health and behavior.
Due to the prevalence of confirmed cases of RMSF from southern Indiana, the
D. variabilis ticks sampled in this project were all collected from southern Indiana
counties. However, D. variabilis has previously been recorded from all 92 counties of
Indiana. A significant number of cases of RMSF has been confirmed in residents of the
northern-most counties and the densely populated, centrally-located Marion County (R.R.
Pinger, unpublished data). Further studies should include D. variabilis ticks from other
parts of the state in order to compare differences in the number of positive samples and
-
.
the type of SFG rickettsiae that infest these ticks .
31
--
It should also be noted that the positive control used for this project, R. slovaca, is
not a SFG rickettsiae native to North America. Additional research should incorporate a
known positive SFG rickettsiae that is in existence in the North America, particularly the
United States. Use of this positive control may provide further understanding of the
particular SFG rickettsiae infesting these D. variabilis ticks.
In conclusion, this project has provided preliminary results about SFG rickettsiae
in D. variabilis ticks from southern Indiana and points to the need for further studies for
species identification of these microorganisms.
-
-
32
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