Canine vector-borne disease in domestic dogs on Isla Santa Cruz, Galápagos

Journal Pre-proof Canine vector-borne disease in domestic dogs on Isla Santa Cruz, Galápagos

Isabel Angelica Jimenez, Patricio Alejandro Vega Mariño, G. Sean Stapleton, Jennifer Battista Prieto, Dwight Douglas Bowman PII:

S2405-9390(19)30227-8

DOI:

https://doi.org/10.1016/j.vprsr.2020.100373

Reference:

VPRSR 100373

To appear in:

Veterinary Parasitology: Regional Studies and Reports

Received date:

27 August 2019

Revised date:

13 January 2020

Accepted date:

16 January 2020

Please cite this article as: I.A. Jimenez, P.A.V. Mariño, G.S. Stapleton, et al., Canine vector-borne disease in domestic dogs on Isla Santa Cruz, Galápagos, Veterinary Parasitology: Regional Studies and Reports(2019), https://doi.org/10.1016/ j.vprsr.2020.100373

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© 2019 Published by Elsevier.

Journal Pre-proof Canine vector-borne disease in domestic dogs on Isla Santa Cruz, Galápagos Isabel Angelica Jimenez, DVMa,b,* [email protected], Patricio Alejandro Vega Mariñoc, G. Sean Stapletona, Jennifer Battista Prieto, DVM, PhDa, Dwight Douglas Bowman, MS, PhDa a

Cornell University, College of Veterinary Medicine, 602 Tower Road, Ithaca, NY 14850

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Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos,

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Animal Medical Center, 510 E. 62nd Street, New York, NY 10065 (present address)

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Avenida Baltra, Puerto Ayora, Santa Cruz, Galápagos, 200102 *

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Corresponding author.

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ABSTRACT

Vector-borne diseases result in significant morbidity and mortality in domestic dogs in

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tropical and subtropical regions and also pose a potential threat to wildlife species and

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humans. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis (CME), has a high reported seroprevalence in dogs on Santa Cruz in the Galápagos Islands,

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Ecuador. Veterinary diagnostic and treatment resources are often scarce and clinical

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follow-up is lacking in the Galápagos. This study evaluated 58 dogs presenting to the Darwin Animal Doctors clinic in the city of Puerto Ayora on Santa Cruz Island during August of 2018. The seroprevalence of E. canis/E. ewingii (48.3%), Anaplasma phagocytophilum/A. platys (12.1%), and Borrelia burgdorferi (0%), as well as the proportion of dogs actively infected with E. canis (12.1%) and E. ewingii (0%), are reported. Active infection was defined as the identification of antigen by PCR. Dogs with a packed cell volume (PCV) ≤ 30% had a 10-fold risk of active infection with E. canis compared to dogs with a PCV ≥ 31% (p = 0.0124). A PCV cutoff of 30% may be a useful

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Journal Pre-proof screening tool for active E. canis infection in regions with high Ehrlichia seroprevalence, in the absence of other apparent causes of anemia. Dirofilaria immitis antigen was present in 6.9% of examined dogs, with the highest prevalence in the barrio Las Ninfas. PCR and Sanger sequencing were used to provide the first molecular identification of D. immitis in the Galápagos. This study updates the seropositivity and prevalence data of these canine vector-borne pathogens and highlights the need for continued surveillance in

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the region.

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Key words: Dirofilaria immitis; Ehrlichia canis; heartworm; mosquito; Rhipicephalus

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sanguineus; tick

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1. INTRODUCTION

The Galápagos Islands, part of the Republic of Ecuador, are located in the Pacific

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Ocean west of the mainland. The city Puerto Ayora on Santa Cruz Island is home to the

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majority of the archipelago’s inhabitants (DPNG, 2014; Toral-Granda et al., 2017). The Galápagos Islands contain one of the highest densities of plant and animal biodiversity in

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the world (Porter, 1984; Phillips et al. 2012). Native species are those that naturally

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inhabit a region without anthropogenic introduction, while endemic species are native species that are unique to the Galápagos. Introduced species have been subject to intentional or accidental introduction to a region as a result of anthropogenic intervention. Introduced species threaten the survival of native and endemic species through predation, competition, habitat destruction, and infectious disease transmission (Barnett, 1985; Van’t Woudt et al., 1990; Causton et al., 2006; Phillips et al., 2012). Domestic dogs (Canis lupus familiaris) and cats (Felis catus) have established feral populations on multiple islands in the Galápagos (Barnett and Rudd, 1983; Barnett and Salmon, 1986).

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Journal Pre-proof In 2016, the canine population of Santa Cruz Island was estimated at 4,200 dogs (Rousseaud et al., 2017). Pet dogs often live outdoors or are allowed to roam freely, receive little to no veterinary care, and infrequently or inconsistently have ectoparasite preventive medication (Gingrich et al., 2010, Adams et al., 2016). Pets are therefore poised to contact arthropod vectors of disease, serve as a reservoir of infection in proximity to wildlife and humans, and further contribute to feral populations.

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Transportation of domestic dogs to or within the Galápagos archipelago and the

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use of canine vaccines have been prohibited since 1999, effectively creating a closed and

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susceptible population of domestic dogs (SICGAL, 1999, Rousseaud et al., 2017).

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Despite these regulations, illegal transport of pet dogs to the Galápagos is suspected to frequently occur (Karez et al., 2006). The Agency for the Regulation and Control of

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Biosecurity and Quarantine for Galápagos (ABG; Agencia de Regulación y Control de la

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Bioseguridad y Cuarentena para Galápagos) manages the threat of invasive species (Rousseaud et al., 2017). In the organization’s first 3 years, 2,149 pet cats and dogs were

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sterilized in collaboration with organizations including Darwin Animal Doctors (DAD)

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(Rousseaud et al., 2017). DAD is a non-profit organization providing free veterinary primary care and emergency services to the city of Puerto Ayora. In the past 30 years, only four peer-reviewed studies surveying two islands have characterized the prevalence of canine vector-borne pathogens in the Galápagos; these pathogens are summarized in Table 1 (Levy et al., 2008; Gingrich et al., 2010; Adams et al., 2016; Diaz et al., 2016). The most common canine vector-borne pathogen on Santa Cruz Island is canine ehrlichiosis (Adams et al., 2016; Diaz et al., 2016).

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Journal Pre-proof The genus Ehrlichia comprises gram-negative obligate intracellular rickettsial bacteria that infect mammalian leukocytes. In domestic dogs, E. canis causes canine monocytic ehrlichiosis (CME) and E. ewingii causes canine granulocytic ehrlichiosis (CGE) (Anderson et al., 1992; Harrus et al., 1997b). Both agents can infect humans (Buller et al., 1999; Ismail et al., 2010). Acutely infected dogs present with lethargy, inappetence, fever, and weight loss. Mild non-regenerative anemia and thrombocytopenia

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are common findings and may lead to hemorrhage (Stockham et al., 1992; Neer et al.,

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2002; Bulla et al., 2004). Recovery without treatment occurs in a subset of individuals.

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The risk factors for the development of subclinical infection have not been well-

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characterized (Waner et al., 1997). Subclinically infected dogs undergo chronic antigenic stimulation with an ineffective immune response and may then clear the infection or

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progress to chronic disease (Reardon and Pierce, 1981; Mylonakis et al., 2004; Harrus

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and Waner, 2011). Dogs with chronic disease have a poor prognosis for survival, particularly without aggressive treatment (Harrus et al., 1997b).

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A. phagocytophilum infects neutrophils and eosinophils, causing canine

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granulocytic anaplasmosis (CGA) (Dumler et al., 2005; Kohn et al., 2008). Affected dogs present with fever, anorexia, weight loss, and polyarthropathy (Eberts et al., 2011). A. platys infects platelets and causes canine infectious cyclic thrombocytopenia (Harrus et al., 1997a; Harvey et al., 1978). Clinical signs are often mild and non-specific, including lethargy and generalized lymphadenopathy, and do not typically include petechiae or ecchymoses (Baker et al., 1987; Harrus et al., 1997b; Harvey et al., 1978). The canine heartworm (D. immitis) is a parasitic filarial nematode transmitted by mosquitoes, for which the domestic dog is the definitive host, although patent infection

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Journal Pre-proof has been documented in other mammals (Kazacos and Edberg, 1979; Barrett et al., 2004; Sacks et al., 2004; Alho et al., 2017). Adult worms reside in the pulmonary arteries (American Heartworm Society, 2018). The most common clinical signs in dogs are coughing and exercise intolerance, but many dogs are subclinically infected (American Heartworm Society, 2018). Diagnosis of vector-borne disease typically relies on a combination of patient

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history and risk factors, clinical signs, and serological or molecular testing. The SNAP

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4Dx Plus test (Idexx Laboratories, Totowa, NJ 07512, USA) is a commercial qualitative

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ELISA test for antibodies against E. canis/E. ewingii, A. platys/A. phagocytophilum, and

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Borrelia burgdorferi, and D. immitis antigen, and is often utilized as a screening tool prior to advanced diagnostics. The persistence of antibodies following clearance of

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infection and the ability of E. canis to cause subclinical infection complicates

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differentiation of prior exposure from active infection in seropositive but clinically healthy animals. In the Galápagos, presumptive diagnosis of canine ehrlichiosis and

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anaplasmosis is often made based on clinical findings and response to treatment.

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Serologic testing is only sporadically available; from January to July 2018, only two SNAP 4Dx Plus tests were used at the DAD clinic on Santa Cruz Island (J. Keulen, pers. comm.). No studies have utilized PCR to evaluate the presence of canine vectorborne pathogens on Santa Cruz Island. The objective of this study was to implement ELISA and PCR testing to characterize the prevalence of vector-borne pathogens and their associated risk factors in dogs presenting to the DAD veterinary clinic on Santa Cruz Island.

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Journal Pre-proof 2. MATERIALS AND METHODS 2.1. Study Site and Population The study population consisted of 58 dogs (24 males, 34 females) that were presented to the DAD veterinary clinic in Puerto Ayora on Santa Cruz Island, Galápagos, between August 6, 2018 and August 24, 2018. The population ranged in age from 2 months to 15 years, with a median age of 24 months, and included both intact (13 males,

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11 females) and sterilized (11 males, 22 females) dogs. Of all dogs presenting to the

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clinic during the study period, 58 were included in the study based on convenience

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sampling, without consideration of the patients’ presenting complaint.

2.2. Client Consent

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Screening for canine vector-borne pathogen exposure using the SNAP 4Dx

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Plus test was recommended for all dogs presenting to DAD during the study period. A verbal explanation was provided in Spanish and a form was signed to provide consent for

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collection of 1-3 mL of blood for the study. No owners approached for the study declined

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testing. Three dogs were removed from the study as blood collection was unsuccessful and deemed stressful to the animal; questionnaires from these animals were not included during data analysis.

2.3. Client Questionnaire All participating clients filled out a questionnaire in Spanish regarding their pet’s lifestyle, prior medical history, and any clinical signs at the time of veterinary evaluation. Examples of questions include: neighborhood, time spent outside per day, use of tick

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Journal Pre-proof control products, and number of times in the last year that the owner found a tick on the dog, as well as the dog’s approximate age, sex, reproductive status (intact or sterilized), presenting complaint, and any history of vomiting, hematemesis, diarrhea, hematochezia, melena, epistaxis, lethargy, weight loss, inappetence, coughing, or sneezing. Additional verbal history was collected from owners of all dogs in the study. In households with more than 1 dog, clients filled out a survey for each pet.

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The questionnaire was prepared following a modification of the methods

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described by Bullinger et al. (1995). Briefly, two independent native Spanish speakers

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with excellent knowledge of English translated the original English questionnaire into

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Spanish, and then agreed upon a common Spanish translation. Two independent native Spanish speakers with excellent knowledge of English compared the original English

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questionnaire to the Spanish translation and rated each question and response choice for

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clarity, common language, and conceptual equivalency to assess the quality of translation. Two independent native English speakers with excellent knowledge of

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Spanish then translated the Spanish questionnaire back to English and then agreed upon a

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common English version.

2.4. Clinical Evaluation Due to the high daily caseload, history collection and physical examination were performed by different veterinarians or veterinary students at the clinic. Clinical examination included evaluation of weight, temperature, heart rate, respiratory rate, mucous membrane color and consistency, number of ticks found on the animal at the time of presentation, and the presence of petechiation, ecchymoses, bruising,

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Journal Pre-proof lymphadenopathy, organomegaly, lameness or joint pain, ocular or nasal discharge, or any additional physical examination findings.

2.5. Sample collection For patients already undergoing intravenous catheter placement (e.g. prior to surgery [16 animals] or blood transfusion [3 animals]), peripheral venous blood was

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collected into two to three EDTA Microtainer tubes (BD Biosciences, San Jose, CA

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95131, USA) from the cephalic vein at the time of catheter placement. For all other

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patients, 1-3 mL of peripheral venous blood was collected from the cephalic vein or

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lateral saphenous vein and stored in two to three EDTA Microtainer tubes. One EDTA tube was stored at room temperature for up to 2 hours, during which time the SNAP

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4Dx Plus test was performed according to manufacturer instructions, blood smears were

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prepared, and packed cell volume (PCV) and total solids (TS) were measured. Any additional EDTA tubes were stored in the clinic freezer for up to 48 hours and then

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transported to the ABG laboratory for storage at -20ºC. For dogs testing SNAP-positive

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for canine heartworm antigen, two direct wet mounts, two blood smears stained with Diff-Quik (Siemens Healthcare Diagnostics, Tarrytown, NY 10591, USA), and two buffy coat smears stained with Diff-Quik were examined for the presence of microfilariae. 2.6. SNAP 4Dx Plus tests Sixty SNAP 4Dx Plus tests were donated by Idexx Laboratories. The tests were refrigerated at 4ºC until the study period, maintained at room temperature during transport to the Galápagos, then stored at 4ºC until use.

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2.7. DNA Extraction DNA extraction was carried out on EDTA blood samples stored at -20ºC using the DNeasy Blood and Tissue Kit (QIAGEN Sciences, Germantown, MD 20874, USA), according to manufacturer instructions. The longest interval between sample collection

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and DNA extraction was 11 days. DNA samples were stored at -20ºC.

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2.8. PCR primers

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For each PCR primer, the primer sequence, target gene, amplicon size, and

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sequence source are listed in Table 2. Primers 8F, 1448F, ECA, and HE3 were manufactured by Life Technologies Corporation (Thermo Fisher Scientific, Grand Island,

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NY 14072, USA). Primers HE3-R, E. ewingii 2, Nem_18S-F, and Nem_18S-R were

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manufactured by Venegas Representaciones (Quito, 17-16-304, Ecuador). 2.9. Nested PCR for the detection of Ehrlichia spp.

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Primers 8F and 1448F for the Ehrlichia genus-level PCR were reconstituted with

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TE buffer (QIAGEN Sciences) to a final concentration of 100 µM, and diluted 1:10 in TE buffer for a 10 µM working solution. Mastermix was prepared using the following recipe: 12.5 µL DreamTaq Green MasterMix 2X (Thermo Fisher Scientific; active ingredients: 0.4 mM DreamTaq DNA Polymerase, 2X DreamTaq Green buffer, dATP, dCTP, dGTP, and dTTP; 4 mM MgCl2), 0.5 µL 10 µM forward primer, 0.5 µL 10 µM reverse primer, 9 µL sterile water. Template DNA (2.5 µL) was added to each well for a final reaction volume of 25 µL. Thermal cycling conditions were as follows: 1 cycle [Initial denaturation: 94ºC for 5 minutes], 35 cycles [Denaturation: 94ºC for 30 seconds,

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Journal Pre-proof Annealing: 50ºC for 30 seconds, Extension: 72ºC for 1 minute], 1 cycle [Final Extension: 72ºC for 10 minutes], hold at 4ºC. The results were read on a 1.5% UltraPure agarose gel (Invitrogen, Thermo Fisher Scientific) in TBE buffer (QIAGEN Sciences) at 90 V and visualized with ethidium bromide (Promega Corporation, Madison, WI 53711, USA). TrackIt 100 bp DNA ladder (Invitrogen, Thermo Fisher Scientific) was used. Primers ECA and HE3 for the species-level E. canis PCR and primers HE3-R and

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E. ewingii 2 for the species-level E. ewingii PCR were reconstituted with TE buffer to a

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final concentration of 100 µM and diluted 1:10 in TE buffer for a 10 µM working

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solution. The products from the genus-level PCR were used as the samples in the nested

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species-level PCR. Positive control amplicons were diluted in UltraPure distilled water

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(Invitrogen, Thermo Fisher Scientific). Mastermix was prepared using the following recipe: 12.5 µL DreamTaq Green MasterMix 2X, 0.5 µL 10 µM forward primer, 0.5 µL

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10 µM reverse primer, 10.5 µL sterile water. PCR product (1 µL) was added to each well

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for a final reaction volume of 25 µL. Thermal cycling conditions were as follows: 1 cycle [Initial denaturation: 94ºC for 5 minutes], 35 cycles [Denaturation: 94ºC for 30 seconds,

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Annealing: 55ºC for 30 seconds, Extension: 72ºC for 1 minute], 1 cycle [Final Extension: 72ºC for 10 minutes], hold at 4ºC. The results were read on a 1.5% agarose gel at 90 V and visualized with ethidium bromide. Primers against E. canis failed to amplify the E. ewingii positive control and primers against E. ewingii failed to amplify the E. canis positive control, demonstrating specificity. 2.10. PCR for the detection of D. immitis DNA samples for all patients testing SNAP-positive for D. immitis antigen underwent conventional PCR amplification of the gene encoding the nematode 18S

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Journal Pre-proof rRNA small ribosomal subunit. Primer sequences Nem_18S-F and Nem_18S-R were reconstituted with TE buffer and diluted in TE buffer for a 10 pM working solution. Mastermix was prepared using the following recipe: 12 µL DreamTaq Green MasterMix 2X, 1 µL 10 pM forward primer, 1 µL 10 pM reverse primer, 9 µL sterile water. Template DNA (2 µL) was added to each well for a final reaction volume of 25 µL. Thermal cycling conditions were as follows: 1 cycle [Initial denaturation: 94ºC for 5

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minutes], 35 cycles [Denaturation: 94ºC for 30 seconds, Annealing: 54ºC for 30 seconds,

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Extension: 72ºC for 1 minute], 1 cycle [Final Extension: 72ºC for 10 minutes], hold at

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4ºC. The results were read on a 1.5% agarose gel at 90 V and visualized with ethidium

2.11. Sanger sequencing of D. immitis

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bromide.

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PCR products obtained from the protocol in section 2.10 were sent to IDGen

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Laboratory (Avenida de los Granados E14-285 y Elroy Alfaro, Quito 170513, Ecuador) for sample purification, and then sent to Macrogen (10F, 254, Beotkkot-ro, Geumcheon-

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gu, Seoul 08511, Republic of Korea) for Sanger sequencing. The resulting sequence was

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deposited in the GenBank database under accession number MN241432. 2.12. Statistical analysis

Based on the number of dogs presented to the DAD clinic during August 2017, a sample size of 60 dogs was anticipated during the dates of this study. Based on a previously reported seroprevalence of 38% for E. canis / E. ewingii, 22 seropositive dogs were anticipated (Adams et al., 2016). Based on reported 22.5% agreement between Ehrlichia seropositivity and PCR positive results, an estimated 5 seropositive dogs would also be actively infected (Maazi et al., 2014). PCR was also expected to identify

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Journal Pre-proof additional acutely infected dogs that had not yet seroconverted. The expected PCR-based prevalence of Ehrlichia spp. infection in this population was therefore anticipated to be 8% (5/60) or higher. For an expected prevalence of 8%, a precision-based sample size of 55 dogs was targeted to allow estimation of the apparent prevalence of Ehrlichia infection with a 3.5% margin of precision, at a 95% confidence level. Statistical analysis of risk factors was carried out using GraphPad Prism (Version

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8.10, GraphPad Software, Inc., San Diego, CA 92108, USA). A positive serology result

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was interpreted as evidence of prior exposure, while a positive PCR result was interpreted

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as evidence of active infection. Quantitative continuous data were assessed using a two-

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tailed unpaired t-test with Welch’s unequal variances correction, to a 95% confidence level. Categorical data was assessed for the impact of historical risk factors and clinical

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examination findings on the prevalence of Ehrlichia spp. exposure or active E. canis

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infection using 2x2 contingency tables and a two-tailed Fisher’s exact test. Odds Ratio (OR) was defined as the ratio between the proportion of dogs with

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active infection (positive on PCR) that were exposed to the parameter of interest (a)

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relative to the proportion of dogs with active infection that were not exposed to the parameter of interest (b), and the proportion of dogs without active infection that were exposed to the parameter of interest (c) relative to the proportion of dogs without active infection that were not exposed to the parameter of interest (d) (Viera, 2008). OR was calculated from 2x2 contingency tables (Eq. 1; Viera, 2008). 𝑂𝑅 =

𝑎/𝑏 𝑐/𝑑

(1)

Relative Risk (RR) was defined as the proportion of dogs with active infection (positive on PCR) that were positive for the parameter of interest, relative to the

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Journal Pre-proof proportion of dogs with active infection that were negative for the parameter of interest (Viera, 2008). RR was calculated from 2x2 contingency tables (Eq. 2; Viera, 2008). 𝑎

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𝑅𝑅 = (𝑎+𝑏)/(𝑐+𝑑)

(2)

3. RESULTS Seroprevalence was defined as the proportion of animals that tested positive for

antigen of D. immitis on the SNAP 4Dx Plus test.

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antibodies against E. canis/E. ewingii, A. phagocytophilum/A. platys, B. burgdorferi, or

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This study reports the seroprevalence of Ehrlichia spp. (48.3%; 28/58),

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Anaplasma spp. (12.1%; 7/58), and Borrelia burgdorferi (0%; 0/58). Active infection is

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defined as the proportion of samples with positive antigen (PCR for E. canis/ewingii,

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ELISA for D. immitis) results for the targeted pathogen. PCR demonstrated active infection with E. canis in 12.1% (7/58) of dogs and no active infection with E. ewingii.

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Active infection with D. immitis was documented in 6.9% (4/58) of dogs. The proportion of dogs seropositive for both E. canis/E. ewingii and A. phagocytophilum/A. platys was

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6.9% (4/58). Of dogs positive for Ehrlichia, 14.3% (4/28) were also positive for

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Anaplasma; of dogs positive for Anaplasma, 57.1% (4/7) were also positive for Ehrlichia. A total of 1,037 ticks were removed from dogs in this study, with a range of 1 to 944 ticks per dog. Tick infestation occurred with higher frequency among dogs seropositive for Ehrlichia or Anaplasma (22.6% (7/31)) compared to seronegative animals (3.7% (1/27)), but this difference was not significant (p=0.746). Of dogs actively infected with E. canis, 14.3% (1/7) were infested with at least one tick. The number of ticks per dog did not have an effect on E. canis PCR result or E. canis/E. ewingii ELISA result.

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Journal Pre-proof There was no effect of PCV, age, or time spent outside per day on E. canis PCR result or E. canis/E. ewingii ELISA result. Dogs with a PCV ≤ 30% had a 10-fold risk of active infection compared to dogs with a PCV ≥ 31% (p = 0.0124). Dogs with a clinical history of lethargy had a 4.6-fold risk of active infection compared to dogs with no clinical history of lethargy (p = 0.05). Risk factors for infection are described in Table 3. Treatment with doxycycline (10 mg/kg once daily for 30 days) was initiated in

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four dogs based on clinical suspicion of tick-borne disease and positive SNAP tests (two

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positive for E. canis/E. ewingii and two positive for both E. canis/E. ewingii and A.

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phagocytophilum/A. platys). Three of the treated animals were severely anemic (PCV

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≤20%) and were successfully treated with blood transfusions at the clinic. One of these animals was later determined to have been actively infected with E. canis on PCR. Two

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dogs presenting with acute onset of lethargy, depression, and gastrointestinal signs that

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were also SNAP positive for A. phagocytophilum/A. platys and/or E. canis/E. ewingii were not treated, but were later found to be positive for E. canis on PCR.

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Four of the 58 dogs (6.90%) tested SNAP-positive for D. immitis antigen. Three

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dogs were amicrofilaremic, a state that has been previously reported in up to 30% of dogs with known heartworm infection (Courtney and Zeng, 1989). Circulating microfilariae were visualized in one D. immitis-positive dog. PCR for the presence of nematode 18S rRNA was carried out on DNA extracted from blood samples of these four dogs and was positive only in the microfilaremic patient. The amplicon was sequenced and compared by NCBI BLAST against all 35 available GenBank sequences of the D. immitis 18S rRNA (Madden, 2002; Benson et al., 2017). The sequences with the closest similarity to as determined based on percent homology, percent query cover, and Expect (E) value are

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Journal Pre-proof listed in Table 6. This represents the first genetic identification of D. immitis in the Galápagos archipelago. All four dogs testing positive for D. immitis had lived their entire lives on Santa Cruz Island. All dogs positive for D. immitis were reported by their owners to spend 1224 hours outside per day, compared to 24% of dogs negative for D. immitis. Of dogs positive for D. immitis, three dogs lived in Las Ninfas and one lived in Mirador. Of dogs

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presenting to the clinic whose owners reported living in Las Ninfas, 42.9% (3/7) were

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positive for D. immitis. Living in Las Ninfas conferred a significantly higher risk of D.

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immitis infection compared to other neighborhoods (Odds Ratio: 19.71, Risk Ratio: 14.1,

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Confidence Interval: 1.791 to 217.1, p=0.0159). Two heartworm positive dogs were clinically healthy with an unremarkable physical examination and no history of cough,

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elevated respiratory rate, exercise intolerance, or collapse; one of these dogs had

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circulating microfilariae. One dog had a week-long history of coughing while sleeping, and a second dog presented with lethargy, weakness, weight loss, inappetence, and a

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grade 4/6 systolic heart murmur, pale mucous membranes, severe anemia (PCV 5%),

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thrombocytopenia, and neutrophilia requiring treatment with a blood transfusion. None of the heartworm positive dogs were actively infected with E. canis. 4. DISCUSSION As expected, more dogs were exposed to than actively infected with E. canis/E. ewingii. Two dogs were seronegative for Ehrlichia canis/E. ewingii but tested PCR positive for E. canis, indicating early infection in which seroconversion had yet to occur. Two E. canis seropositive dogs were clinically healthy but were found to have moderate anemia (PCV 21-30%) and were PCR positive for E. canis, indicating subclinical

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Journal Pre-proof infection. A PCV cutoff of 30% may be a useful screening tool for active E. canis infection in regions with high Ehrlichia seroprevalence, in the absence of other apparent causes of anemia. Some proportion of seropositive dogs in this study may have been actively infected with unidentified species of Ehrlichia. Cross-reactivity on the SNAP 4Dx Plus test has been reported between E. canis and E. chaffeensis and between E. canis and A.

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phagocytophilum (Waner et al., 2001; O’Connor et al., 2006; Solano-Gallego et al., 2006;

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Stillman et al., 2014). The sensitivity of PCR may additionally be lower in pancytopenic

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dogs with chronic E. canis infection, yielding false negatives (Mylonakis et al., 2004).

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Serologic and molecular testing used in parallel is therefore recommended to maximize diagnosis of tick-borne diseases. Future studies sequencing Ehrlichia 16S rRNA may

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identify novel members of the Ehrlichia genus.

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Tick infestation, male sex, older age (>1 year old), and urban neighborhood have been reported as risk factors for exposure to Ehrlichia or Anaplasma spp. in dogs on

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Santa Cruz Island (Adams et al., 2016). There was no effect of sex, the presence of ticks,

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or the number of ticks on the proportions of exposure or infection with Ehrlichia spp. in the current study. Older age (≥1 year old) was a risk factor for Ehrlichia spp. seropositivity (Table 4), but age had no effect on active E. canis infection (Table 3). Older animals are more likely to have been exposed to vector-borne pathogens and therefore have a higher proportion of seropositivity but have the same risk of active infection as younger animals. There was no association between the number of hours spent outside daily and Ehrlichia spp. exposure or infection (Table 3, Table 4). No analysis could be made regarding the distribution of exposed or infected individuals

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Journal Pre-proof across urban versus rural neighborhoods due to a small sample size of dogs from rural neighborhoods. Certain risk factors could not be statistically evaluated due to incomplete or inconsistent survey results (e.g. owner indicated use of a tick control product but did not provide the name or type of product). Owners were often not specific about the brand or composition of the product, and home remedies were also occasionally listed, such as

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talcum powder. The authors also acknowledge that “tick control product” was ambiguous

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and while the intent was to determine which clients consistently used preventative

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products aimed at preventing tick infestation, clients also indicated intermittent use of

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products to treat active infestation, such as sprays and powders. Subjectively, owners were more likely to use acaricide products after noticing ticks on the dog, but did not use

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or inconsistently used products to prevent infestation.

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The primary vector of E. canis is the brown dog tick (Rhipicephalus sanguineus) (Groves et al., 1975), a monotropic three-host tick uniquely able to survive indoors for

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extended periods of time. The Charles Darwin Foundation lists 11 known species of hard

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ticks (Ixodidae) in the Galápagos – 3 introduced and 8 endemic (Table 5) (Schatz, 1991; Baert et al., 2018). Of these, only R. sanguineus has been documented as a competent vector of E. canis and E. ewingii. All ticks collected by Diaz et al. (2016) were identified as R. sanguineus. The ticks of the genera Amblyomma, Dermacentor, and Ixodes previously identified in the Galápagos (Table 5) are not documented to parasitize dogs and no studies have been performed to assess their competence as vectors of Ehrlichia spp. However, given the relative dearth of research on these tick species and the experimental competence of other members of these genera as vectors of Ehrlichia spp.,

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Journal Pre-proof it remains possible that ticks in the Galápagos other than R. sanguineus may have the potential to contribute to Ehrlichia spp. transmission (Johnson et al., 1998; Ferrolho et al., 2016). The majority of the ticks in this study were identified by one author as R. sanguineus, but due to the large number of ticks collected (1,037 ticks), not every tick could be examined for identification. This study represents the first report of D. immitis, the causative agent of canine

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heartworm disease, in dogs on Santa Cruz Island, Galápagos. It is also the first study to

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utilize molecular testing to identify D. immitis in the Galápagos. D. immitis microfilariae

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were reported in 87% of domestic dogs on Floreana Island in the 1980’s (Barnett and

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Rudd, 1983; Barnett, 1985) and more recently, D. immitis antigen was detected in 34% of dogs on Isabela Island (Levy et al., 2008). However, studies on Santa Cruz Island in 2014

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and 2015 did not find any dogs positive for D. immitis antigen (Adams et al., 2016; Diaz

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et al., 2016). D. immitis has also been isolated from endemic Galápagos wildlife, including Galápagos sea lions, a Galápagos penguin (Spheniscus mendiculus), and a

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and Merkel, 2018).

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Humboldt penguin (S. humboldti) (Barnett, 1985; Sano et al., 2005; Santiago-Alarcon

D. immitis infection has been documented in domestic dogs in Guayaquil, a major city in Ecuador from which depart multiple daily flights to Santa Cruz (Fernandez et al., 2017). Fernandez et al. (2017) also references several doctoral theses from the 1980s that document the presence of D. immitis in Ecuador. These reports, like the work of Barnett (1985), are relatively inaccessible to the scientific community as they are not readily available online and, in some cases, have no published English translation. Mainland Ecuador continues to be reported as free of heartworm disease in reviews of canine vector

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Journal Pre-proof borne disease (Labarthe and Guerrero, 2005; Maggi et al., 2019), despite local veterinarians commonly diagnosing and treating the disease (C. Gonzalez-Lorza, pers. comm.). These barriers present a challenge to achieving comprehensive disease surveillance. A positive SNAP result for D. immitis antigen represents active infection, and treatment is recommended even in dogs without clinical signs (American Heartworm Society, 2018). However, access to the gold standard treatment, melarsomine, is limited

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in the Galápagos (J. Keulen, pers. comm.). Infected dogs may also have negative SNAP

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results during early infection (American Heartworm Society, 2018; Weil et al., 1985).

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Mosquitoes of the genera Anopheles, Aedes, Culex, and Psorophora are

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competent vectors of D. immitis (Ledesma and Harrington, 2011; Montarsi et al., 2015). In the Galápagos, D. immitis larvae have been isolated from the endemic black salt marsh

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mosquito, Aedes taeniorhynchus (Wiedemann, 1821), and the introduced southern house

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mosquito, Culex quinquefasciatus (Say, 1823), which has a worldwide sub-tropical distribution and is now naturalized in the Galápagos (Barnett, 1985; Bataille et al., 2009a;

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Bataille et al., 2009b; Whiteman et al., 2005). Aedes aegypti is an introduced and

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naturalized species in the Galápagos that is known to be a competent vector of canine heartworm (Hendrix et al., 1986; Sinclair et al., 2017). C. quinquefasciatus breeds in standing freshwater around human dwellings, while A. taeniorhynchus breeds in salt or brackish water (Barnett, 1985; Manimegalai and Sukanya, 2014). Due to the relatively few sources of fresh water in the archipelago, A. taeniorhynchus has been suggested as the principal vector of D. immitis in the Galápagos (Barnett, 1985). The majority of heartworm positive results in this study occurred in dogs living in the barrio Las Ninfas, which borders a large brackish water lagoon. Diaz et al.

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Journal Pre-proof (2016) randomly selected 8 of the 16 urban neighborhoods in Puerto Ayora for sampling and did not include Las Ninfas; the presence of D. immitis may therefore have gone undetected for lack of investigation in proximity to mosquitoes. This site could be targeted for further investigation, including testing of dogs living in Las Ninfas, and collection of mosquitoes for species identification and analysis for the presence of D. immitis by compound microscopy or PCR.

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Heartworm disease on Santa Cruz Island may alternatively be a result of transport

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of an infected dog or infected mosquitoes from Floreana or Isabela Islands within the

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archipelago, or from the mainland (Maggi et al., 2019). Importation of infected dogs has

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been documented as an important means of introduction of canine vector-borne disease (Menn et al., 2010). Both A. taeniorhynchus and C. quinquefasciatus have been caught in

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airplanes landing at Baltra Airport in the Galápagos (Bataille et al., 2009b). Microsatellite

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genotyping suggests that C. quinquefasciatus has been frequently introduced into the archipelago by anthropogenic transport and that newly introduced mosquitos breed with

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established island populations (Bataille et al., 2009b). Air travel is considered the highest

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risk for the introduction of new mosquitoes potentially carrying pathogens (Kilpatrick et al., 2006). Cargo boats traveling to the Galápagos have also been implicated as a source of introduction of invasive insects, although the contribution of this method of transport to genetic diversity of C. quinquefasciatus on Santa Cruz Island is considered minimal compared to air transport (Causton et al., 2006; Bataille et al., 2009b). Puerto Ayora is located over 670 miles away from mainland Ecuador, and at its closest points is located over 30 miles from Floreana Island and 17 miles from Isabela Island – too far for an

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Journal Pre-proof infected mosquito to fly, even aided by wind (Lapointe, 2008; Ciota et al., 2012; Verdonschot and Besse-Lototskaya, 2014). 5. CONCLUSION This study represents the first identification of E. canis and E. ewingii using PCR on Santa Cruz Island and updates the seropositivity and prevalence data of these pathogens. This study also represents the first report of D. immitis-infected dogs on Santa

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Cruz Island, and the first identification of D. immitis using PCR and Sanger sequencing

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in the Galápagos archipelago. These findings emphasize the public health importance of

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disease surveillance and the importance of year-round vector control for dogs, and

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suggest avenues for ongoing research in the region. Ethics

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This study received approval from the Cornell University College of Veterinary Medicine

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Veterinary Clinical Studies Committee (CUVCSC) and exemption from Institutional Animal Care and Use Committee (IACUC) review (CUVCSC Protocol ID# 62618-09).

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Funding

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This study was funded by the Cornell University College of Veterinary Medicine Expanding Horizons grant program with assistance from the ABG laboratory in Santa Cruz, Galápagos. Acknowledgements The authors would like to thank the Cornell University Expanding Horizons Grant Program for supporting and funding this project, IDEXX Laboratories for donating SNAP 4Dx Plus tests, and Darwin Animal Doctors and Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos, particularly director Dr.

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Journal Pre-proof Marilyn Cruz and Alberto Velez, for approving and aiding in this study. The authors also thank Dr. Ricardo Maggi (North Carolina Vector-Borne Disease Laboratory) for providing positive controls of E. canis and E. ewingii, Dr. Amy Glaser (Cornell Animal Health Diagnostic Center) for providing PCR primer sequences and protocols, Dr. David Erikson and Dr. Ryan Snodgrass (Cornell University Sibley School of Mechanical and Aerospace Engineering) for assistance in investigating the suitability of LAMP primers

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for this study, and the following translators: Jose Oyola Morales, Jacqueline Joya, Josue

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San Emeterio, Eva Quijano Carde, Chistopher Peritore Galve, Maria Juarez, Emma

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Castillo, and Katherine Schuhmacher Vissio. The authors are grateful to the Charles

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Darwin Foundation G.T. Corley Library on Santa Cruz Island, Galápagos for providing research support.

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Declarations of interest:

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Japanese woman after a tour to Europe. Parasite. 22, 2. Toral-Granda, M.V., Causton, C.E., Jäger, H., Trueman, M., Izurieta, J.C., Araujo, E., Cruz, M., Zander, K.K., Izurieta, A., Garnett, S.T., 2017. Alien species pathways to the Galápagos Islands, Ecuador. PLoS One. 12, e0184379. Van’t Woudt, B.D., 1990. Roaming, Stray, and Feral Domestic Cats and Dogs as Wildlife Problems. In: Davis, L., Marsh, R. (Eds.), Proc. 14th Vertebr. Pest C. University of California, Davis. pp. 291–295.

31

Journal Pre-proof Verdonschot, P.F.M., Besse-Lototskaya, A.A., 2014. Flight distance of mosquitoes (Culicidae): A metadata analysis to support the management of barrier zones around rewetted and newly constructed wetlands. Limnologica. 45, 69–79. Viera, A. J, 2008. Odds ratios and risk ratios: what’s the difference and why does it matter? South Med. J. 101, 730-734. Waner, T., Harrus, S., Bark, H., Bogin, E., Avidar, Y., Keysary, A., 1997.

ro

infected beagle dogs. Vet. Parasitol. 69, 307–317.

of

Characterization of the subclinical phase of canine ehrlichiosis in experimentally

-p

Waner, T., Harrus, S., Jongejan, F., Bark, H., Keysary, A., Cornelissen, A.W.C.A., 2001.

re

Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia

lP

canis. Vet. Parasitol. 95, 1–15.

na

Watts, K.J., Coutney, C.H., Reddy, G.R., 1999. Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria

ur

immitis, in its mosquito intermediate host. Mol. Cell. Prob. 14, 425-430.

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Weill G.J., Malane, M.S., Powers, K.G., Blair, L.S., 1985. Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs. J Immunol. 135, 1185-1191. Whiteman, N.K., Goodman, S.J., Sinclair, B.J., Walsh, T., Cunningham, A.A., Kramer, L.D., Parker, P.G., 2005. Establishment of the avian disease vector Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) on the Galápagos Islands, Ecuador. Ibis. 147, 844–847. Table 1: Review of canine vector-borne diseases detected on Isabela and Santa Cruz Islands

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Journal Pre-proof Santa Cruz Island Pathogen

Targeta

Percentage

Reference

Ehrlichia canis/ Ehrlichia ewingii

Antibody

39%

Diaz et al., 2016

Ehrlichia canis/Ehrlichia ewingii

Antibody

37.84%

Adams et al., 2016

Anaplasma

Antibody

24%

Diaz et al., 2016

Antibody

13.51%

Adams et al., 2016

Borrelia burgdorferi

Antibody

0%

Borrelia burgdorferi

Antibody

0%

Adams et al., 2016

Dirofilaria immitis

Antigen

0%

Diaz et al., 2016

Dirofilaria immitis

Antigen

Anaplasma

re

-p

ro

phagocytophilum/Anaplasma platys

of

phagocytophilum/Anaplasma platys

Diaz et al., 2016

Adams et al., 2016

Percentage

Reference

Antigen

33.7%

Levy et al., 2008

Antigen

13.7%

Levy et al., 2008

Antibody

4.2%

Levy et al., 2008

Anaplasma platys

Antigen

1.0%

Levy et al., 2008

Mycoplasma haemocanis

Antigen

1.0%

Levy et al., 2008

Borrelia burgdorferi

Antibody

0%

Levy et al., 2008

Ehrlichia canis

Antibody

0%

Levy et al., 2008

Ehrlichia spp.

Antigen

0%

Levy et al., 2008

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0%

Isabela Island

Targeta

na

Pathogen

Bartonella spp.

Jo

Leishmania infantum

ur

Dirofilaria immitis

a

Antibody detection was carried out by ELISA in all studies; antigen detection was carried out by PCR in all studies except those involving D. immitis, in which antigen detection was carried out by ELISA. Table 2: Ehrlichia spp. and Nematode PCR primers Primer Sequence (5’-3’) Target gene Amplicon Sequence source size (bp)

33

Journal Pre-proof

ECA HE3 HE3-R E. ewingii 2 Nem_18S-F

Glaser, Cornell Animal Health Diagnostic Center

Glaser, Cornell Animal Health Diagnostic Center Breitschwerdt et al., 1998

Floyd et al., 2005

-p

Nem_18S-R

16S ribosomal RNA of 1,370 alpha-proteobacteria (including Ehrlichia spp., Anaplasma spp., Rickettsia spp., Wolbachia spp.) CAA TTA TTT ATA GCC TCT 16S ribosomal RNA of 389 GGC TAT AGG A Ehrlichia canis TAT AGG TAC CGT CAT TAT CTT CCC TAT CTT CTA TAG GTA CCG TCA 16S ribosomal RNA of 395 TAT CTT CCC TAT Ehrlichia ewingii CAA TTC CTA AAT AGT CTC TGA CTA TT GGT CAA CAA ATC ATA 18S rRNA small 900 AAG ATA TTG G ribosomal subunit of nematodes TAA ACT TCA GGG TGA CCA AAA AAT CA

of

1448F

AGT TTG ATC ATG GCT CAG CCA TGG CGT GAC GGG CAG TGT G

ro

8F

ORb

RRc

95% CI

p-valued

Sex (female vs. male)

ur

Signalment

na

lP

Parameter

re

Table 3: Odds Ratios for Ehrlichia canis infectiona in dogs on Santa Cruz Island

Age (<1 year old vs. ≥1 year old)

Jo

Diagnostic results

Ehrlichia spp. antibody status

2.067

1.889

0.418 to 10.23

0.374

1.136

1.118

0.195 to 6.210

>0.999

2.054

1.894

0.364 to 11.59

0.415

13.75

10.00

1.450 to 165.6

0.0124

0.081 to 8.079

>0.999

(negative vs. positive) Anemia (PCV ≥ 31% vs. PCV ≤ 30%) Clinical history and physical examination findings Ticks found on dog during physical

1.048

1.042

examination (none vs. at least one)

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Journal Pre-proof Ticks found on dog by owner in the

2.000

1.857

0.278 to 25.32

>0.999

2.318

2.036

0.408 to 14.31

0.629

Vomiting (No vs. Yes)

2.044

1.855

0.358 to 13.14

0.597

Diarrhea (No vs. Yes)

6.133

4.080

0.874 to 34.52

0.113

Lethargy (No vs. Yes)

6.750

4.594

1.323 to 34.90

0.050

Inappetence (No vs. Yes)

1.867

1.709

0.326 to 12.07

0.607

Bleeding tendencye (No vs. Yes)

8.333

4.667

0.3780 to 160.2

0.229

Lymphadenopathy

1.350

1.292

0.306 to 5.556

0.697

past year (none vs. at least one) Fever (No (100.5-102.5ºF) vs. (Yes

ro

-p

Infection was defined as a positive PCR result for E. canis

re

a

of

(<102.5ºF))

b

lP

Odds Ratio (OR) was defined as the ratio between A) the proportion of dogs with active

infection (positive on PCR) that were exposed to the parameter of interest relative to the

na

proportion of dogs with active infection that were not exposed to the parameter of interest, and B) the proportion of dogs without active infection that were exposed to the parameter of interest

ur

relative to the proportion of dogs without active infection that were not exposed to the parameter

c

Jo

of interest. OR was calculated from 2x2 contingency tables (Eq. 1; Viera, 2008). Relative Risk (RR) was defined as the proportion of dogs with active infection (positive on PCR)

that were positive for the parameter of interest, relative to the proportion of dogs with active infection that were negative for the parameter of interest. RR was calculated from 2x2 contingency tables (Eq. 2; Viera, 2008). The significance level was =0.05

d

e

Bleeding tendency was defined as the presence of one or more of the following criteria on

physical examination: petechiae, ecchymoses, bruising, hematemesis, hematochezia, melena, epistaxis

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Journal Pre-proof Table 4: Odds Ratios for Ehrlichia spp. exposure in dogs on Santa Cruz Island ORb

RRc

95% CI

p-valued

Sex (female vs. male)

0.964

0.980

0.295 to 2.967

>0.999

Age (<1 year old vs. ≥1 year old)

16.63

8.143

2.320 to 184.2

0.0016

0.590

0.737

0.172 to 2.027

Parameter

Signalment

PCV ≤ 30%)

0.778

a

0.778

0.875

0.232 to 2.657

0.746

na

past year (none vs. at least one)

0.746

lP

examination (none vs. at least one) Ticks found on dog by owner in the

0.232 to 2.657

0.875

re

Ticks found on dog during physical

-p

Clinical history and physical examination findings

0.528

ro

Severe anemia (PCV ≥ 31% vs.

of

Diagnostic results

Exposure was defined as a positive SNAP test result for antibody against Ehrlichia canis/ewingii

b

ur

Odds Ratio (OR) was defined as the ratio between A) the proportion of dogs with evidence of

Jo

Ehrlichia exposure (positive on SNAP) that were exposed to the parameter of interest relative to the proportion of dogs with Ehrlichia exposure that were not exposed to the parameter of interest, and B) the proportion of dogs without evidence of Ehrlichia exposure that were exposed to the parameter of interest relative to the proportion of dogs without evidence of Ehrlichia exposure that were not exposed to the parameter of interest. OR was calculated from 2x2 contingency tables (Eq. 1; Viera, 2008). c

Relative Risk (RR) was defined as the proportion of dogs with evidence of exposure (positive on

SNAP) exposed to the parameter of interest, relative to the proportion of dogs with evidence of

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Journal Pre-proof exposure unexposed to the parameter of interest. RR was calculated from 2x2 contingency tables (Eq. 2; Viera, 2008). The significance level was =0.05

d

Table 5: Ixodid Ticks of the Galápagos archipelago First described in Galápagos

Definitive host

Rhipicephalus sanguineus (Latreille,1806)

Neumann, 1905

Dog

ro

Rhipicephalus microplus (Canestrini, 1888) Minning, 1934

of

Species

Cattle

Neumann, 1897

Horse

Ixodes galapagoensis

Clifford and Hoogstraal, 1980

Galápagos rice rat

re

-p

Dermacentor nitens

Jo

Amblyomma pilosum

ur

Amblyomma macfarlandi

na

Amblyomma darwini wollebaecki

Hirst and Hirst, 1910

lP

Amblyomma darwini darwini

Amblyomma boulengeri

Schulze-Rostock, 1936

Marine iguana

Marine iguana

Keirans, Hoogstraal and Clifford, Giant tortoise 1973 Neumann, 1899

Giant tortoise

Hirst and Hirst, 1910 and Keirans, Giant tortoise 1973

Amblyomma usingeri

Keirans, Hoogstraal and Clifford, Giant Tortoise 1973

Amblyomma williamsi

Banks, 1924

Marine iguana Land iguana

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Journal Pre-proof Table 6: Sequence homology for 18S rRNA isolate of D. immitis Homologya

Query Coverb

E valuec

Isolate source

Reference

100.00%

36%

2 x 10-155

Atas et al., 2018

AF182647

99.43%

66%

0.0

AB973231

99.10%

98%

0.0

AB973230

98.97%

98%

0.0

AF036638

96.95%

98%

0.0

D. immitis worms collected from stray dogs in Turkey D. immitis infected mosquitos from Colorado Male D. immitis worm collected from Japanese dog Female D. immitis worm collected from Japanese dog Not specified

Watts et al., 1999 Suzuki et al., 2015 Suzuki et al., 2015

ro

of

GenBank accession number KJ183078

-p

Blaxter et al., 1998 a Percent homology describes the quantity of identical base pairs between each target sample and

Query cover represents the percentage overlap between the target sequence and the queried

lP

b

re

the query sample

sequence; 100% indicates that the target sequence completely covers the queried sequence E value represents the number of sequence matches with comparable homology and query cover

na

c

HIGHLIGHTS

This study documents the first report of Dirofilaria immitis in dogs on Santa Cruz. This study reports seroprevalence of Ehrlichia (48.3%) and Anaplasma (12.1%). This study reports the proportion of active infection with E. canis (12.1%). Dogs with PCV ≤ 30% had a 10-fold risk of active Ehrlichia infection (p = 0.0124)

Jo

   

ur

that could occur by chance, given the size of the available database

38