Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

Two studies were conducted to evaluate the presence of tick-borne bacterial agents in Amblyomma americanum, lone star tick (LST), and various wildlife in Mississippi. Adult LSTs had DNA evidence of Ehrlichia chaffeensis (3.7%), E. ewingii (6.3%), and Borrelia lonestari (2.6%), while both larval (24.3%) and adult (43.5%) LSTs were positive for a Rickettsia spp. by polymerase chain reaction (PCR). White-tailed deer (WTD) were the only wildlife PCR positive for E. chaffeensis (18.8%), B. lonestari (3.1%), and Anaplasma phagocytophilum (3.1%). In addition, WTD had the highest seroprevalence to B. lonestari (19.3%) and E. chaffeensis (43.9%) antigens while raccoons had the highest seroprevalence to spotted fever group rickettsiae (R. parkeri antigen) (73.7%). These studies demonstrate evidence of potentially zoonotic tick-borne agents in LSTs and wildlife in Mississippi underscoring the importance of monitoring these agents for human and animal health.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

Infestation of cattle by Lone Star ticks, Amblyomma americanum, leads to damage of hides intended for leather, weight loss, infertility, and potentially death of cattle, which contribute to production losses for farmers. Public concerns regarding chemical residues in food and the environment necessitate development of chemical-free alternative tick controls, such as breeding for tick-resistant phenotypes and developing anti-tick vaccines. Thus, the goal of this study was elucidation of mechanisms that mediate immune responses in cattle infested with A. americanum using gene expression techniques. Methods for isolation of total RNA from bovine tick bite-site biopsies and blood leukocytes were optimized to provide RNA suitable for gene expression studies. Tick bite-site biopsies (6 mm) and blood leukocytes were collected from a total of 13 calves (N=6, Group 4 and N=7, Group 5 calves) during experimental tick infestations to determine A. americanum tick-susceptible and -resistant phenotypes. Microarray experiments compared gene expression in tick bite-sites from tick-susceptible, moderately tick-resistant, and highly tick-resistant calves. A total of 35 genes were profiled in tick bite-site biopsies and 12 genes were evaluated in blood leukocytes via gene-specific qRT-PCR assays, and analyzed for each phenotype and for each group of calves as a whole. Analysis of microarray data revealed differential expression of IL-1R-mediators among the three cattle phenotypes. Expression profiles generated by qRT-PCR for TLR-mediating genes such as TLR2, TLR4, CD14, and MyD88 suggest that a MyD88-dependent signaling pathway may mediate the development of acquired immunity in cattle infested with Lone Star ticks. Additionally, increased expression for IL12, IFNgamma, and TNFalpha suggests that a Th1-type cell-mediated reaction may be activated, whereas increased expression of IL6, IL10, and IGHG1 supports the involvement of a Th2-type humoral-mediated response at tick bite-sites in cattle infested with at A. americanum. Regression analyses identified strong correlations between factors involved in pattern recognition in tick bite-site biopsies, including associations between TLR4 and IL1alpha, and between IL1alpha and IL1RN. In conclusion, this dissertation reports optimal methodology for gene expression studies in tick-infested cattle and provides preliminary data concerning the underlying mechanisms associated with the immune response in Lone Star tick-infested cattle.

Human Monocytic Ehrlichiosis (HME) is an emerging disease first described in 1987 and is transmitted by the bite of Amblyomma americanum. Over the past 10 years, the CDC has documented increasing ehrlichiosis case reports nationwide. Our study site is a golf-oriented retirement community located in the Cumberland Plateau of Tennessee. In 1993, four men at the study site had symptoms consistent with HME which prompted a CDC outbreak investigation and led community managers to mitigate ticks feeding on deer. The objectives of this study were to measure the efficacy of current tick mitigation attempts, to determine the level of infection and composition of tick-borne disease in the study area, and to assess which wildlife species are potentially acting as reservoirs for disease. Ticks were sampled in the community at eight sites of ‘4-poster’ acaricide applicator utilization and at seven untreated sites. Close to the ‘4-poster’ devices, larval, nymphal, and adult tick abundances were reduced by 90%, 68% and 49% respectively (larval p<0.001, nymphal p<0.001, adult p=0.005) relative to the untreated areas. We extracted DNA from A. americanum ticks collected at the treatment and non-treatment sites and tested for Ehrlichia spp. infections. Of 253 adult and nymphal A. americanum tested, we found 1.2% to be positive for Ehrlichia chaffeensis, 4.7% positive for Ehrlichia ewingii, and 1.6% positive for Panola Mountain Ehrlichia; in combination this prevalence is similar to that reported in other Ehrlichia-endemic areas of the eastern U.S.. We also performed blood meal analysis on DNA from A. americanum ticks and the results suggest that the most significant reservoir hosts for Ehrlichia spp. are white-tailed deer, turkeys, grey squirrels, and Passeriformes. We conclude that while the ‘4-poster’ acaricide applicators reduce the number of ticks close to treatment, at the density at which they are currently being used (8 applicators per 52.6 km2, average distance between applicators = 6.6km) they will have no large-scale effect on the community’s tick population. In order to accomplish area-wide reduction of A.americanum and Ehrlichia spp. in this locale, community managers should develop an integrated management strategy that utilizes other techniques in addition to ‘4-poster’ devices.

Amblyomma americanum is a hard tick species found in southern Indiana. Once a notorious pest to humans and livestock, A. americanum has now taken on a role as vector to pathogenic organisms. This study aimed to detect and quantify three microbes in A. americanum: Rickettsia amblyommii, Ehrlichia chaffeensis, and Borrelia lonestari. A primary objective of this study was to determine microbial interaction within a single A. americanum tick through quantification of each microbe within a co-infected tick. A second objective was to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. Infection rates were 44%, 1%, and 0% for R. amblyommii, E. chaffeensis, and B. lonestari respectively. This study found no co-infected ticks, therefore no microbial interaction was determined. This study also found multiple drawbacks with utilizing quantitative real-time PCR to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. / Department of Biology

Human monocytotropic ehrlichiosis (HME) is a tick-borne infectious disease caused by the bacterium Ehrlichia chaffeensis and transmitted by the lone star tick, Amblyomma americanum. The disease was recognized in Indiana for the first time in 1994. Since 1999, 11 cases have been confirmed in Indiana and two additional cases are under investigation. In the past five years, the cases have been reported from Crawford, Harrison, Warrick, Martin, Perry, Spencer, and Madison counties.A total of 2765 adult Amblyomma americanum ticks were collected from eight counties in southern Indiana during two field trips in May 2000. Ticks were pooled and examined for the presence of Ehrlichia chaffeensis using nested PCR with primers HE1 and HE3, specific for the 16S rRNA gene of the pathogen. Ninety-six pools of A. americanum specimens tested positive for E. chaffeensis DNA. This represented a minimum infection rate (MIR) of 3.5%.To identify different genetic forms (strains) of E. chaffeensis, the positive tick pools were probed for the Variable Length PCR Target (VLPT) gene of E. chaffeensis. The data were used to develop a geographic map of the distribution of the different strains of the pathogen. Overall, nine different genetic variants (91HE17, Arkansas, Jax, Liberty, Osceola, Sapulpa, St. Vincent, Wakulla, West Paces) of E. chaffeensis were identified from pools of ticks collected in four counties (Harrison, Perry, Pike, Warrick). All samples positive for the 16S rRNA were also positive for the VLPT gene.E. chaffeensis isolates are polymorphic in the number of repetitive sequences within the genes encoding the VLPT, and the isolates obtained illustrate this phenomenon. The high concordance rate between the 16S rRNA and the VLPT gene reveals that the VLPT gene is a very sensitive tool for detecting E. chaffeensis in the lone star ticks. We found no clear correlation between geographic distribution of different genetic variants of E. chaffeensis and the genetic polymorphism of the VLPT gene. Further study with a relatively larger sample size from a wider geographical area might be able to detect such a pattern. / Department of Physiology and Health Science

The aim of the study was to analyze the infectious potential of the newly described species, B. americana and B. carolinensis, studied on the laboratory model mammals mice. Our goal was to analyze and compare the vectorial capacity of two different tick vectors, Amblyomma americanum and Ixodes ricinus, in acquiring and transmition of both spirochete species to the host. The results of this study confirmed that ticks A. americanum and I. ricinus are capable to maintain and transmit B. americana and B.carolinensis.We confirmed that both analysed spirochete species, B. carolinensis and B. americana, showed the potential to develop the disease in laboratory model mammal, which indirectly support the fact that both spirochete species might be concidered as the risk factors in the area where they are distributed. Our results shows that A. americanum is able to transmit both spirochete species, which increases that risk of acquiring the Lyme disease to human population in the area of distribution of A. americanum

Ticks are hematophagous ectoparasites capable of transmitting various pathogens, including bacteria, protozoa, and viruses, to vertebrates. In the United States, tick-borne pathogens are responsible for around 95% of arthropod-borne diseases. Lyme disease is the most common tick-borne illness. However, emerging tick-borne viruses such as Bourbon virus (BRBV), Powassan virus (POWV), and Heartland virus (HRTV) can cause more severe health problems, including death and neurological abnormalities. The reports of molecular detection of viral RNA in field-collected ticks and serological evidence in a pilot study of wildlife species suggest the presence of these emerging viruses in Virginia. The presence poses a serious health threat, but the extent of their presence or circulation in Virginia is unknown. The objectives of the research are (1) to determine the evidence of circulation of POWV, HRTV, and BRBV in Virginia through serological assessment of domestic and wild animals in Virginia and (2) estimate transmission parameters and the basic reproduction number underlying tick-borne virus distribution and prevalence via a mathematical model. Here, we discuss the known literature relevant to tick-borne virus emergence; we assessed the presence of specific neutralizing antibodies against POWV, HRTV, and BRBV in wildlife and livestock sera collected from different health planning regions in Virginia. We used a susceptible-infected-susceptible (SIS) ordinary differential equation model to estimate transmission parameters that best describe the disease dynamics of emerging tick-borne viruses in Virginia. In our study, wildlife sera were seropositive against POWV (18%), BRBV (8%), and HRTV (5%). A wide range of different wildlife species were shown to be exposed to each virus examined. Livestock are also exposed to tick-borne viruses, with seroprevalences of 1%, 1.2%, and 8% detected in cattle for POWV, BRBV, and HRTV, respectively. We estimated the transmission rate and basic reproduction number to be 1.57 and 0.645, respectively. In conclusion, there is a widespread circulation of tick-borne viruses in western and northern Virginia within diverse species of animal populations. / Master of Science in Life Sciences / Ticks are blood-sucking ectoparasites that can transmit various pathogens, including bacteria, protozoa, and viruses, to humans and other vertebrates. In the United States, tick-borne pathogens are responsible for about 95% of all arthropod-borne disease cases. Lyme disease is the most common tick-borne illness. However, emerging tick-borne viruses such as Bourbon virus (BRBV), Powassan virus (POWV), and Heartland virus (HRTV), can cause more severe health problems, including potentially death or neurological abnormalities. The reports of molecular detection of viral RNA in field-collected ticks and serological evidence in a pilot study of wildlife species suggest the presence of these emerging viruses in Virginia. However, there is a lack of knowledge on the extent of their circulation. Firstly, this study aims to determine the evidence of circulation of POWV, HRTV, and BRBV in Virginia through serological assessment of domestic and wild animals. Secondly, this study aims to estimate transmission parameters and calculate the basic reproduction number of emerging tick-borne viruses. Evidence of prior infection against all three tick-borne viruses was detected in both wild and domestic animal species from the five Virginia health planning regions, with most samples in the study coming from southwestern and northwestern regions. In conclusion, there is a circulation of tick-borne viruses in Virginia, which is a potential threat to the public health.