Kyasanur Forest disease virus (KFDV) of the Flaviviridae virus family has caused seasonal infections and periodic outbreaks in Karnataka, India. First identified in 1957, KFDV annually infects 400-500 people and has a fatality rate of 3-5%; there are no approved antivirals and the existing licensed vaccine’s effectiveness appears to be questionable. Many tools for KFDV research are limited and this work sought to develop methods for analysing antivirals, including interferon (IFN)-α/β species. The BHK-21 (ATCC) cell line allowed for high virus propagation and distinguishable cytopathic effects (CPE) for determining antiviral effectiveness. The additional tool of a reverse genetics system expressing a full-length cDNA KFDV genome with a GFP reporter failed to propagate, despite numerous GFP genome-insertion strategies. The clinically approved IFN-α2a or IFN-α2b has had variable success at combatting flavivirus diseases in people, especially in the immuno-compromised. The continued passaging of KFDV-infected cells with repeated IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production. IFN-αspecies, αWA and α were more effective than IFN-α2a and α2b at reducing KFDV; however dose ranges indicated that while low concentrations could limit CPE, higher concentrations were needed to inhibit virion release. Avoidance of IFN-α/β through Jak/STAT signalling repression was attributed to the NS5 protein, specifically the RdRp domain based on data obtained with luciferase and vesicular stomatitis virus (VSV) recovery assays. However, the mechanism appears to act subsequently to STAT1/2 activation without NS5 binding to any Jak/STAT components. A non-infectious, replicative system serving as a platform for antiviral drug testing against KFDV in a high throughput manner could only provide luciferase signals when the NS proteins capable of driving replication, were supplied in cis (subgenomic) but not in trans (antigenome). To conclude, IFN-α species such as IFN-αWA may be better suited than the licensed IFN-α2a for treatment of KFDV infections; however, IFN effects appear to be subdued in vitro due to the actions of the NS5 protein. While IFN may not be a successful antiviral against KFDV, the work in this thesis provides a foundation for evaluating other potential anti-KFDV therapeutics. / February 2016

All wildlife populations harbour parasites. However, seabirds are likely to play a particularly important role in the maintenance and dispersal of infectious agents as a result of their colonial breeding habits. Seabird colonies are also known to be highly spatially structured, but little is known about the effects of this spatial structuring on seabird parasite dynamics. In this thesis, I use a tick-borne virus, Great Island virus (GIV), found in a large common guillemot (Uria aalge) colony bordering the North Sea as a model system to explore this relationship. I use a multidisciplinary approach, framed by a simple epidemiological model of the guillemot-tick-virus system. In Chapter 2, I describe a novel epidemiological model and parameterise it using the existing literature. The model suggests the importance of spatial structure within the guillemot colony, but also identifies a key missing parameter, the rate of virus transmission between pre-breeding and breeding areas. In Chapter 3, I go on to test the potential role of independent tick movement in driving transmission between these two areas, by quantifying the mobility of host-seeking seabird ticks, Ixodes uriae. I show the potential for ticks to walk ranges described anecdotally in the literature, in just a few minutes, but stress the importance of further experiments in the field. Chapter 4, I test the potential role of guillemot-mediated tick movement between pre-breeding and breeding areas. I show that pre-breeding guillemots spend a limited proportion of time ashore during daylight hours, which increases significantly as the season progresses and varies between individuals. A similar pattern is observed when considering how often they enter breeding areas when ashore; generally infrequently but varying spatiotemporally and between individuals. In Chapter 5, I apply finite mixture modelling techniques to improve existing estimates of age- and strain-specific GIV seroprevalence and force of infection in the guillemot colony. I also provide the first estimates of these parameters for eight strains, and highlight the importance of understanding strain-specific differences in GIV dynamics in future studies. Finally, I bring all four data chapters together in Chapter 6, by inputting my new parameter estimates (Chapters 3-5) into my existing model (Chapter 2). Taken together, my results suggest that GIV transmission within the guillemot colony may increase in the future as a result of declining breeding abundance and success, with more frequent or extreme disruption leading to a higher risk of infection within the colony. More generally, my results suggest that seabird colonies can be highly sensitive to changes in their spatial structure, and that endemic parasites have the potential to substantially impact, and hence to be an added threat to, their seabird hosts.

Leishmaniasis is a parasitic disease that affects humans and animals in more than 98 countries across the globe placing more than 1 billion people at risk for the disease and killing more than 20,000 people per year. In the United States the disease is enzootic within the hunting dog population and vertical transmission has been identified as the primary route of transmission in this population. In Brazil the disease is endemic in the human population and enzootic in the dog population with vector and vertical transmission having been reported. In many diseases reports have found there is increased disease severity when an individual is co-infected with another organism. Case reports have suggested this may also occur with tick borne diseases and leishmaniosis in dogs but there is limited longitudinal data to support this relationship. Even less is known and understood regarding the risk factors and basic reproduction number, number of secondary cases one infected individual can cause in a susceptible population, of leishmaniosis in regards to vertical transmission. The goal of the work presented in this thesis is to address host factors related to the transmission of L. infantum and the way in which co-infections affect the progression of the disease both in the U.S. and in Brazil. Understanding the risk factors associated with the transmission of the parasite Leishmania infantum, the causative agent of the disease, are necessary to controlling and potentially elimination the disease. Utilizing a large prospective cohort and both active and passive surveillance it was identified that leishmaniosis can be maintained in a population via vertical transmission at prevalence rates similar to other endemic countries, 20%. With this knowledge an additional study examining a longitudinal cohort and assessing the impact of tick borne disease co-infections upon disease transmission was performed. It was identified that dogs exposed to three or more tick borne diseases were 11x more likely to progress to clinical disease (Adjusted RR: 11.64 95% CI: 1.22-110.99 p-value: 0.03) than dogs with no tick borne disease exposures. Furthermore, dogs with Leishmania and tick borne disease were 5x more likely to die within the study (RR: 4.85 95% CI: 1.65-14.24 p-value: 0.0051). When examining this relationship in a cross-sectional study in Brazil it was found that dogs with multiple tick borne disease exposures had 1.68x greater risk of being positive for Leishmania (Adjusted RR: 1.68 95% CI: 1.09-2.61 p-value: 0.019). Using a retrospective cohort of dogs and information regarding their dam’s diagnostic status near the time of pregnancy risk factors associated with vertical transmission and the basic reproduction number were calculated. It was found that dogs who were born to dams that were ever diagnostically positive for exposure and/or infection with L. infantum were 13.84x more likely become positive for L. infantum within their lifetime (RR: 13.84 95% CI: 3.54-54.20 p-value < 0.0001). The basic reproduction number for vertically transmitted L. infantum within this cohort was 4.16. The results of these studies suggest that leishmaniosis can be maintained in a population through vertical transmission. Furthermore, the studies show the risk factors associated with vertical transmission relate to the mother’s diagnostic status at time of pregnancy. The results of the co-infection studies highlight the importance of tick prevention in order to reduce disease progression. With increased disease severity associated with increased transmission to potential vectors these studies underline the need for immunotherapies and prevention measures to reduce disease progression in order to reduce transmission. Furthermore, these studies highlight the need for public health control and prevention programs to address vertical transmission if elimination of the disease is to ever be successful.

The incidence of tick-borne zoonoses such as Ehrlichiosis, Rocky Mountain Spotted Fever, and Lyme disease has steadily increased in the southeastern United States in recent years. According to the Centers for Disease Control and Prevention (CDC), the southeastern states accounted for 1,200 of the 27,000 total cases of Lyme disease reported in the U.S. in 2007. Although Ixodes scapularis is the most commonly recognized vector for the Lyme disease spirochete Borrelia burgdorferi, Dermacentor variabilis (a common vector for Rocky Mountain Spotted Fever) also has been shown to be a viable host for this pathogen. The purpose of the present study was to use PCR and DNA sequencing technologies to determine if Borrelia burgdorferi sensu lato is present in ticks and whole blood samples removed from raccoons and opossums trapped in south-central Kentucky. Raccoons and opossums were trapped in Barren and Warren counties of Kentucky between June 2007 and June 2008. Ticks were removed and stored in 70% ethanol. Sterile blood samples were collected into three 10 ml tubes containing the anticoagulant K2EDTA and stored at 4°C. Genomic DNA was extracted from ticks and blood samples using a QIAamp DNA mini kit and a QIAamp DNA blood mini kit (Qiagen) respectively. DNA samples were analyzed by polymerase chain reaction (PCR) for the presence of B. burgdorferi using oligonucleotide primers specific for the OspA gene. A total of 976 ticks were collected. Three different species were obtained from raccoons; Dermacentor variabilis, Amblyomma americanum, and Ixodes sp. Dermacentor variabilis was the only tick species found on opossums. Twenty-five percent (163/642) of the tick DNA samples were positive for Borrelia burgdorferi. Prevalence of B. burgdorferi by tick species was 24.4% (141/577) in D. variabilis, 40.6% (13/32) in A. americanum, and 27.6% (8/29) in I. scapularis. In the present study, 15.7% (8/51) of the total raccoon blood samples examined by PCR were positive for B. burgdorferi, while no opossum blood samples were positive. The high prevalence of B. burgdorferi in ticks common to raccoons and opossums observed in this study, as well as in a tick species that aggressively bites humans in the southeast U. S. (A. americanum), creates concern that there are ample opportunities for people to come in contact with the infected ticks on these animals. Future studies are urgently needed to fully assess the presence and prevalence of B. burgdorferi in Kentucky and other southeastern states in the U. S.

Thesis (MSc (Veterinary Tropical Diseases)--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Erkinis encefalitas (EE) yra dažniausia ir sunkiausia virusinė nervų sistemos infekcija, neturinti specifinio priešvirusinio gydymo. Vienintelė efektyvi šios ligos profilaktikos priemonė yra vakcinacija. Nesant galimybių įvesti visuotinę vakcinaciją nuo EE mūsų šalyje, ypač svarbu nustatyti didžiausią šios ligos riziką turinčias gyventojų grupes, kurioms būtų teikiamas vakcinacijos prioritetas. Pagal tarptautines Laimo boreliozės (LB) diagnostikos ir gydymo gaires, klinikiniai sindromai ir jų trukmė yra svarbiausias šios ligos diagnostikos kriterijus. Serologiniai tyrimai vaidina tiktai pagalbinį vaidmenį patvirtinant diagnozę. Pagrindinis serologinių tyrimų trūkumas yra tas, kad jie neleidžia atskirti aktyvios infekcijos nuo buvusio kontakto su šios ligos sukėlėjais. Laimo boreliozės atveju tai labai svarbu, kadangi endeminiuose rajonuose nuo 1,8 iki 26 proc. sveikų žmonių turi šios ligos sukėlėjų antikūnus dėl buvusios besimptomės infekcijos praeityje. Neįmanoma teisingai interpretuoti LB serologinių tyrimų, nežinant LB paplitimo regione, kuriame šie tyrimai taikomi LB diagnostikai. Šis darbas yra pirmasis Lietuvoje LB sukėlėjų antikūnų paplitimo tyrimas, kuriame nustatytas dviejų pagrindinių erkių pernešamų ligų sukėlėjų antikūnų serokonversijos dažnis tyrimo laikotarpiu, didelio endemiškumo regione, tiriamųjų imtį sudarius atsitiktinės sluoksniuotos atrankos pagal amžių ir lytį principu. Šiame darbe ištirtos įvairių rizikos veiksnių sąsąjos su EE ir LB sukėlėjų paplitimu... [toliau žr. visą tekstą] / TBE is the most common and severe CNS affecting zoonosis. No specific treatment for TBE exists. Tick-borne encephalitis can be prevented by active immunization. Due to governmental financial shortages this vaccination is not included in the national program of imunoprophylaxis for the years 2014-2018. It is very important to find out groups for priority vaccination according to living area, age, leisure activities and occupational risk. Lyme borreliosis diagnosis should be based primarily on the clinical presentation and an assessment of tick exposure risk. In most cases laboratory diagnosis is only supporting clinical picture. Antibody assays still fall short of being completely sensitive and specific to distinguish acute and chronic infection. The diagnostic specificity of serum antibody tests is low because seropositivity in normal population ranges from 1.8% to 26% and LB IgM and IgG may persist for years. The interpretation of serological tests may also lead to misdiagnosis not knowing the seroprevalence of LB in the area. This is the first study of seroprevalence of LB and incidence of TBE and LB during a three year period in Lithuania. The study was conducted in high endemic areas and random samples stratified by gender and age groups were made. Only a few studies perform neutralization tests for detecting TBEV antibodies. Assumed risk factors for the prevalence of TBE and LB were established.

Erkinis encefalitas (EE) – pati dažniausia ir sunkiausia virusinė nervų sistemos infekcija Lietuvoje, kuria per metus vidutiniškai suserga 400 žmonių. Užsikrėtus EE virusu (EEV), galima besimptomė arba klinikinius požymius sukelianti ligos eiga, turinti platų požymių spektrą – nuo lengvos, meningitinės ligos formos iki sunkaus encefalito. Nors mirštamumas nuo EE yra nedidelis, svarbiausia problema – ilgai trunkantis sveikimo laikotarpis ir ilgalaikiai liekamieji reiškiniai, kurie būdingi 26–46 proc. persirgusiųjų. Neaišku, kodėl užsikrėtę identiško virulentiškumo virusu, vieni žmonės perserga besimptome arba lengva EE forma, o kitiems atsiranda sunkus nervų sistemos pažeidimas. Manoma, kad didelę reikšmę turi žmogaus genetiniai veiksniai. Nesant specifinio priešvirusinio EE gydymo, šios sunkios centrinės nervų sistemos infekcijos patogenezės ir genetinių rizikos veiksnių tyrimai yra ypač svarbūs, siekiant nustatyti galimas veiksmingo gydymo paieškos kryptis ateityje. Šio darbo tikslas – nustatyti CCR5 ir TLR3 genų polimorfizmų paplitimą tarp EE sirgusių asmenų ir šių genų polimorfizmų reikšmę polinkiui sirgti EE bei skirtingoms EE klinikinėms formoms išsivystyti. Šis darbas yra didžiausias iki šiol atliktas genetinių veiksnių reikšmės EEV infekcijos metu tyrimas ir pirmasis tokio pobūdžio vaikų tyrimas. Tyrimo rezultatai patvirtino hipotezę, kad nefunkcionuojantis CCR5 ir funkcionuojantis TLR3 yra reikšmingi simptominės EE formos išsivystymo veiksniai, užsikrėtus EEV. / Tick-borne encephalitis (TBE) is the most common and severe viral infection of the central nervous system in Lithuania, with the average number of 400 cases per year. The clinical spectrum of TBE virus infection varies considerably from asymptomatic to mild meningitis or severe encephalitis. Although the mortality of TBE is relatively low, as many as 26–46% of the patients experience long-lasting sequelae. No specific treatment for TBE exists. A most intriguing question is why certain individuals respond with seve¬re clinical symptoms after infection with TBEV while the majority either remains asymptomatic or develops only mild disease. Studies of host genetic susceptibility to infectious diseases aim to in¬crea¬se our understanding of why some individuals are more susceptable than others. Knowledge of genetic susceptibility may be used in develope¬ment of new therapeutic means and also to recognize individuals who are at increased risk of severe symptoms if infected with a pathogen. The aim of this study – to establish the prevalence of the polymorphisms in CCR5 and TLR3 genes in TBE patients and their role in susceptibility to clinical TBE and disease severity. This study is the largest study on the host genetic risk factors predis¬posing to TBEV infection, and the first study of such kind performed in the pediatric population. The results of this study confirmed that a non¬func¬tional CCR5 protein and a functional TLR3 receptor are associated with the clinical expression... [to full text]

Thesis (MSc (Veterinary Science)--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (MSc (Production Animal Studies)--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 284-323.