Point Prevalence Survey for Tick-Borne Pathogens in Military Working Dogs, Shelter Animals, and Pet Populations in Northern Colombia

Point Prevalence Survey for Tick-Borne Pathogens in Military Working Dogs, Shelter Animals, and Pet Populations in Northern Colombia M. E. McCown, DVM, MPH, DACVPM; A. Alleman, DVM, PhD, DABVP, DACVP; K. A. Sayler, PhD; R. Chandrashekar, PhD; B. Thatcher, BS; P. Tyrrell, BS; B. Stillman, PhD; M. Beall, DVM, PhD; A. F. Barbet, PhD ABSTRACT Background: Based on the high tick-borne pathogen results from a 2011 surveillance study in three Colombian cities, an in-depth point prevalence survey was conducted to determine the seroprevalence of tick-borne pathogens at a specific point in time in 70 working dogs, 101 shelter dogs, and 47 client-owned dogs in Barranquilla, Colombia. Results: Of the 218 serum samples, 163 (74%) were positive for Ehrlichia canis and 116 (53%) for Anaplasma platys. Exposure to tick-borne pathogens was highest in shelter and working dogs where more than 90% of the samples were seropositive or positive on polymerase chain reaction for one or more organisms as compared to 51% in client-owned animals. Conclusion: Surveillance for exposure to tickborne pathogens provides vital information necessary to protect and conserve the health of local humans and animals, deployed military service members, and working dogs in various parts of the world. This study and resultant data demonstrate the value of following a broadbased surveillance study with a more specific, focused analysis in an area of concern. This area s high levels of exposure warrant emphasis by medical planners and advisors on precautionary measures for military dogs, Special Operations Forces personnel, and the local public. Keywords: tick-borne pathogens, point prevalence, surveillance, US Military SOF, military working dogs, Colombia Introduction Vector-borne diseases (VBD), and more specifically tickborne diseases, continue to increase over time in global distribution and emergence 1 and are increasingly described as primary causes of zoonotic and infectious diseases. 2 Canine populations, susceptible to most of the tick-borne pathogens that readily infect humans, are documented reservoirs and sentinels for zoonotic and infectious diseases in many parts of the world. 3 10 Canine diseases caused by tick-borne pathogens include ehrlichiosis (Ehrlichia canis), Lyme disease (Borrelia burgdorferi), and anaplasmosis (Anaplasma spp.) and should be continually surveyed due to their veterinary and public health significance. The true value of ongoing, continual surveillance for these and other diseases is the resultant decreased risk of disease exposure and occupational and environmental hazards. 3,11 13 Additional value gained from surveillance studies is the disease prevalence rates in the local canine population, information about the local vectors present, and the risk of disease spread to the human population. This information is critical to medical planners and public health leadership for preserving the health of deployed Servicemembers and working dogs. In a previous study, we presented preliminary data regarding the seroprevalence of Ehrlichia spp., Anaplasma spp., B. burgdorferi, and Dirofilaria immitis (heartworm antigen prevalence) in dogs from three cities in Colombia. 10 The overall sample prevalence of heartworm and seroprevalence of B. burgdorferi in the feral, shelter, and pet populations of dogs was quite low, 1.6% and 0%, respectively. However, the seroprevalence of Ehrlichia spp. and Anaplasma spp. in the three cities was alarmingly high, at 62% and 33%, respectively. These data were collected using a point-of-care diagnostic test, IDEXX SNAP 4Dx Plus Test (IDEXX Laboratories Inc., Westbrook, ME, USA; https://www.idexx.com). While very sensitive in detecting antibodies to both Ehrlichia spp. and Anaplasma spp., peptides used in this assay contain shared antigens that can result in cross-reactivity between antibodies to E. canis and E. chaffeensis as well as A. phagocytophilum and A. platys. 14 16 In addition, while our previous study defined exposure to Ehrlichia spp. and Anaplasma spp., it did not indicate how many of the animals tested were actively infected (Figures 1 4). The objective of this work was to conduct a point prevalence survey in canines from Barranquilla, Colombia, to determine seroprevalence and provide molecular 53

Figure 1 Barranquilla working dog unit. Figure 2 Blood draw. evidence of tick-borne pathogens in three populations: working dogs, shelter dogs, and client-owned/pet dogs. For this study, point prevalence is defined as the proportion of the Barranquilla canine population surveyed that was positive for one or more tick-borne pathogens at a specific point in time: July 2012. In this study, we used species-specific peptide antigens to investigate the seroprevalence of specific Ehrlichia spp. (E. canis, E. chaffeensis, and E. ewingii) and specific Anaplasma spp. (A. phagocytophilum and A. platys) in military working dogs, shelter animals, and client-owned pets in and around the northern Colombian city of Barranquilla. We also used nucleic acid based molecular diagnostics to elucidate the prevalence of rickettsemia present in the sample population. Methods Figure 4 Ticks under a paw. Figure 3 Map of Colombia. Sample Population and Preparation Blood samples (n = 218) were collected from clientowned dogs presenting to local veterinary clinics (n = 47), animals housed in regional community shelters (n = 101), and from military working dogs (n = 70) assigned to the area for duty. Blood was collected via cephalic venipuncture directly into 2mL vacutainer tubes with no anticoagulant for harvesting serum and into 2mL vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Monoject ; Tyco, Mansfield, MA, USA; http://www.tyco.com/). All study protocols were approved through the University of Florida institutional animal care and use committee (protocol no. 201207460). Whole blood samples collected in EDTA were stored at 20 C, while blood collected without additives was immediately centrifuged; serum was harvested and was stored at 20 C. The samples were then transported under cold conditions and stored at 20 C in the laboratory of the investigators at the University of Florida, College of Veterinary Medicine for analysis. The samples were shipped in accordance with guidelines set forth in Permit no. 2012-05-164 from the Public Health Service at the US Centers for Disease Control and Prevention in Atlanta, GA, USA. Enzyme-Linked Immunosorbent Assay Serum samples were analyzed for the simultaneous detection of antibodies against five different tick-borne pathogens, Anaplasma phagocytophilum, A. platys, Borrelia burgdorferi, Ehrlichia canis, and E. chaffeensis using a research SNAP (IDEXX Laboratories, Inc., Westbrook, ME, USA; www.idexx.com) Multianalyte Prototype Test. 17 Briefly, three drops of patient serum was mixed with four drops of conjugate and applied to the flow matrix in an identical fashion as performed on the commercially available ELISA assay SNAP 4Dx Plus (IDEXX Laboratories, Inc., Westbrook, ME, USA; 54 Journal of Special Operations Medicine Volume 14, Edition 4/Winter 2014

www.idexx.com), according to manufacturer s recommendations. Peptide-specific antibody to A. phagocytophilum, A. platys, E. canis, E. chaffeensis, or B. burgdorferi present in the patient sample binds to the peptide-hrp conjugate. Immune complexes that form are bound to peptide conjugates adhered to the membrane on the flow matrix. Unbound antibodies and peptide-hrp conjugates were washed and substrate reagents were applied. Color development in the area of the deposition species-specific peptides and immune complexes indicated a positive result. DNA Extraction and Real-Time Polymerase Chain Reaction Amplification DNA was extracted from canine whole blood using a commercially available kit (High Pure PCR Template Preparation Kit, Roche Applied Science, Madison, WI, USA; lifescience.roche.com) according to the manufacturer s instructions. Genomic DNA was stored at 20 C until testing. Real-time polymerase chain reaction (PCR) hybridization probe assays were used that detect the disulfide oxidoreductase gene (GenBank AF403710) of E. canis and the p44 gene of A. platys. These two assays were used based on the high seroprevalence of exposure to these agents in the sample population. The real-time PCR assays were performed with the LightCycler 480 instrument (Roche Applied Science, Madison, WI, USA; lifescience.roche.com). PCR was carried out in a total reaction volume of 20μL containing LightCycler 480 Genotyping Master mix (Roche Applied Science, Madison, WI, USA; lifescience.roche.com), assay specific primers and probes, and 5μL of template DNA. Cycling parameters for the E. canis PCR consisted of a denaturation cycle of 95 C for 10 minutes, followed by a 55-cycle amplification profile (95 C for 20 seconds, 60 C for 30 seconds with a single data acquisition, 72 C for 20 seconds), a melting curve profile (95 C for 1 minute, 45 C for 1 minute, and 80 C continuous with a ramp rate of 0.14 C per second and four data acquisitions per C). The A. platys PCR cycling parameters were a denaturation cycle of 95 C for 10 minutes, followed by a 50-cycle amplification profile (95 C for 30 seconds, 58 C for 25 seconds with a single data acquisition, 72 C for 20 seconds), a melting curve profile (95 C for 1 minute, 45 C for 1 minute, and 80 C continuous with a ramp rate of 0.4 C per second and four data acquisitions per C), and a cool cycle of 40 C for 30 seconds. In each run, 105 and 102 copies of recombinant plasmids containing an insert of either the E. canis or A. platys amplicons, respectively, were tested as positive controls. PCR grade water (Roche Applied Science, Madison, WI, USA; lifescience.roche.com) was tested as the negative control. Analytical sensitivity was determined to be 10 gene copies using the assay-specific plasmids. Statistical Analysis Local prevalence for single disease status was calculated as the proportion of positive samples from the total of the samples tested. The odds ratios (ORs) for single diseases among the three populations were compared by logistic regression analysis using the PROC LOGISTIC function of the Statistical Analysis System (SAS version 9.2; SAS Institute Inc., Cary, NC, USA; http://www.sas.com/). Results Of the 218 serum samples tested, 62 (28.4%) had antibodies only to E. canis and 15 (6.9%) had antibodies only to A. platys. However, 101 (46.3%) samples had antibodies to both E. canis and A. platys. Therefore, the total number of E. canis positive samples was 163 (74.4%) and the total number of A. platys positive samples was 116 (53.2%). Of the 218 DNA samples, 46 (21.1%) were positive for E. canis DNA, 19 (8.7%) were positive for A. platys DNA and 16 (7.3%) were positive for both E. canis and A. platys DNA. Therefore, 62 samples (28.4%) were positive for the presence of E. canis DNA and 35 samples (16.1%) were positive for the presence of A. platys DNA. Of the 163 dogs seropositive for E. canis, 60 (36.8%) were also positive by PCR analysis. Two samples were PCR positive for E. canis but seronegative. Of the 116 dogs seropositive for A. platys, 26 (22.4%) were also positive by PCR analysis. Nine samples were PCR positive but seronegative. Of the 101 samples that were seropositive for both E. canis and A. platys, 10 (10.0%) were also PCR positive for both organisms. All 218 serum samples were seronegative for E. ewingii, E. chaffeensis, and B. burgdorferi. Two samples were seropositive for A. phagocytophilum, and both of these were PCR negative. These data are outlined in Table 1. Exposure to tick-borne pathogens was highest in shelter animals and military working dogs: More than 90% of the samples were seropositive or PCR positive for one or more organisms as compared to 51% in client-owned animals. Table 1 Number of Positive Samples to Antibody in Canine Blood Samples Collected in Barranquilla, Colombia Pathogen Antibody Positive/All Tested Dogs Point Prevalence, % At least one organism 186/218 85.3 Ehrlichia canis, total 163/218 74.4 Anaplasma platys, total 116/218 53.2 E. canis, alone 62/218 28.4 A. platys, alone 15/218 6.9 E. canis + A. platys, coinfection 101/218 46.3 Borrelia burgdorferi 0/218 0 Tick-Borne Pathogens in Dogs in Northern Colombia 55

The overall canine tick-borne pathogen point prevalence (infection with one or more tick-borne pathogens) in Barranquilla, Colombia, was 85.3% (186 of 218 samples). Comparing the three populations, 98% (99 of 101) of the canines from the regional community shelters, 90% (63 of 70) of the military working dogs, and 51% (24 of 47) of client-owned canines were seropositive and/or PCR positive for E. canis and/or A. platys (Table 2). Table 2 Number of Canines Seropositive and/or PCR Positive for Ehrlichia canis and/or Anaplasma platys Canine Population Seropositive and/ or PCR Positive/ All Tested Dogs Point Prevalence, % Shelter dogs 99/101 98 Military working dogs 63/70 90 Client-owned dogs 24/47 51 Note: PCR, polymerase chain reaction. Discussion The Atlántico Department of northern Colombia and its capital city, Barranquilla, was selected to provide a greater level of tick-borne surveillance data from this region of the country. Barranquilla (latitude: 10 53 N, longitude: 74 46 47 W, elevation: 98 feet [30m]) is Colombia s largest Caribbean coastal city and port located near the air base in Malambo, not far from the Venezuelan border. This region is home to one of the country s largest working-dog training sites and kennels. Colombian working dogs are trained at this site and deployed throughout the country for explosive detection and counter-narcotic duties. As Colombia s most populous city of the Caribbean Coast region and bolstering one of the country s highest population densities, Barranquilla is at sea level with a hot, humid, year-round tropical climate. Tropical humid climates provide an adequate environment for the presence of vectors such as ticks and mosquitoes. 11,18 In South America, tick vectors such as Rhipicephalus sanguineous (brown dog tick) and Ixodes spp. ticks have been documented. 13 The documented presence of these tick vectors is significant, as R. sanguineus ticks are primary vectors for E. canis and A. platys, and Ixodes spp. ticks are vectors for A. phagocytophilum and B. burgdorferi. 19 21 These favorable climatic conditions and vectors known to be present make VBD more likely. 22 Published findings from people and surveillance in dogs in South America reinforce this further. 12,23 26 Barranquilla was included for these and the other outlined reasons in the initial 2011 surveillance study of three Colombian cities. 3 The 2011 study determined Barranquilla to have the highest sample prevalence of Ehrlichia spp. and a high sample prevalence of Anaplasma spp. 10 A limitation of the 2011 study was that the IDEXX SNAP 4Dx Test was used, which could not differentiate between A. phagocytophilum and A. platys. In this study, we used the research SNAP Multianalyte Prototype Test 17 combined with advanced laboratory methods to differentiate the two Anaplasma species. These methods demonstrate that the Anaplasma spp. infections are primarily A. platys. The results from this current point prevalence survey expound upon the results from the earlier general surveillance study. These data elucidate the seroprevalence of specific species of tick-borne pathogens in this region of Colombia and, importantly, identify significant levels of bacteremia in this canine population. This indicates that these dogs serve as a substantial reservoir for these rickettsial agents and could serve as a source for increasing pathogen prevalence in tick vectors and elevating the risk of contracting vector-borne agents in people and dogs. Results concluded this to be most critical in dogs in kennel environments such as shelters or military working dog kennels. Tick-borne pathogens E. canis and A. platys are primarily associated with clinical disease in dogs. This high level of exposure along with the documentation of E. canis infection and infection with an A. platys-like organism in people in South America warrants precautionary measures for both dogs and people. Conclusion The results of this tick-borne pathogen, point prevalence survey reinforce the prior surveillance data demonstrating the presence of tick-borne pathogens in Barranquilla, Colombia. Findings such as these and others confirm the critical need for continual field surveillance for VBD present in animals. 27 These types of surveillance studies for tick-borne pathogens and the resultant data continually demonstrate the value of determining disease prevalence to develop risk assessments and implement timely preventive medicine and other control measures with the intent of preserving health. The two studies in Colombia, back-to-back and in conjunction with each other, substantiate the importance of conducting initial baseline surveillance studies in areas of concern and following up with more in-depth point prevalence surveys and advanced laboratory analyses of collected samples. Ultimately, the immediate importance to medical and Special Operations Forces (SOF) planners is the knowledge that this area s high levels of exposure warrant precautionary measures for military dogs, SOF personnel, and the local public. Disclosures The authors have nothing to disclose. 56 Journal of Special Operations Medicine Volume 14, Edition 4/Winter 2014

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E-mail: michael.mccown@us.army.mil. Dr Alleman is at the University of Florida, College of Veterinary Dr Sayler is at the University of Florida, College of Veterinary Dr Chandrashekar is at IDEXX Laboratories, Westbrook, Maine. Mr Thatcher is at IDEXX Laboratories, Westbrook, Maine. Ms Tyrrell is at IDEXX Laboratories, Westbrook, Maine. Dr Stillman is at IDEXX Laboratories, Westbrook, Maine. Dr Beall is at IDEXX Laboratories, Westbrook, Maine. Dr Barbet is at the University of Florida, College of Veterinary Tick-Borne Pathogens in Dogs in Northern Colombia 57