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Active surveillance of Anaplasma marginale in populations of arthropod vectors (Acari: Ixodidae; Diptera: Tabanidae) during and after an outbreak of bovine anaplasmosis in southern Manitoba, Canada Matthew E.M. Yunik, Terry D. Galloway, L. Robbin Lindsay

Abstract Bovine anaplasmosis is the disease caused by the bacterium Anaplasma marginale. It can cause production loss and death in cattle and bison. This was a reportable disease in Canada until April 2014. Before then, infected herds were quarantined and culled, removing infected animals. In North America, A. marginale is biologically vectored by hard ticks (Acari: Ixodidae), Dermacentor variabilis and D. andersoni. Biting flies, particularly horse flies (Diptera: Tabanidae), can also act as mechanical vectors. An outbreak of bovine anaplasmosis, consisting of 14 herds, was detected in southern Manitoba in 2008. This outbreak lasted multiple rounds of testing and culling before eradication in 2011, suggesting local maintenance of the pathogen was occurring. We applied novel approaches to examine the vector ecology of this disease in this region. We did not detect A. marginale by screening of 2056 D. variabilis (2011 and 2012) and 520 horse flies (2011) using polymerase chain reaction (PCR).

Résumé L’anaplasmose bovine est une maladie causée par la bactérie Anaplasma marginale. Elle peut être responsable pour la perte de production et entrainer la mort du bétail et des bisons. La maladie devait être obligatoirement déclarée jusqu’en avril 2014. Avant cette date, les troupeaux infectés étaient mis en quarantaine et abattus pour éliminer les animaux contaminés. En Amérique du Nord, A. marginale est transmise par des vecteurs biologiques; les tiques dures (Acari: Ixodidae), Dermacentor variabilis et D. andersoni. Les mouches piquantes, en particulier les mouches à cheval (Diptera: Tabanidae), peuvent aussi agir comme vecteurs mécaniques. Une épidémie d’anaplasmose bovine touchant 14 troupeaux, a été détectée au Sud du Manitoba en 2008. Suite à une série de tests et d’abattages, cette épidémie fut éradiquée en 2011, suggérant qu’il se produisait localement un entretien du pathogène. Nous avons appliqué de nouvelles approches pour examiner l’écologie du vecteur de cette maladie dans cette région. Lors des tests de dépistage par réaction en chaîne pas polymérase (PCR), nous n’avons pas détecté A. marginale sur 2056 D. variabilis (2011 et 2012) et 520 mouches du cheval (2011). (Traduit par Dre Florence Huby-Chilton)

The Gram-negative bacterium, Anaplasma marginale, is the causative agent of bovine anaplasmosis, a disease that affects ruminants worldwide (1). Anaplasma marginale is an obligate intra-erythrocytic bacterium that chronically infects wildlife, including: bison (Bison bison), some cervid species including elk (Cervus canadensis), whitetailed deer (Odocoileus virginianus), and mule deer (O. hemionus). Infections in wildlife may not cause any measurable level of morbidity or reach parasitemia levels that allow them to serve as reservoirs for the pathogen (1). Clinical signs can develop in cattle including anemia, splenomegaly, enlargement of the gallbladder, icterus, reduced weight gain, abortion, and in severe cases, death (2). Animals that survive acute infections of A. marginale may become chronically infected (1). All species of Anaplasma typically use a tick as a biological vector, undergoing a migration and maturation in the tick before becoming infectious to the definitive vertebrate host (1). Ticks of the genus Dermacentor are the main biological vectors of A. marginale in North America (3). In Canada, the 2 key tick vectors are the Rocky Mountain wood tick, D. andersoni, which ranges from British Columbia to south-central Saskatchewan, and the American dog tick,

D. variabilis (3). The range of D. variabilis extends from Saskatchewan east to Nova Scotia and is found throughout the southern portion of Manitoba, often in high densities (4). Adult male D. variabilis and D. andersoni are known to take multiple blood meals and even feed on multiple hosts (5). Anaplasma marginale may also be transmitted by other means. Vertical transmission can occur between an infected cow and naïve calf through trans-placental infection or through blood contact during the birthing process (6). Additionally, the infection can be spread mechanically by contaminated veterinary tools, an important mode of transmission in some regions (7). Under some circumstances, biting flies, specifically deer flies and horse flies (Diptera: Tabanidae), may serve as mechanical vectors, transferring infected blood cells on their mouthparts between animals with varying degrees of success (8,9). Anaplasma marginale is of economic importance in Canada, and has resulted in trade restrictions being imposed on Canadian cattle for export (1,10). Bovine anaplasmosis was a reportable disease in Canada until 2014 and, under the Health of Animals Act, required producers with infected herds to undertake a quarantine, test, and cull

Department of Entomology, University of Manitoba, Winnipeg, Manitoba R3T 2N2 (Yunik, Galloway); Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba R3E 3R2 (Lindsay). Address all correspondence to Matthew E.M. Yunik; telephone: 204-474-9257; fax: 204-474-7628; e-mail: [email protected] 2016;80:171–174

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program until there were no longer infected animals in the region (10,11). Vaccines are not currently available in North America and no antibiotics are currently licensed for use in Canada. The Canadian Food Inspection Agency (CFIA) conducted a Bovine Serological Survey (BSS) every 3 to 5 y to affirm that the national herd remained free of A. marginale as well as other pathogens. In 2007–2008, the BSS revealed that the prevalence of infection in the Canadian herd was higher than the acceptable 0.02% for A. marginale (11). A portion of the positive samples was traced to Manitoba. Enhanced surveillance efforts led to the detection of animals infected with A. marginale from herds belonging to 13 beef producers and 1 bison producer. A quarantine, test, and cull program was initiated in 2009 and concluded in the summer of 2011 (11). From a Canadian perspective, this outbreak was unique because infected animals were detected during multiple summers, requiring multiple rounds of culling to end local transmission. At the initiation of the program, producers who participated in the study had a cumulative beef cattle herd size of approximately 1278 animals (range of head per farm: 40 to 373), 15% of which were culled after the first round of testing by the CFIA. Prevalence of A. marginale infection ranged from 2.7% to 32.0% per herd. In the spring of 2011, 7 additional animals were culled from a herd of 115, while one additional animal was culled in the summer from a herd of approximately 30. These data are based on results from a questionnaire distributed by the CFIA to participating producers in 2011. The exact role that arthropods, especially ticks, play in the epidemiology of bovine anaplasmosis is understudied (1). Certain strains of A. marginale have varying ability to infect vertebrate and arthropod hosts (12–14). Additionally, the behavior, physiology, and life history of the tick vectors can vary throughout their geographic range (15). Only recently have we had the molecular tools to conduct large-scale, active surveillance of arthropod vectors in a pastoral setting, which has been suggested as a key element to understanding the ecology of bovine anaplasmosis. The objective of this study was to assess what role, if any, ticks and horse flies had in the potential maintenance A. marginale in the outbreak region in Manitoba. In April 2011, we contacted CFIA personnel who were the primary responding veterinarians responsible for the control of the anaplasmosis outbreak in southeastern Manitoba. Staff from the CFIA provided essential historical background on the presence of A. marginale in the region over previous years and initiated contact with producers who owned the pastures in which infected animals had been detected. Of those contacted, 9 producers participated in the study. Ticks were collected by drag sampling using a piece of white flannel (1.3 3 0.70 m), spread by a plastic spar, dragged by researchers at a leisurely pace through the pastures. Tick dragging was conducted on non-rainy days when the air temperature was above 10°C; after snow had melted in April until August when questing ticks were no longer present. The flannel, along with researchers’ clothes, was examined for ticks approximately every 10 m. Collected ticks were then placed in a 56-mL plastic vial with a perforated snap-top lid. Upon leaving the pasture, the date and location of the collection were recorded, and the vials were placed in a plastic bag with a moistened paper towel, and deposited in an insulated container for transport. The time allotted for drag sampling the over 18 km2 of pasture, distributed among the 9 par-

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ticipating producers, was based on suitability of tick habitat, recent herd infection history, including number of animals culled per herd and pasture utilization, and producer interest. At least 100 ticks were collected from land owned by each producer involved in the study. Once returned to the lab, ticks were identified based on morphological characteristics to be D. variabilis before being surface-­ sterilized in 4 different solutions, and grouped according to collection site. The first solution consisted of one drop of Tween 80 per 10 mL of 0.5% bleach. The second solution was 0.5% benzalkonium chloride. The third solution was 70% ethanol. The final solution was filtered water. All ticks collected from the same pasture on the same day were placed in 50 mL centrifuge tubes and approximately 45 mL of the first solution was added. The tubes were then sealed and slowly and repeatedly inverted for 3 min. The solution was decanted and the next solution added. This process was repeated for all solutions. After decanting the filtered water, the ticks and centrifuge tube were dried with a paper towel; the ticks were replaced into the tube and frozen at 280°C for storage. Horse flies were collected using a Manitoba Horse Fly Trap (16). In 2011, a trap was operated for 4 days from 9 a.m. to 8 p.m. in the first week of June when fly populations appeared to be at their highest. The trap was placed for the first 2 d on a pasture where fly intensity appeared to be the highest and where previously infected animals had been maintained in 2009–2010. On subsequent days, the trap was placed in close proximity to the herd that contained a cow which had an active A. marginale infection approximately one month prior. Once removed from the field, flies were killed by being placed in a freezer at 25°C for approximately 20 min before being transferred to a sterile plastic bag and placed in a freezer at 280°C. Ticks were removed from storage at 280°C and sorted by location and date collected, as well as gender, and placed into pools of no more than 5. Each tick was then cut in half sagittally using a scalpel on a sterile Petri dish in a biosafety cabinet. The scalpel was disinfected between ticks by washing in 10% chlorine bleach solution followed by a rinse in 90% ethanol. Half of each tick was frozen individually in a 2 mL microtube (Sarstedt AG and CO, Newton, North Carolina, USA), while the other half remained in a designated pool. This ensured that individual positive ticks found in pools which tested positive for the presence of A. marginale could be identified. The ticks in each pool were then cut further into smaller pieces using a sterile scalpel to enhance DNA extraction. Extraction of DNA from the pools was conducted using DNAeasy Blood and Tissue Kits (Qiagen, Austin, Texas, USA) following the blood and tissue protocol. All extracts were then stored in a freezer at 280°C. Extractions were screened for A. marginale DNA using real-time polymerase chain reaction (RT-PCR) with primers and a probe that had been successfully used in previous studies targeting the 16S rRNA gene (17). The RT-PCR was conducted using 96-well fast plates on a VIIATM 7 RT-PCR system (Applied Biosystems, Foster City, California, USA). The thermocycling regime consisted of an activation stage lasting for 2 min at 50°C, an initial denaturation lasting 10 min at 95°C, and 40 cycles of 95°C, and 60°C lasting for 15 s and 1 min, respectively. Each reaction contained 12.5 mL of TaqMan Universal Master Mix (2X) (Applied Biosystems), forward and reverse primers at a concentration of 0.667 mM each, a probe at a

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concentration of 0.125 mM, and 5 mL of DNA extract. The remaining volume consisted of nuclease-free water. An isolate of A. marginale from an infected cow from the Manitoba outbreak in 2008 was used as a positive control. No template controls were also used. Horse flies are potential mechanical vectors; therefore, only the mouthparts were tested for A. marginale as bacteria in the remaining part of the fly body should not be transmittable. Flies were sorted by the date collected and to the lowest taxonomic level possible. Mouthparts were removed from the flies and pooled in groups of no more than 5, keeping each trap date and taxonomic group separate. The pools of mouthparts were subjected to the same DNA extraction and PCR protocols that were used to screen the ticks for A. marginale. In total, 1013 (487 males; 526 females forming 217 pools) and 1043 (493 males; 550 females forming 224 pools) D. variabilis were collected in 2011 and 2012, respectively. A total of 520 horse flies collected in 2011 were placed into 110 pools for testing. The total collection consisted of 2 genera: Tabanus (n = 116) and Hybomitra (n = 404). Tabanus similis was the most abundant species of the genus Tabanus; however, 3 T. vivax were also collected. There were 6 species of Hybomitra identified, H. illota (n = 61) and H. pechumani (n = 10). In order to minimize the amount of time between collection and testing for A. marginale, the remaining 4 Hybomitra species (n = 333) were not sorted to species level. However, H. nuda, H. lasiophthalma, H. frontalis, and H. epistates were all present in the samples. Because of the success of the CFIA’s quarantine, test, and cull program, the probability of collecting infectious horse flies in the summer of 2012 was seen as negligible; therefore, no flies were collected. The potential for A. marginale to be harbored in overwintering adult ticks from one vector season to the next (18) or in potential wild reservoir hosts justified collecting ticks in 2012. All ticks and flies tested were negative for A. marginale; positive controls always tested positive while no template controls always tested negative. As part of another study, a proportion of tick extracts were positive for the presence of a related tick-associated bacterium, Rickettsia montanensis, confirming that our DNA extractions were successful (19). The producers involved were informed of the results as they became available. There are numerous factors that may have been responsible for our failure to detect A. marginale. The most likely factor was the success of the quarantine, test, and cull program that the CFIA enacted in 2009 and concluded in 2011, when all herds that had been infected, and neighboring herds, were deemed free of anaplasmosis. This success by the CFIA dramatically reduced the probability that A. marginale would be detected in the arthropod populations, particularly the biting flies. Vector competency might have also been an issue. Although Dermacentor ticks collected in Canada have been experimentally shown to transmit A. marginale (13) some strains are not vectored by ticks (12,15). Finally, the lack of detection could also be caused by the absence of infected wild reservoirs that were previously in the area. Although the exact role wild reservoirs play in the maintenance of bovine anaplasmosis is not well-understood, O. virginianus and C. canadensis are present in southeastern Manitoba and are known to come in close proximity to livestock, providing a potential spatial link for pathogen transmission. These animals may also frequently cross the international border into Minnesota where anaplasmosis is classified as an endemic, non-regulated pathogen

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(11). However, the herd of C. canadensis that routinely migrates between Minnesota and the outbreak region in Manitoba has been monitored for A. marginale in the past. On the basis of serological tests, the herd has been considered free of anaplasmosis since at least 2004 (20). Although the results of this study were negative there are many aspects of this work that are novel and noteworthy. This was the first large-scale study to use field-collected ticks and flies from pastoral settings to examine their potential role in the transmission of A. marginale in North America. This was accomplished through the use of molecular techniques not previously applied to such specimens. The use of fly mouthparts as opposed to the whole fly, demonstrating the potential role of the fly as a mechanical vector, is also very important. In a preliminary study, whole horse fly heads were used for DNA extraction. A component of the fly’s large compound eyes inhibited conventional and real time PCR. This is also the first time the Manitoba Horse Fly Trap has been used for surveillance of A. marginale. This trap has been used to evaluate fly population composition and density, and to reduce fly pressure (16). Using this trap to study the epidemiology of a mechanically vectored disease agent spread by tabanids may provide data that are more reflective of what is occurring in nature rather than other methods, such as sweep netting near the cattle, because only unfed or partially fed host seeking flies are caught in the trap. Finally this work demonstrates the effectiveness of the CFIA’s former control measure for managing outbreaks of bovine anaplasmosis in areas where potential vectors are present.

Acknowledgments Antonia Dibernardo of the Public Health Agency of Canada at the National Microbiology Laboratory, Winnipeg, provided training on laboratory techniques. Diana Dunlop assisted with fieldwork in 2012. Manitoba Conservation provided research permits for collecting ticks from crown land. Dr. Alvin Gajadhar of the CFIA, Saskatoon, Saskatchewan, provided the A. marginale isolate that served as the positive control. Staff from the CFIA in Winnipeg, Dr. Lynn Bates, Dr. Krista Howden, and those at the Steinbach district office, including Drs. Max Popp and Jolene Byers, offered insight and support. Dr. Wayne Tomlinson, Dr. Glen Dizer, and Peter Veldhuis, Manitoba Agriculture, Food and Rural Initiatives, provided advice and support. Drs. Kateryn Rochon and Kim Ominsky, University of Manitoba, offered their expertise. Dr. Neil Chilton, University of Saskatchewan provided feedback on the manuscript. This research was funded by Growing Forward, a program jointly funded by Manitoba Agriculture Food and Rural Initiatives, and Agriculture and Agri-Food Canada. Additional and special thanks go to the producers who allowed access to their pastures and offered firsthand experience of the Anaplasma problem in southeastern Manitoba.

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  2. Jones EW, Brock WE. Bovine anaplasmosis — Its diagnosis, treatment and control. J Am Vet Med Assoc 1966;149:1624–1633.   3. Kocan KM, Hair JA, Ewing SA, Stratton LG. Transmission of Anaplasma marginale Theiler by Dermacentor andersoni Stiles and Dermacentor variabilis (Say). Am J Vet Res 1981;42:15–18.   4. Dergousoff SJ, Galloway TD, Lindsay LR, Currry PS, Chilton NB. Range expansion of Dermacentor variabilis and Dermacentor andersoni (Acari: Ixodidae) near their northern distributional limits. J Med Entmol 2013;50:510–520.   5. Lysyk TJ. Movement of male Dermacentor andersoni (Acari: Ixodidae) among cattle. J Med Entomol 2013;50:977–985.   6. Grate HEG, da Cunha NA, Pappens FG, Farias NAD. Trans­ placental transmission of Anaplasma marginale in beef cattle chronically infected in southern Brazil. Rev Bras Parasitol Vet 2013;22:189–193.   7. Reeves JD, Swift BL. Iatrogenic transmission of Anaplasma marginale in beef cattle. Vet Med Small Anim Clin 1977;72:911–914.   8. Foil LD. Tabanids as vectors of disease agents. Parasitol Today 1989;5:88–96.   9. Scoles GA, Broce AB, Lysyk TJ, Palmer GH. Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae). J Med Entomol 2005;42:668–675. 10. Aubry P, Geale DW. A review of bovine anaplasmosis. Transbound Emerg Dis 2011;58:1–30. 11. Howden KJ, Geale DW, Paré J, Golsteyn-Thomas EJ, Gajadhar AA. An update on bovine anaplasmosis (Anaplasma marginale) in Canada. Can Vet J 2010;51:837–840. 12. Scoles GA, McElwain TF, Rurangirwa FR, Knowles DP, Lysyk TJ. A Canadian bison isolate of Anaplasma marginale (Rickettsiales: Anaplasmataceae) is not transmissible by Dermacentor andersoni (Acari: Ixodidae), whereas ticks from two Canadian D. andersoni populations are competent vectors of a U.S. strain. J Med Entomol 2006;43:971–975.

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13. Lankester MW, Scandrett WB, Golsteyn-Thomas EJ, Chilton NC, Gajadhar AA. Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada. Can J Vet Res 2007;71:271–277. 14. Kocan KM, de La Fuente J, Blouin EF, Garcia-Garcia JC. Anaplasma marginale (Rickettsiales: Anaplasmataceae): Recent advances in defining host-pathogen adaptations of a tick-borne rickettsia. Parasitology 2004;129:S285–S300. 15. Scoles GA, Ueti MW, Palmer GH. Variation among geographically separated populations of Dermacentor andersoni (Acari: Ixodidae) in midgut susceptibility to Anaplasma marginale (Rickettsiales: Anaplasmataceae). J Med Entomol 2005;42: 153–162. 16. Thorsteinson AJ, Hanec W, Bracken GK. The Manitoba horse fly trap. Can Entomol 1964;96:166. 17. Reinbold JB, Coetzee JF, Sirigireddy KR, Ganta RR. Detection of Anaplasma marginale and A. phagocytophilum in bovine peripheral blood samples by duplex real-time reverse transcriptase PCR assay. J Clin Microbiol 2010;48:2424–2432. 18. Yunik MEM, Galloway TD, Lindsay LR. Ability of unfed Dermacentor variabilis (Acari: Ixodidae) to survive a second winter as adults in Manitoba, Canada, near the northern limit of their range. J Med Entomol 2015;52:138–142. 19. Yunik MEM, Galloway TD, Lindsay LR. Assessment of prevalence and distribution of spotted fever group rickettsiae in Manitoba, Canada, in the American dog tick, Dermacentor variabilis (Acari: Ixodidae). Vector Borne Zoonotic Dis 2015;15:103–108. 20. Hildebrand E, Carstensen M, Butler E, Cornicelli L. Preliminary results of herd health assessment for northwestern free-ranging elk from 2004–2009. Minnesota Department of Natural Resources Summaries of Wildlife Research Findings 2010;2009:135–149.

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Active surveillance of Anaplasma marginale in populations of arthropod vectors (Acari: Ixodidae; Diptera: Tabanidae) during and after an outbreak of bovine anaplasmosis in southern Manitoba, Canada.

L’anaplasmose bovine est une maladie causée par la bactérie Anaplasma marginale. Elle peut être responsable pour la perte de production et entrainer l...
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