SURVEY OF EHRLICHIA AND ANAPLASMA SPECIES IN WHITE TAILED DEER AND IN TICKS BY REAL-TIME RT-PCR/PCR AND DNA SEQUENCING ANALYSIS by CHAKRAVARTHY KATRAGADDA B.S., Andhra University, 2007 A THESIS Submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Diagonostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2011 Approved by: Major Professor Dr. Roman R. Ganta, M.S., Ph. D. Abstract Ehrlichia and Anaplasma species are rickettsial organisms which infect a variety of mammalian species. The organisms are transmitted from ticks and are maintained in reservoir hosts. Several pathogens have been identified in recent years as the causative agents for emerging infections in people. One of the primary reservoir hosts for the pathogens is the white tailed deer. In this study, 147 deer blood samples and 37 ticks were evaluated for the prevalence of Ehrlichia/Anaplasma species by TaqMan-based real time amplification assay and DNA sequence analysis. One hundred and thirteen (74%) samples tested positive with the Ehrlichia/Anaplasma genera-specific probe. Further analysis of the samples with the probes specific for human ehrlichiosis agents, E. chaffeensis and E. ewingii identified 4 (2.7%) and 7 (4.7%) positives, respectively. Test positives from 24 randomly selected samples were further evaluated by sequence analysis targeting to a 450 bp segment of 16S rRNA gene. All 24 samples were confirmed as positive for the Ehrlichia GA isolate # 4 (GenBank #U27104.1). DNAs from 37 pools of ticks collected from the white tailed deer were also evaluated. The TaqMan-based real time PCR assay with Anaplasma/Ehrlichia common probe identified 29 (78%) tick pools as positives whereas E. chaffeensis- and E. ewingii-specific probes identified three (8%) and one (3%) positives, respectively. The PCR and sequence analysis of tick samples identified Gram- negative bacteria species which included one endosymbiont of Rickettsia species (one tick pool), one Alcaligenes faecalis strain (three tick pools), five different Pseudomonas species (9 tick pools) and five different uncultured bacteria organisms (7 tick pools). Although the pathogenic potential of the white-tailed deer isolates of Anaplasma and Ehrlichia agents remains to be established, their high prevalence and the presence of human ehrlichiosis pathogens in white- tailed deer is similar to earlier findings. The high prevalence of the deer isolates of Anaplasma and Ehrlichia species demonstrates the need for further assessment of the pathogenic potential of these organisms to people and domestic animals. (This study was supported by the grant #AI070908 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.) TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………………………….v LIST OF TABLES………………………………………………………………………………..vi CHAPTER ONE: LITERATURE REVIEW ……………………………………………….…...1 CHAPTER TWO: Survey of Ehrlichia and Anaplasma species in white tailed deer and ticks by real-time RT-PCR/PCR and DNA sequencing analysis…………………………………………19 REFERENCES………………………………………………………………………………......76 iv LIST OF FIGURES 1. The general life cycle of ticks…………………………………………………………….7 2. Comparision between number of tested positive for E.chaffeensis, E.ewingii and Ehrlichia/Anaplasma species specific probes in deer blood samples……………………30 3. Comparision between number of tested positive for E.chaffeensis, E.ewingii and Ehrlichia/Anaplasma species specific probes in tick samples…………………………...36 4. Amplified PCR products of 450 bp fragment 16S rRNA gene of Ehrlichia /Anaplasma species of deer blood samples resolved on 1% agarose gel…………………...................38 5. Amplified PCR products of 450 bp fragment 16S rRNA gene of Ehrlichia /Anaplasma species of tick samples resolved on 1% agarose gel…………………………..…..……..40 6. The BLAST search results of two sequences derived from deer blood samples………...44 7. Alignment of Anaplasma sps. WTD 81 and Ehrlichia sps. GA isolate No. 4…………...47 8. The BLAST search results of all 20 sequences derived from tick samples……………...49 9. Fluorescence emission peaks diagrams of DNA sequence analysis……………………..70 v LIST OF TABLES 1. Few examples of vectors and the pathogens transmitted by them………………………..3 2. Few examples of ticks and diseases in Dogs and Cats………………………….………..8 3. List of some Ehrlichia and Anaplasma species and diseases…………………………….18 4. Primers and Probes used in the Real time PCR and RT-PCR and PCR and RT-PCR assays…………………………………………………………………………………..25 5. Ct-values given by E.chaffeensis, E.ewingii and Ehrlichia/Anaplasma species specific probes for deer blood samples…………………………………………………………...31 6. Ct-values given by E.chaffeensis, E.ewingii and Ehrlichia/Anaplasma species specific probes for tick samples…………………………………………………………………..37 7. List of the bacterial species identified by the tick sample sequences in BLAST search...69 vi CHAPTER ONE LITERATURE REVIEW 1 Vector-borne diseases The diseases which spread through the means of vectors are classified under vector- borne diseases (1). The vector-borne diseases are caused by viruses, bacteria, protozoa and helminth parasites. The name ‘vector’ is referred to an organism which transmit a pathogenic organism to a host (1). Arthropods are the most common disease vectors for vector borne diseases. Among arthropod vectors, mosquitoes and ticks are the most common vectors (1). The common process through which the vector-borne pathogens are transmitted is by blood feeding of vectors on their hosts (2). For example, mosquitoes are responsible for transmitting agents of diseases such as malaria, dengu fever, West Nile virus (WNV), while they feed on vertebrate hosts. Many vector-borne diseases are known to have a significant impact on the health of humans for over centuries (3). For example, plague which is caused by a Gram-negative bacterium, Yersinia pestis killed a millions of people over centuries (5). The infected rodents and rodent fleas help in transmission of this disease causing agent from one host to another. The rate of transmission of vector-borne disease agents to their hosts depends on three different factors: the pathologic agent; its arthropod vector and vertebrate host (4). 2 Table 1: Examples of pathogens and their vectors Pathogenic organism Vectors Virus West Nile Virus Mosquitoes Rift Valley Fever Virus Mosquitoes Yellow Fever virus Mosquitoes Dengue virus Mosquitoes Bacteria Rickettsia akari House mouse mite Rickettsia mooseri Body louse Rickettsia prowazokii Body louse Rickettsia rickettsia Tick Rickettsia typhi Fleas Rickettsia tsutsugamuchi Rat Mite Ehrlichia spp. Tick Yersinia pestis Fleas Borrelia burgdorferi Tick Borrelia recurrentisa Body louse Protozoan Babesia microti Tick Babesia divergens Tick Plasmodium falciparum Mosquitoes Plasmodium malariae Mosquitoes Plasmodium ovale Mosquitoes Plasmodium vivax Mosquitoes 3 Leishmania braziliensis Sandflies Leishmania chagasi Sandflies Leishmania donovani Sandflies Leishmania infantum Sandflies Leishmania major Sandflies Leishmania mexicana Sandflies Leishmania tropica Sandflies Trypanosoma cruzi Kissing Bugs Trypanosoma brucei gambiense Tsetse Flies Trypanosoma brucei Tsetse Flies rhodesiense Helminth Wuchereria bancrofti Mosquitoes Brugia malayi Mosquitoes Mansonella ozzardi Blackflies Onchocerca volvulus Blackflies Mansonella perstans Biting Midges Mansonella streptocerca Biting Midges Loa loa Tabanid Flies 4 The majority of vector-borne disease pathogens survive in nature in invertebrate vectors and their vertebrate hosts. Some intermediary animal hosts (generally wild animals) often serve as reservoir of infection. A vector acquires a pathogen from an infected host and transmits to another vertebrate host during its blood feeding cycle (2). Vector-borne disease pathogens depend on their hosts for their survival. Some of the pathogens undergo various modifications in different stages in their life cycle. In order to complete their life cycle, some vector-borne disease agents must be transmitted from one host to another. For example, Plasmodium species requires mosquito for sexual reproduction; the gametocytes will transform to gametes in mosquitoes and gametes form zygote. Zygotes then transform to sporozoites, which are transferred to vertebrate host during mosquito blood feeding on another host. There are many factors that determine the prevalence of vector-borne diseases. The abundance of vectors and reservoir hosts plays a major role in the prevalence of vector-borne diseases. Local environmental conditions, especially temperature and humidity may also influence the rate of prevalence of a pathogen in a given geographical area (4). The patterns of a vector-borne disease occurrence cannot be predicted (4). Many researches worked on various vector-borne diseases to develop drugs, vaccines and also to establish the preventive measures against them. The military activities performed due to interferences of various countries in many parts of the world may also contribute for the emergence or re-emergence of vector borne diseases (6). Since 1970s, the incidence of vector- borne diseases in the world is mostly increasing or maintaining at constant level, but certainly not declining (3). Some of the examples include malaria, dengu, yellow fever, louse-borne typhus, plague, leishmaniasis, sleeping sickness, West Nile encephalitis, Lyme disease, Japanese encephalitis, Rift Valley fever, and Crimean-Congo hemorrhagic fever (3). The reasons for the 5 increased