Highlights of tick-borne disease research at Mississippi State University.
Ticks are second only to mosquitoes as vectors of human disease agents, being responsible for thousands of cases of human illness worldwide annually. In fact, they transmit more cases of human disease than any other arthropod in Europe and North America. Lyme disease alone is considered responsible for perhaps as many as several hundred thousand clinical cases each year in Europe (Ginsberg and Faulde 2008). There are at least 20,000 cases of the illness reported in the U.S. annually, with over 27,000 reported in 2007 (CDC 2008). In addition, many tick-borne diseases are quite severe clinically, with sudden onset of high fever, headache, and myalgias, often accompanied by nausea and other symptoms. Rocky Mountain spotted fever is fatal in as many as 5% of cases, even with treatment, while Crimean-Congo hemorrhagic fever, caused by a virus, is the most severe tick-borne disease in Europe with a fatality rate of about 10% (Ginsberg and Faulde 2008).
The ecology of infectious diseases, and especially that of emerging tick-borne diseases, is a crucial component of public health and safety in the 21st century. Many tick-borne human diseases, especially those that are zoonotic, have complex life cycles wherein different stages of the tick feed on different hosts, become infected, and (later) infect humans or other animals. One flaw of modern medicine is to depend too heavily on pharmacologic agents for disease prevention and control without understanding the natural life cycles of the causative agents and using this knowledge to intervene and mitigate those negative health effects. The relationship of diseases to the environment has been neglected in recent decades, making us vulnerable to widespread and potentially devastating outbreaks. This paper highlights past and current research at Mississippi State University concerning ticks, their relationship to the environment, tick ecology, tick-borne disease transmission, and the factors affecting that transmission.
TICK BIOLOGY AND ECOLOGY
Basic Tick Biology. Ticks are arachnids in the Phylum Arthropoda. Within this phylum, three families of ticks are currently recognized in the world: 1) Ixodidae (hard ticks), 2) Argasidae (soft ticks), and 3) Nuttalliellidae (a small, curious, little-known group with some characteristics of both hard and soft ticks). The terms hard and soft refer to the presence of a dorsal shield or scutum in the Ixodidae, which is absent in the Argasidae (Figure 1). Hard ticks display sexual dimorphism, whereby males and females look obviously different, and the blood-fed females are capable of enormous expansion. In some species of hard ticks the males do not feed; others imbibe only small quantities. Hard tick mouthparts are anteriorly attached and visible from dorsal view (Figure 1A). If eyes are present, they are located dorsally on the sides of the scutum (Figure 2). Soft ticks are leathery and nonscutate (no shield), without sexual dimorphism (Figure 1B). Their mouthparts are subterminally attached in adult and nymphal stages and not visible from dorsal view. Eyes, if present, are located laterally in folds above the legs.
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Hard ticks have three feeding stages in their life cycle (larvae, nymphs, and adults) which normally utilize a different host animal during each stage (Figure 3). An example of such a "three-host" tick is the lone star tick, Amblyomma americanum (Figure 2). Modifications of this feeding pattern occur. For example, Rhipicephalus evertsi (not a North American species) uses only two hosts, and the cattle tick, Rhipicephalus (Boophilus) annulatus (essentially eradicated from the United States in the 1940s), parasitizes only one. In the former case, the larvae and nymphs feed on the same animal, and in the latter case, all three stages feed on the same animal. Except for some Ixodes spp., hard tick adults mate on a host and, after the fully fed female drops from the host animal to the ground, she lays from 2000 to 18,000 eggs and subsequently dies. Many hard tick species "quest" for hosts, by climbing vegetation and remaining attached, forelegs outstretched, awaiting a passing host. They may travel up a blade of grass (to quest) and back down to the leaf litter where humidity is high (to rehydrate) several times a day. This useful physiological adaptation allows them to recover moisture from the air. Also, some hard ticks are considered "hunters," traveling a short distance toward host cues, such as a C[O.sub.2] source. Adult ticks are more adept at traveling through vegetation than the minute larvae.
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Ticks feed exclusively on blood, and begin the process by cutting a small hole into the host epidermis with their chelicerae and inserting the central hypostome into the cut, thereby attaching to the host. Blood flow is presumably maintained with the aid of anticoagulants from the salivary glands. Some hard ticks secure their attachment to the host by forming a cement cone around the mouthparts and surrounding skin. Two phases are recognized in the feeding of nymphal and female hard ticks: 1) a growth feeding stage characterized by slow continuous blood uptake and 2) a rapid engorgement phase occurring during the last 24 h or so of attachment.
Ecology of ticks. Hard ticks and soft ticks occur in different habitats. In general, most hard ticks are non-nidicolous, occurring in brushy, wooded, or weedy areas containing numerous deer, cattle, dogs, small mammals, or other hosts. Soft ticks are generally nidicolous, being found in animal burrows or dens, bird nests, bat caves, dilapidated or poor-quality human dwellings (huts, cabins, and so forth), or animal rearing shelters. Many soft tick species thrive in hot and dry conditions, whereas ixodids which are more sensitive to desiccation are usually found in areas providing protection from high temperatures, low humidities, and constant breezes.
Being sensitive to desiccation, most hard ticks must practice water conservation and uptake. Their epicuticle contains a wax layer, which prevents water movement through the cuticle. Water can be lost through the spiracles; therefore, resting ticks keep their spiracles closed most of the time, opening them only one or two times an hour. Tick movement and its resultant rise in C[O.sub.2] production cause the spiracles to open about 15 times an hour with a corresponding water loss.
Development, activity, and survival of hard ticks are influenced greatly by temperature, humidity, and host availability within the tick microhabitat. Because of their temperature and high humidity requirements, as well as host availability, most hard ticks tend to congregate in areas providing those factors. Ecotonal areas (interface areas between major habitat types) are excellent habitats for hard ticks. For example, open meadows/prairies, along with climax forest areas, support the fewest lone star ticks, but ecotone areas and small openings in the woods are usually heavily infested. Deer and small mammals thrive in ecotonal areas, thus providing blood meals for ticks. In fact, deer are often heavily infested with hard ticks in the spring and summer months. The optimal habitat of white-tailed deer has been reported to be the forest ecotone, since the area supplies a wide variety of browse and frequently offers the greatest protection from their natural enemies. Many favorite deer foods are also found in the low trees of an ecotone, including greenbrier, sassafras, grape, oaks, and winged sumac. On the other hand, some ticks, such as the American dog tick, Dermacentor variabilis, concentrate along paths and roads, presumably because their hosts spend more time along roads than along any single comparable line in the surrounding fields. This concentration is believed to be a result of movements of ticks from adjacent fields to the roads, where they remain.
CURRENT RESEARCH ON TICKS IN MISSISSIPPI
A seven-member team is currently investigating tick-borne diseases at Mississippi State University (Figure 4). Prior to joining the faculty of the College of Veterinary Medicine at Mississippi State University, Dr. Andrea Varela-Stokes concentrated mainly on Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), and Borrelia lonestari (Figure 5), putative agent of "southern tick-associated rash illness" (STARI) in her PhD and post-doctoral work. However, since her arrival in summer of 2007 and her collaborations with Dr. Jerome Goddard, the laboratory has explored various aspects of tick-borne disease, including, but not limited to, the above agents.
One of the initial objectives of the lab was to begin to evaluate the presence of tick-borne agents in the lone star tick, Amblyomma americanum, and selected wildlife in Mississippi. As it is the most common tick in the state and in the Southeast, determining the prevalence of disease agents in the lone star tick will help us understand the importance of tick-borne diseases here and potential risk of human exposure. In addition, our objective in monitoring tick-borne disease exposure and infection in wildlife is to begin to understand the maintenance of these organisms in nature.
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Ashley Harris is currently working towards an MS degree in Veterinary Science and is testing ticks and wildlife collected from several places around Mississippi. With the help of a summer vet student worker, Jamesia Showers, a survey of Mississippi lone star ticks was conducted last summer. A total of 192 adult ticks were collected by drag cloth or dry ice traps from four regions of Mississippi: East (Noxubee National Wildlife Refuge and Lowndes County, MS), Northwest (Wall Doxey State Park), Northeast (Tishomingo State Park), and Southeast (Natchez State Park). In addition, 42 pools of larval ticks were collected from Southwest Mississippi (Copiah Co.). DNA from adult ticks and larval pools were tested for E. chaffeensis, Borrelia spp., Francisella tularensis, and Rickettsia spp., as well as for the presence of tick-specific DNA. All ticks tested were positive for tick-specific DNA, demonstrating that DNA was successfully extracted from the tick tissues. Thus far, Borrelia sp. DNA was amplified from 5/192 (2.6%) of adult ticks tested, 7/192 (3.7%) had evidence of E. chaffeensis, and 34/192 (17.7%) were positive for a Rickettsia species. Nine of forty-two (21.4%) of the pools of larval ticks were positive for a Rickettsia species. No ticks have been found co-infected with any of these organisms. The region with the highest prevalence of ticks infected with E. chaffeensis was the Northeast (Tishomingo SP), however because the sample size at that location was very small, this prevalence may not reflect true prevalence in nature. The Northwest (Wall Doxey SP) had the highest prevalence of Borrelia and Rickettsia sp. infections. We are currently in the process of sequencing these products to determine the Borrelia species as well as the Rickettsia species involved. The sequence of one of the E. chaffeensis amplicons was identical to that of E. chaffeensis. In addition to the PCR assays of ticks, 2 pools of ten ticks were dissected and cultured in an attempt to cultivate rickettsial or Borrelia species. Organisms resembling Rickettsia spp. were visualized in stained slides from two flasks of the same set of cultivated ticks (Figure 6). PCR for Rickettsia spp. revealed two amplicons of appropriate sizes. We are currently working to sequence these amplicons.
The prevalence of known and putative zoonotic, tick-borne agents is also being assessed in white-tailed deer (Odocoileus virginianus), feral swine (Sus scrofa), raccoons (Procyon lotor) and opossums (Didelphis virginiana) in the state of Mississippi. Animals are being tested for exposure to or infection with six tick-borne agents: Borrelia spp., E. chaffeensis, E. ewingii, Anaplasma phagocytophilum, Francisella tularensis, and Rickettsia species. Both whole blood and serum from white-tailed deer and feral swine are being tested, while only serum is being tested from raccoons and opossums. DNA from these samples is being tested by single or nested PCR for the above-mentioned organisms. An indirect immunofluorescent antibody assay (IFA) using antigen from E. chaffeensis, B. lonestari, and Rickettsia parkeri is being used to test for antibodies against these organisms. Thus far, molecular evidence of infection to B. lonestari, E. chaffeensis, and A. phagocytophilum has been detected only in deer; all swine have been negative. In general, deer have had the greatest evidence of exposure to E. chaffeensis and B. lonestari, which supports previous work suggesting that deer are primary reservoirs for these two agents (Dawson et al. 1994, Lockhart et al. 1997, Moore et al. 2003, Moyer et al. 2006). However, interestingly, the other animal species also showed evidence of exposure to most agents. These results demonstrate that wildlife in Mississippi are exposed to tick-borne diseases, suggesting that ticks carry and have the potential to transmit these agents to humans in Mississippi as well.
In addition to the tick-borne diseases mentioned above, we have recently become interested in the newly emerged pathogen, R. parkeri. Dr. Kristine Edwards is a DVM, MPH, who is currently working on her PhD in medical entomology in our laboratory. She is testing the hypothesis that cattle play a central role in the maintenance of R. parkeri as a host for the Gulf Coast tick (Amblyomma maculatum). An experiment was designed using healthy Holstein bull calves from the Mississippi State University. One group comprised calves injected with R. parkeri organism cultured in Vero cells and one negative control calf injected with Vero cells containing no organism. Another group comprised calves infested with adult A. maculatum ticks injected with cultured R. parkeri placed on each right ear and one negative control calf with adult A. maculatum ticks injected with phosphate-buffered saline (PBS) placed on the right ear. Ticks were confined to the feeding site by a sock fitted over the calf's ear (Figure 7). Dr. Edwards found that in addition to successful tick feeding on the calves, a transient rickettsemia was demonstrated by PCR in some exposed calves and evidence of organisms in biopsies was demonstrated by immunohistochemistry. Also, the calves produced antibodies to R. parkeri as revealed by IFA. Hematologic changes were not conclusive and the calves were not clinically ill for the duration of the study. However, a condition known as "gotch" ear was manifest in all the tick-infested calves wherein the ears became edematous and erythemic near sites of tick attachment.
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Dr. Edwards has also been investigating R. parkeri in cattle naturally infested with Gulf Coast ticks in Mississippi. Beginning in July 2008, blood and ticks were collected from cattle from sale barns in Tupelo, West Point, Macon, Tylertown, Natchez, and Meridian. Cattle were bled either from the tail vein or by jugular venipuncture and samples for PCR and serology were obtained. Animals were examined rapidly for ticks as they were held in the chute. Blood was obtained from all cattle regardless of whether ticks were present. Sites of tick attachment were biopsied whenever possible. Blood samples, biopsy samples, and tick specimens were all designated with a unique identifier associated with the animal and recorded on each individual animal's chart as well as the animal's signalment (age, gender, breed, use, approximate weight). Hemolymph tests and IFA tests are currently being performed on these samples (Figures 8). DNA extracts are being evaluated using nested PCR assays. Preliminary results of cattle from sale barns in Mississippi naturally infested with A. maculatum ticks revealed Spotted Fever Group (SFG) rickettsiae in some of the ticks as demonstrated by hemolymph tests. Further PCR and IFA analyses are currently underway to confirm these findings.
We have recently expanded our studies of R. parkeri with the addition of two other graduate students to the lab. Gail Moraru is a student working on both her PhD and DVM, while Flavia Girao is working on her MS degree in Veterinary Science. Gail's project will concentrate on the ecological aspects of the natural history of R. parkeri. Because R. parkeri is carried by a vector to its host, the vector largely determines in which host the bacterium ends up. Therefore, it is important to know what animal hosts the Gulf Coast tick prefers. One objective of her proposal is to learn which host(s) the larvae and nymphs prefer to feed on, feeding success on available host(s), and potentially which host(s) the bacterium grows best in. In one experiment, a "choice" of host will be offered to ticks to determine host preference, while in another experiment, feeding success will be determined by placing the ticks directly on several different hosts. In addition, infection with R. parkeri might be performed on the various animal hosts if time allows. Further, field work will be necessary to help put the pieces together of where both the ticks and the rickettsiae are found. Both animals and ticks will be collected from at least two sites in Mississippi and blood samples will be taken from the animals and used in serologic (IFA) and PCR assays. DNA from ticks will be tested for Rickettsia parkeri by PCR and sequencing of a key gene. The information gathered from this study should help uncover missing information about the natural history of R. parkeri and A. maculatum. Ideally, information pertaining to potential reservoir host(s) might be obtained; however, this will have to be determined by combining results from several different components of her study. Through Gail's work, we anticipate gaining better understanding of the natural history of R. parkeri as it involves potential wild reservoirs.
Flavia's project is currently investigating R. parkeri and A. maculatum populations as a model for the movement of the foreign animal disease, heartwater, in the event of an accidental or intentional introduction to the United States. Approximately 7001000 ticks will be collected from ten sites within Mississippi and DNA from these samples will be tested by PCR targeting the tick mitochondrial 16S rRNA gene (Qiu et al. 2002) and the Rickettsial ompA gene as described by Paddock (Paddock et al. 2004) to generate specific sequence products for single stranded conformational polymorphism (SSCP) analysis. We intend to use SSCP data to estimate gene frequencies of individual alleles in the R. parkeri and A. maculatum populations. The comparison of these frequencies will provide information regarding variation within and among geographically distinct populations and determine the amount of movement and interbreeding in the tick populations while also relating this to R. parkeri populations. This project will provide the basis for further studies of population structures over time.
Our fifth lab member, Erle Chenney, plays the invaluable role of technician. In addition to maintaining day to day operations of the lab, Erle has also been invaluable in data collection for the tick and wildlife surveys. He handles the task of ordering supplies, equipment, and reagents for use in the lab and makes various media for use in several types of cell culture, which he also maintains. Erle is familiar with all the research projects of the graduate students as well as other projects ongoing in the lab. His work is essential to the survival of the laboratory.
Dr. Jerome Goddard has been conducting ecological studies of ticks in Mississippi for twenty years. Ticks are not evenly distributed in the wild; but instead, they are localized in areas providing their necessary temperature, humidity, and host requirements. Earlier ecological studies performed by Dr. Goddard included studying the effect of weather on questing populations of hard ticks (Goddard 1992, 2001) as well as mark-release-recapture experiments which estimated tick populations in Mississippi forests (Goddard 1993, Goddard and Goddard 2008). Studies on the spatial and geographic distribution of the tick, A. americanum, revealed that they "cluster" in spots in the woods, and are not evenly distributed (Goddard 1997). For example, in one study, the majority of adult and nymphal ticks were collected by drag cloth in only 17.7% and 9.7% of the field plots, respectively (Figure 9) (Table 1) (Goddard 1997). In addition, ticks, being subject to desiccation, are found in predominantly shady spots. In field plots with the amount of shade ranging widely from 0 to 90 percent, we have found 21/31 (68%) of adult ticks and 24/33 (73%) of nymphs were collected in areas of 71% and 65% shade, respectively (Table 2) (Goddard, 1997).
Oddly, current preliminary data with the Gulf coast tick, A. maculatum, reveals just the opposite behavior. Careful collections of this tick along the Mississippi Gulf Coast have shown that adults of this tick are found in grasslands in open sunshine, with little relation to shade or soil moisture. One 0.5 ha collection site in open sunshine within the Grand Bay National Wildlife Refuge yielded over 100 adult A. maculatum. Also, systematic drag cloth collections within the Sandhill Crane National Refuge revealed that most A. maculatum were found along the road, in bright sunshine, as opposed to wooded areas.
Further ecological research is needed to determine the variables predicting A. maculatum questing and host-finding activity, especially in relation to the changing climate and habitat transformation. Knowledge of these variables could theoretically lead to precision-targeting of pesticides or other pest control interventions to lower human exposure to the disease-bearing ticks.
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Tick-borne diseases continue to emerge. Therefore, ticks will remain an important threat to human health for the foreseeable future. For example, in the 1970's, Lyme disease was virtually unknown; now it is the most important tick-borne disease in the United States, if not the world (Bonnefoy et al. 2008). Other such disease entities likely occur but have yet to be recognized. For example, a brand new tick-borne agent called the Panola Mountain ehrlichia was recently found to infect humans (Loftis et al. 2008, Reeves et al. 2008). Changes in pathogens, increased human populations, environmental and ecological changes, and other such factors are contributing to the emergence of these and other vector-borne diseases. University researchers, public health officials, and clinicians all play important roles in the control of tick-borne diseases and must work together to find, treat, manage, and/or prevent such diseases in the future. We are hopeful that the contributions of Drs. Goddard and Varela-Stokes to the field of tick-borne research will serve to heighten our understanding of the natural history of tick-borne disease in such a way as to help prevent further transmission.
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Reeves, W. K., A. D. Loftis, W. L. Nicholson, and A. G. Czarkowski. 2008. The first report of human illness associated with the Panola Mountain Ehrlichia species: a case report. J. Med. Case Rep. 2: 139-142.
Andrea Varela-Stokes, (1) Jerome Goddard, (2) Kristine T. Edwards, (2) Ashley Harris, (1) Gail Moraru, (1) Flavia Girao, (1) and Erle Chenney (1)
(1) Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, and (2) Department of Entomology and Plant Pathology, Mississippi State University
Corresponding Author: Jerome Goddard (JGoddard@entomology.msstate.edu)
Table 1. Clustering of lone star ticks in study sites in central Mississippi. Site Tick stage Percent of area where majority ticks found 1 Adult 17.7 1 Nymph 9.7 2 Adult 14.5 2 Nymph 25.8 Table 2. Percent shade in study sites in central Mississippi where majority of lone star ticks were collected. Site Tick stage Number of LSTs Percent Shade collected 1 Adult 21/31 (68%) 71 1 Nymph 24/33 (73%) 65 2 Adult 31/44 (70%) 45 2 Nymph 81/113 (72%) 71
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|Author:||Varela-Stokes, Andrea; Goddard, Jerome; Edwards, Kristine T.; Harris, Ashley; Moraru, Gail; Girao, F|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Apr 1, 2009|
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