EPIDEMIOLOGY OF BARTONELLA INFECTIONS IN DOGS AND MOOSE: Animals as sentinels for human disease

................................................................................................................ 6 ABBREVIATIONS ...................................................................................................... 7 LIST OF ORIGINAL PUBLICATIONS .................................................................. 8 1. REVIEW OF THE LITERATURE ................................................................... 9-15 1.1 Arthropod borne diseases in dogs 1.2 Bartonella infection in dogs 1.3 Deer keds as possible vectors of Bartonella spp 1.4 Bartonella spp. infection in moose 2. AIMS OF THE STUDY ....................................................................................... 16 3. MATERIALS AND METHODS ........................................................................ 17-29 3.1. Survey of selected arthropod borne diseases in Finland 3.2. Diagnosis and clinicopathologic findings in dogs with Bartonella infection 3.3. Detection of Bartonella spp in deer ked pupae and winged adult keds 3.4. Bartonella infection in moose from Finland 3.5. Statistical analyses 4. RESULTS ............................................................................................................ 30-50 4.1. Survey of selected arthropod borne diseases in Finland 4.2. Diagnosis and clinicopathologic findings in dogs with Bartonella infection 4.3. Detection of Bartonella spp in deer ked pupae and winged adult keds 4.4. Bartonella infection in moose from Finland 5. DISCUSSION ....................................................................................................... 51-66 5.1 Survey of selected arthropod borne diseases in Finland 5.2. Diagnosis and clinicopathologic findings in dogs with Bartonella infection 5.3. Detection of Bartonella spp in deer ked pupae and winged adult keds 5.4. Bartonella infection in moose from Finland 6. CONCLUSIONS ................................................................................................. 67-68 7. FUTURE STUDIES ............................................................................................. 69 8. ACKNOWLEDGEMENTS ............................................................................... 70-71 9. REFERENCES ................................................................................................... 72-79 _________________________________________________________________________________________________ 5 ABSTRACT The incidence of arthropod-borne infections is increasing worldwide and Fennoscandia is no exception. In the last decades, infections transmitted by ticks are being diagnosed more frequently in people living in the Nordic countries. Ixodes ricinus, the sheep or castor bean tick, which is the most common tick in North-Western Europe, is widely distributed in Finland. Ixodes ticks are vectors of a broad spectrum of pathogens of medical and veterinary importance, such as Babesia spp., Borrelia spp., Anaplasma phagocytophilum (Ap), Bartonella spp., tick-borne encephalitis virus (TBEV), and Francisella tularensis. To date, there is limited information regarding the prevalence of many vector borne diseases in companion animals in Finland, and therefore the majority of available data come from human medicine studies. Infections caused by Bartonella species are considered an emerging zoonosis. One peculiarity of this genus of bacteria is its ability to cause long lasting bacteremia in reservoir hosts. Also, it appears that no other infectious agent is transmitted by more vectors. The deer ked, Lipoptena cervi, is an ectoparasite of moose (Alces alces), which carries Bartonella DNA. Deer keds, which are a nuisance for people, can occasionally bite humans and cause deer ked dermatitis. Whether or not the deer ked can successfully transmit bartonellae to ruminants or humans has not been determined. Because many of the arthropod-borne infections that affect dogs can cause serious disease in people, dogs are considered to be effective sentinel animals to assess the risk of human infection. Also, pets represent a large reservoir for human Bartonella infection because most of the species that infect them are zoonotic. The objective of the present research project was threefold: first, to establish the serological and molecular prevalence of selected tick borne diseases in a large group of dogs in Finland; second, to retrospectively compare different diagnostic approaches and clinicopathologic findings in dogs infected with Bartonella spp.; and third, to explore the role of the deer ked in the transmission of Bartonella spp. to Finnish moose. The serological results from dogs in this study indicate that Finnish dogs are exposed to at least one of four tested arthropod borne pathogens. Dogs were most frequently exposed to Ap (5.3%) followed by Bb (2.9%). Exposure rates were significantly higher in dogs living in Åland. No Finnish dog in this study was infected with Bartonella spp, based on PCR. Bartonella-infected dogs from the USA were most often infected with B. henselae, based on BAPGM enrichment PCR. Interestingly, for most of these dogs, no positive antibodies against Bartonella spp were detected. Clinicopathologic abnormalities in dogs with Bartonella infection were similar to those dogs suspected to have other vector-borne infection. The presence of Bartonella DNA (B. schoenbuchensis and B.bovis) was demonstrated in deer ked pupae samples and in one winged adult, which indicates transstadial transmission of this bacterium in the deer ked. The same Bartonella species were identified in blood samples from free ranging moose in Finland. Furthermore, a high prevalence of Bartonella infection was found in moose, which was significantly lowest in northern Lapland, a region considered deer-ked free. These findings further support the potential of L.cervi as vector of Bartonella. _________________________________________________________________________________________________ 6 ABBREVIATIONS Ap Anaplasma phagocytophilum Bb Borrelia burgdorferi Ec Ehrlichia canis ELISA Enzyme-linked immunosorbent assay TBEV Tick-borne encephalitis virus IFA Immunofluorescent antibody assay PCR Polymerase chain reaction Bvb Bartonella vinsonii subsp. berkhoffii Bh Bartonella henselae VBDDL Vector Borne Diseases Diagnostic Laboratory NCSU-CVM North Carolina State University, College of Veterinary Medicine BAPGM Bartonella alpha-Proteobacteria growth medium EDTA Ethylene diaminetetraacetic acid MRI Magnetic resonance imaging SPG Sucrose-phosphate glutamate PBS Phosphate buffered saline DPBS Dulbecco’s phosphate buffered saline rpoB beta subunit of the RNA polymerase gene OD Odds ratio CI Confidence Intervals Bk Bartonella koehlerae Bvl Bartonella volans-like _________________________________________________________________________________________________ 7 LIST OF ORIGINAL PUBLICATIONS I. C. Pérez Vera, S.Kapiainen, S.Junnikkala, K.Aaltonen, T.Spillmann and Olli Vapalahti. Survey of selected tick-borne diseases in dogs in Finland. Parasites and Vectors, 2014 Jun 23;7(1):285. II. C. Pérez, R. G. Maggi, P. P. V. P. Diniz, and E. B. Breitschwerdt. Molecular and Serological Diagnosis of Bartonella Infection in 61 dogs from the USA. Journal of Veterinary Internal Medicine 2011 Jul-Aug; 25(4): 805-10 III. C. Pérez Vera, P. P. V. P. Diniz, Elizabeth L. Pultorak, R. G. Maggi, and E. B. Breitschwerdt. An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection. Comparative Immunology and Microbiology of Infectious Diseases. 2013 Sep; 36(5):481-7. IV. E.M. Korhonen*, C. Pérez Vera*, A.T. Pulliainen, T. Sironen, K. Aaltonen, R. Kortet, L. Härkönen, S. Härkönen, T. Paakkonen, P. Nieminen, A-M. Mustonen, H. Ylönen and O. Vapalahti. Molecular detection of Bartonella spp. in deer ked pupae, adult keds and moose blood in Finland. Epidemiology and Infection 2014 Jun 5:1-8. *both authors contributed equally V. C. Pérez Vera*, K. Aaltonen, T. Spillmann, O. Vapalahti and T. Sironen. Geographic distribution and molecular diversity of Bartonella infections in moose, Alces alces, in Finland. Accepted for publication to Journal of Wildlife Diseases,