The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis

Veterinary Parasitology 179 (2011) 335–342

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The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis John W. McCall a , Christine F. Baker b,∗ , Thomas N. Mather c , S. T. Chester d , Scott D. McCall a , Jennifer P. Irwin b , Stephanie L. Young b , Luiz G. Cramer d , Matthias G. Pollmeier e a b c d e

TRS Labs, Inc., PO Box 5112, Athens, GA, USA Merial Limited, 115 Transtech Drive, Athens, GA, USA Center for Vector-Borne Disease, University of Rhode Island, Kingston, RI, USA Merial Limited, 3239 Satellite Blvd, Duluth, GA 30096, USA Merial S.A.S., CRSV-PIPA, 1 allée des Cyprès, 01150 St Vulbas, France

a r t i c l e

i n f o

Keywords: Anaplasma phagocytophilum Borrelia burgdorferi CERTIFECTTM Granulocytic anaplasmosis Ixodes scapularis Lyme borreliosis

a b s t r a c t Healthy, purpose-bred laboratory beagle dogs that had not been exposed to ticks and were seronegative for Borrelia burgdorferi and Anaplasma phagocytophilum were randomly assigned to four groups of eight dogs each. Control group 1 was not treated. Groups 2, 3 and 4 were treated with a single topical application of a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECTTM , Merial Limited, GA, USA) at 28, 21 or 14 days prior to tick infestation, respectively. Each dog was infested with 25 female and 25 male fieldcollected adult Ixodes scapularis ticks that had infection rates of 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum, as determined by polymerase chain reaction. Two and five days after tick infestation, control dogs had an average of 9.5 and 13.9 attached adult female ticks, respectively, whilst the 24 treated dogs remained tick-free aside from a single tick on the 2nd day after infestation. Serial serological tests demonstrated that the ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 with A. phagocytophilum. B. burgdorferi infection also was confirmed in most control dogs by culture (6/8) and PCR (7/8) of skin biopsies. In contrast, CERTIFECT protected all 24 treated dogs against infection by both B. burgdorferi and A. phagocytophilum, as demonstrated by their negative serological tests throughout the study and the absence of any positive skin biopsy culture or PCR in these dogs. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Lyme disease and granulocytic anaplasmosis are ticktransmitted diseases known to affect humans and domestic animals in North America, Europe, and Asia, wherever ticks carry their respective causative agents, Borrelia burgdor-

∗ Corresponding author. Tel.: +1 706 552 2445; fax: +1 706 543 1667. E-mail address: [email protected] (C.F. Baker). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.03.046

feri and Anaplasma phagocytophilum (Littman et al., 2006; Murphree Bacon et al., 2008; Beugnet and Marié, 2009; Carrade et al., 2009). In the United States of America (USA), Lyme disease is the most frequently diagnosed vectorborne disease in people and is caused by B. burgdorferi sensu stricto. In the north-eastern and north-central USA, blacklegged (deer) ticks, Ixodes scapularis, are the principal vectors for B. burgdorferi sensu stricto and A. phagocytophilum which they have been carrying northward into Canada (Lindsay et al., 1995; Dennis et al., 1998; Morshed

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et al., 2003; Morshed et al., 1999, 2000; Artsob et al., 2000; Guerra et al., 2001; Duncan et al., 2004; Beall et al., 2006,2008; Bowman et al., 2009). Although B. burgdorferi and A. phagocytophilum infections can be asymptomatic, each agent is capable of causing severe disease in dogs, and co-infections increase the risk of clinical disease (Breitschwerdt et al., 1998; Littman et al., 2006; Beall et al., 2008; Beall et al., 2006; Kohn et al., 2008). Similarly, the clinical spectrum of granulocytic anaplasmosis in humans ranges from asymptomatic to fatal infections (Bakken and Dumler, 2008). Subclinical infections with A. phagocytophilum can persist in dogs, even after treatment with doxycycline (Egenvall et al., 1997; Alleman et al., 2006; Carrade et al., 2009). Dogs can be protected against the transmission of the agents of Lyme disease and granulocytic anaplasmosis by ectoparasiticides that prevent successful attachment of I. scapularis ticks. The objective of this study was to evaluate the ability of a single topical treatment with a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECTTM , Merial Limited, GA, USA) to protect dogs against transmission of B. burgdorferi sensu stricto and A. phagocytophilum from carrier I. scapularis for at least 28 days after treatment. 2. Materials and methods 2.1. Ticks and tick infection rates Adult male and female I. scapularis ticks were collected from field locations in South Kingstown, Rhode Island between 21 and 27 October 2008. They were maintained refrigerated at 4–5 ◦ C and under saturated humidity conditions until used to infest the dogs. A representative sample of 30 adult I. scapularis female ticks of this field collected population was tested by polymerase chain reaction (PCR) for the presence of B. burgdorferi sensu stricto and A. phagocytophilum DNA. First, DNA was purified using a modification of the DNeasy® blood and tissue kit protocol from Qiagen (Qiagen, Inc., Valencia, CA, USA – DNEASY® is a registered trademark of Qiagen in the United States of America and elsewhere). Individual ticks were placed in 1.5 mL microcentrifuge tubes containing 180 ␮L of tissue lysis buffer. Ticks were crushed using a disposable 1000 ␮L pipette tip with its tip heat sealed. After crushing, 20 ␮L of proteinase K were added and samples were incubated for 2 h in a 55 ◦ C water bath. The DNEASY tissue extraction protocol was then followed, as described by the manufacturer. PCR amplifications to detect B. burgdorferi sensu stricto were performed using Primers A2 [5 GTT TTG TAA TTT CAA CTG CTG ACC 3 ] and A4 [5 CTG CAG CTT GGA ATT CAG GCA CTT C 3 ] following published methods (Nocton et al., 1994). Nested PCR was used to detect A. phagocytophilum. Primers ge3a [5 CAC ATG CAA GTC GAA CGG ATT ATT C 3 ] and ge10 [5 TTC CGT TAA GAA GGA TCT AAT CTC C 3 ] were added to each reaction mixture at a final concentration of 0.5 ␮M for the primary amplification (Massung et al., 1998; Massung and Slater, 2003). Optimized cycling conditions for the primary amplification involved an initial 2-min denaturation at 95 ◦ C, followed by 40 cycles, each consisting of a 30-s denaturation at 94 ◦ C, a 30-s anneal-

ing at 52 ◦ C, and a 45-s extension at 72 ◦ C. These 40 cycles were followed by a 5-min extension at 72 ◦ C, and reaction products were subsequently maintained at 4 ◦ C and used as a primary template in the nested amplification. Nested amplifications used 1 ␮L of the primary PCR product as template in a total volume of 25 ␮L. Primers ge2 [5 GGC AGT ATT AAA AGC AGC TCC AGG 3 ] and ge9 [5 AAC GGA TTA TTC TTT ATA GCT TGC T 3 ] were added at a final concentration of 0.5 ␮M per reaction for the nested amplification. Nested cycling conditions were as described for the primary amplification, except that only 30 cycles were used. Reaction products were maintained at 4 ◦ C until analyzed by agarose gel electrophoresis. 2.2. Animal model and treatment Sixteen male and sixteen female healthy purpose-bred laboratory beagles that had not received any prior ectoparasiticide treatment were used in this single center trial. The study design was blinded and controlled with a randomized block design based on body weight within sex. All animals were managed with due regard for their well being and in accordance with local Institutional Animal Care and Use Committee approvals and requirements, as well as international laws and ethics, including those of the International Guiding Principles for Biomedical Research Involving Animals, as issued by the Council for the International Organizations of Medical Sciences (Anonymous, 1985). At the beginning of the study, the dogs’ age range was 2.8–3.2 months old, and their weight range was 3.14–5.76 kg. Their good health was verified by a physical examination conducted by a veterinarian, and they were observed daily to detect any health changes. The eight dogs in control group 1 were not treated. The eight dogs from each of groups 2, 3 and 4 received a single application of the formulation of fipronil, amitraz and (S)-methoprene on Day 0, 7, or 14, respectively. The novel combination was applied directly on the skin in two spots on the dorsal neck: one spot between the base of the skull and the shoulder blades, and the other at the front of the shoulder blades. Groups 2, 3 and 4 were treated with the recommended commercial dose volume of the novel combination (1.07 mL for dogs ranging from 2 to 10 kg body weight), i.e. with a dose to deliver at least 6.7 mg fipronil, 8.0 mg amitraz and 6.0 mg (S)-methoprene per kg of body weight. After completing treatment, all dogs were observed for any adverse reaction every hour for 4 h. 2.3. Tick infestation, tick counts and choice of skin biopsy sites On study Day 28, each dog was sedated and infested with 25 male and 25 female unfed adult I. scapularis. Live attached female ticks (thought to be the vectors of infection of dogs) were counted on Day 30 (48 ± 3 h after infestation) by parting the hair and visually counting the ticks on the entire body. After allowing five days for pathogen transmission, a final count of live female ticks was conducted on Day 33, and all ticks were removed from the dogs. At each tick count, all sites of tick attachment were marked on a standard drawing of a ventral and dorsal “silhouette” for each

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dog. When present, four tick attachment sites per dog were selected for skin biopsy because they had either the highest number of attached female ticks or the most notable inflammation when a single tick was attached. These sites were clipped and indicated with numbers 1–4 on the dog silhouette, as applicable. To keep sites identified for the skin biopsies, the clipping marks were refreshed on Days 49, 63, 77, and 91.

337

2.5. Blinding All personnel involved with general health observations, post treatment observations, tick counts, silhouette marking, selection of biopsy sites, refreshment of clipping marks, skin biopsy collection, serology, and tissue analyses were blinded to treatment groups. 2.6. Data analysis

2.4. Sample collection and tests 2.4.1. Serum samples and serological tests A blood sample was collected from each dog on Days −8, 27, 49, 63, 77, 91 and 104 (i.e., respectively: before treatments, before tick infestation, and at 21, 35, 49, 63, and 76 days after tick infestation). At each time point, serum was separated from each sample; three drops were assayed by the SNAP® 4Dx® test (IDEXX Laboratories, Inc., Westbrook, Maine, USA – ® SNAP and 4DX are registered trademarks of IDEXX Laboratories, Inc. in the United States of America and elsewhere), and the remaining serum was separated into 3 aliquots, which were stored frozen at −70 ◦ C. The SNAP 4Dx test detects antibodies to B. burgdorferi, A. phagocytophilum and Ehrlichia canis, and detects Dirofilaria immitis (heartworm) antigen in dog serum. The SNAP test has been shown to be 94.4–99.5% sensitive and 100% specific for the detection of B. burgdorferi infection, through the detection of antibodies to the C6 peptide of B. burgdorferi (Liang et al., 2000; Levy et al., 2002; O’Connor et al., 2002; Duncan et al., 2004; Littman et al., 2006; Bowman et al., 2009). For Anaplasma, the SNAP 4Dx test utilizes a synthetic peptide derived from the major A. phagocytophilum outer surface protein (p44/Msp2), providing 99.1% sensitivity and 100% specificity in detecting Anaplasma antibodies when compared to IFA (Chandrashekar et al., 2007; Alleman and Wamsley, 2008). On Day 104, one serum aliquot from each dog at each time point was shipped on dry ice to the Animal Health Diagnostic Center (AHDC), Cornell University College of Veterinary Medicine, Department of Population Medicine and Diagnostic Science, Ithaca, New York, USA. The AHDC tested each serum aliquot for B. burgdorferi antibody by kinetic ELISA (k-ELISA) in a single assay using published methods (Chang et al., 2001; Jacobson et al., 2004). 2.4.2. Skin biopsies On Day 104 (i.e., at 76 days after tick infestation), each dog was sedated. Each marked biopsy site was clipped and thoroughly scrubbed. A pair of adjacent 4 mm skin punch biopsies was collected from each site, using individual, sterile disposable instruments for each biopsy. When a dog had less than four tick attachment sites, the skin biopsies were collected from the sites identified most frequently on other dogs. Each biopsy sample was placed in an individually labelled sterile container, and refrigerated. All biopsies were packed on ice and shipped by overnight courier to the AHDC, where one skin biopsy from each site was cultured for B. burgdorferi re-isolation, whilst its paired biopsy was assayed for B. burgdorferi by polymerase chain reaction (PCR) using published methods (Chang et al., 2001).

The treatment efficacy for each treated group was assessed as the percent reduction in live female tick counts, as compared to counts from the control group, using the following formula (in which T and C were the geometric means of the live female tick counts in the treated and the control group, respectively):



Treatment efficacy (%) = 100 × 1 −

T C



In addition, the expected tick count in control group 1 was compared with the expected tick counts in each of the three treated groups at each counting day using Friedman rank test with the replicate used as the block. The comparison of the proportion of dogs satisfying a criterion in the control group versus each of the treated groups was done using Fisher’s exact test. For the k-ELISA B. burgdorferi antibody titres, equivocal results were classified with the negative titres. The loge [titre + 1] transformed titres were analyzed by a completely randomized design using the MIXED procedure in SAS® . Treatment was the fixed effect, and there was no random effect. At each time point, the expected mean titre of each of the groups treated with fipronil, amitraz and (S)—methoprene was compared with the expected mean titre of the control group. In addition, the proportion of dogs whose k-ELISA results were classified as positive (i.e., “low positive” or “positive”) for each treated group was compared with the proportion classified as positive for the control group by Fisher’s Exact test, which was computed using the FREQ procedure in SAS Version 9.1.3. For the skin biopsy PCR and culture results, the proportion of dogs with at least one positive result for each of the groups treated with the novel combination was compared with the proportion of dogs with at least one positive result for the control group by Fisher’s Exact test, which was computed using the FREQ procedure in SAS. 3. Results 3.1. I. scapularis infection rates Tick infection rates determined by PCR on a representative sample of 30 adult female I. scapularis ticks were 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum. 3.2. Animal health None of the dogs had any health abnormality detected during the qualifying physical examinations. No adverse

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600

Table 1 Attached live female adult Ixodes scapularis tick counts. ∼48 h after tick infestationb

Treatment groupa Group 1 Group 2 Groups 3 and 4

Geometric meanc Geometric mean % reductiond Geometric mean % Reduction

9.35 0.09 99.03% 0.00 100%

a Eight dogs per treatment group. Group 1 control dogs were not treated. Groups 2, 3 and 4 dogs were treated once topically with fipronil, amitraz and (S)-methoprene on Days 0, 7 and 14, respectively, e.g. 28, 21 and 14 days before each dog’s infestation on Day 28 with 25 male and 25 female I. scapularis. b Ticks were “thumb” counted on Day 30, at 48 ± 3 h after infestation. c Geometric mean computed by formula exp[ave] − 1, where “ave” is the average of the loge [count + 1] transformed values. d Percent reduction of tick counts in treated groups 2, 3 and 4 compared to untreated control group 1, computed by formula: 100 × [1 − T/C], where T and C are the geometric means of each treated group (T) and for the control group (C). For each calculated percent reduction, the p value was <0.01, as calculated by two-sided p-value of the Friedman rank test (testing the alternative hypothesis that the expected mean of listed treated groups and the expected mean of treatment group 1 are different).

Titres

338

500

400

300

200

100

0

reaction to treatment and no clinical sign of tick-borne disease were detected in any dog during the study. 3.3. I. scapularis counts On Days 30 and 33, respectively, untreated control dogs averaged 9.5 and 13.9 attached female adult ticks per dog, with ranges of 7–13 and 9–17 ticks per dog, respectively. In contrast, only one attached live female tick was found amongst all 24 treated dogs on Day 30, and no tick was present on any treated dog on Day 33. On Day 30, the efficacy of treatment with a combination of fipronil, amitraz and (S)-methoprene against I. scapularis (percent reduction in live female tick counts from the control group) was 99% for treatment group 2 and 100% for both treatment groups 3 and 4 (Table 1). 3.4. Serology All 32 dogs were seronegative for B. burgdorferi and A. phagocytophilum by the SNAP 4Dx and k-ELISA tests prior to allocation and treatment (Day −8) and prior to tick infestation (Day 27). All 24 treated dogs remained negative for B. burgdorferi and A. phagocytophilum by all tests throughout the study. 3.4.1. Kinetic ELISA test for B. burgdorferi Analyses of k-ELISA titres (see Table 2) showed no difference between the expected mean B. burgdorferi k-ELISA antibody titres prior to tick infestation (Days −8 and 27). From Day 49 (21 days after tick infestations) forward, the control group had a significantly greater proportion of positive k-ELISA tests than any of the groups treated with fipronil, amitraz and (S)-methoprene (p < 0.0001). The control group’s geometric mean titre steadily rose from 59.6 on Day 49 to 472.5 on Day 104, whilst it never exceeded 1.4 in the three treated groups. The continuous rise in k-ELISA antibodies to B. burgdorferi in each of the eight control dogs

20

40

60

Negative titre = 0-99 Equivocal titre = 100-199 Low positive titre = 200-299 Positive titre ≥ 300

80

100

Day

Fig. 1. Borrelia burgdorferi k-ELISA antibody titres of each group 1 untreated Control dog, from Day 27 to Day 104 (tick infestations on Day 28).

is represented in Fig. 1. In addition, at least 50% of dogs in the control group were classified as positive from Day 63 forward, and the expected proportion of positive dogs in the control group was significantly greater than in each of the three treated groups from Day 77 (49 days after tick infestation) forward. 3.4.2. SNAP 4Dx test All 32 dogs remained negative throughout the study for E. canis antibodies and D. immitis antigen. Individual SNAP 4Dx test results for B. burgdorferi and A. phagocytophilum antibodies and their analyses are presented in Table 3. None of the 24 dogs treated with the novel combination were positive for either B. burgdorferi or A. phagocytophilum antibodies at any time during the study. In contrast, 7 of the 8 control dogs became positive for C6 B. burgdorferi antibody from Day 77 forward, and 5 became positive for A. phagocytophilum antibody, giving significantly different (p < 0.026) expected proportions of positive dogs both A. phagocytophilum and B. burgdorferi in each of the 3 treated groups compared to the control group on Days 63, 77, 91, and 104. One control dog was positive for A. phagocytophilum SNAP4Dx tests of Days 49, 63, 77 and 91, but reverted to being seronegative for A. phagocytophilum on Day 104, suggesting that its antibody level had been near the threshold of detection on Days 49–91. The A. phagocytophilum SNAP 4Dx test can cross react and be positive in the presence of antibodies to Anaplasma platys (Yabsley et al., 2008; Carrade et al., 2009), but cross reaction was very

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Table 2 Borrelia burgdorferi kinetic ELISA antibody titres (k-ELISA units).a Treatment groupb

Dog ID

Treatment group 1c

740 742 748 741 757 761 763 758

Treatment groups 2, 3 and 4

Geo Meane Pct Posf Geo Mean Pct Pos p-Valueg p-Valueh

Day 27d

Day 49

0 0 0 0 4 0 25 0

78 188 38 103 36 83 87 10

0.8 0.0 0.3–1.2 0.0 0.434–0.740 1.000

59.6 0.0 0.0–0.9 0.0 <0.01 1.000

Day 63

Day 77

Day 91

Day 104

266 435 119 301 81 136 276 71

404 454 334 356 168 364 365 228

480 472 356 435 342 377 441 410

492 556 371 479 435 469 490 511

175.6 50.0 0.2–0.9 0.0 <0.01 0.077

320.4 87.5 0.0–1.0 0.0 <0.01 ≤0.01

411.2 100.0 0.2–1.4 0.0 <0.01 <0.01

472.5 100.0 0.0–0.5 0.0 <0.01 <0.01

Negative titre = 0–99, equivocal titre = 100–199, low positive titre = 200–299, and positive titre ≥300. Eight dogs per treatment group. Group 1 dogs were not treated (control group). Groups 2, 3 and 4 dogs were treated once topically with fipronil, amitraz and (S)-methoprene on Days 0, 7 and 14, respectively, i.e. 28, 21 and 14 days before each was infested with 25 male and 25 female Ixodes scapularis on Day 28. c Individual k-ELISA titre increases in group 1 also shown in Fig. 1. d Day 27 = day before tick infestations (all dogs infected, each with 25 male and 25 female unfed adult I. scapularis on Day 28). e Geo Mean = geometric mean, computed by averaging logarithm of titre + 1, taking the anti-log and subtracting 1. f Pct Pos = percentage of animals with titre classified as low positive or positive. g p-Value for comparison of group 1 with the Geo Mean titres of groups 2–4 (analyzed using mixed procedure in SAS® Version 9.1.3.). h p-Value for comparison of proportion of positive (low positive or positive) titres in group 1 versus groups 2–4 (analyzed using Fishers Exact test). a

b

the 8 control dogs had at least one positive biopsy PCR result.

unlikely in this study because the PCR conducted on the ticks used for infestation showed that A. phagocytophilum was the infecting species.

3.4.4. Skin biopsy culture and re-isolation of B. burgdorferi The B. burgdorferi culture and re-isolation results from skin biopsies taken on Day 104 (76 days after tick infestation) are provided and analyzed in Table 5. Six of the eight control dogs had 2–3 positive skin biopsy cultures. In contrast, none of the skin biopsy cultures collected from the 24 dogs treated with fipronil, amitraz and (S)-methoprene were positive. The culture collected from the site where a

3.4.3. Skin biopsy PCR for B. burgdorferi The B. burgdorferi PCR results on the skin biopsies collected on study Day 104 (76 days after tick infestation) are provided in Table 4. All B. burgdorferi PCR assays were negative for all of the skin biopsies collected from the 24 dogs treated with the novel combination (including the biopsy collected from the site where a female tick had been attached on Day 30). In contrast, 7 of

Table 3 SNAP® 4Dx® test results for antibodies to Borrelia burgdorferi and Anaplasma phagocytophilum.a Dog ID

Day 27

Day 49

Day 63

Day 77

Day 91

Day 104

Treatment group 1b

ID 740 ID 742 ID 748 ID 741 ID 757 ID 761 ID 763 ID 758

N/N N/N N/N N/N N/N N/N N/N N/N

N/N N/P N/N N/N N/P N/N N/N N/N

P/N P/P N/P P/N N/P P/P P/P P/P

P/N P/P P/P P/N N/P P/P P/P P/P

P/N P/P P/P P/N N/P P/P P/P P/P

P/N P/P P/P P/N N/N P/P P/P P/P

Tally group 1c

B. burgdorferi A. phagocytophilum

0 (0%) 0 (0%)

0 (0%) 2 (25%)

6 (75%) 6 (75%)

7 (88%) 6 (75%)

7 (88%) 6 (75%)

7 (88%) 5 (63%)

Treatment groups 2–4d

N/N – 0 (0%) (all 24 treated dogs negative for both agents at all time points, from Day 27 through Day 104)

p-Valuese B. burgdorferi A. phagocytophilum

1.000 1.000

a

1.000 0.467

<0.01 <0.01

<0.01 <0.01

<0.01 <0.01

<0.01 <0.05

N = negative, P = positive SNAP® 4Dx® test results for B. burgdorferi/A. phagocytophilum. Treatment group 1 dogs were not treated (control group). c Number (percentage) of positive animals. d Treatment groups 2, 3 and 4 dogs were treated once topically with fipronil, amitraz and (S)-methoprene on Days 0, 7 and 14, respectively, i.e. 28, 21 and 14 days before each dog was infested with 25 male and 25 female Ixodes scapularis on Day 28. e p-Values for difference between control group 1 and treated groups 2–4 results (Fishers Exact test). b

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Table 4 Day 104 skin biopsies: Borrelia burgdorferi PCR results.a Treatment Groupb

Dog ID

Site 1

Site 2

Site 3

Site 4

Group 1 Untreated controls

740 742 748 741 757 761 763 758

NEG NEG NEG NEG POS NEG POS POS

POS POS NEG POS POS POS POS POS

NEG POS NEG NEG NEG POS POS NEG

NEG POS NEG NEG POS POS POS NEG

All 24 treated dogs, groups 2–4

ALL

NEG

NEG

NEG

NEG

a

Samples collected on study Day 104. NEG = negative, POS = positive. Four skin biopsy sites were selected because they had either the highest number of attached female ticks or the most notable inflammation when a single tick was attached. For dogs that had less than 4 tick attachment sites, the attachment sites most frequently selected on the control dogs were selected. b 8 dogs per treatment group. Control group 1 dogs were not treated. Groups 2, 3 and 4 dogs were treated once topically with fipronil, amitraz and (S)-methoprene on Days 0, 7 and 14, respectively, i.e. 28, 21 and 14 days before each dog was infested with 25 male and 25 female Ixodes scapularis on Day 28.

dog from group 2 had a tick attached on Day 30 was negative for B. burgdorferi. In about two thirds of the dogs (21 out of 32), one or more of the 4 skin biopsies could not be assessed because of culture contamination. The 4 biopsies were contaminated in 2 dogs only (one each from Treatment groups 2 and 4). Contamination could have occurred during biopsy collection or sample handling in the laboratory, both conducted without knowledge of treatment group assignment. The control dog that remained negative for C6 B. burgdorferi antibody by the SNAP test had a positive skin biopsy PCR and culture, and the only control dog that did not have at least one positive PCR (or culture) was positive for C6 antibody from Day 77 forward. 4. Discussion The combination of tests used in this study showed that the I. scapularis ticks did infect 8/8 control dogs with B.

burgdorferi and 6/8 with A. phagocytophilum, whilst all 24 treated dogs remained free of any trace of infection by either pathogen. Co-infections with B. burgdorferi and A. phagocytophilum have been previously reported and are suspected to make dogs more susceptible to developing clinical disease (Beall et al., 2006; Bowman et al., 2009; Carrade et al., 2009). The time necessary for transmission of infectious agents by ticks likely varies as a function of tick–host–pathogen characteristics and relationships. In laboratory animal studies, I. scapularis only occasionally transmitted B. burgdorferi earlier than 24 h following infestation (Piesman et al., 1987; Kidd and Breitschwerdt, 2003). Although the minimum time necessary for the transmission of infectious agents by ticks to dogs is not known precisely and varies by agent, the ability of the combination of fipronil, amitraz and (S)-methoprene to disrupt or prevent I. scapularis infestations within 18 h (Baker et al., 2011) is likely to pre-

Table 5 Borrelia burgdorferi culture and isolation from Day 104 skin biopsies.a

Treatment group 1b

Treatment groups 2–4b

Dog ID

Biopsy site 1

Biopsy site 2

Biopsy site 3

Biopsy site 4

740 742 748 741 757 761 763 758

NEG POS NEG NEG POS NEG POS POS

POS POS CON NEG POS POS POS POS

POS POS NEG NEG CON POS POS POS

NEG NEG NEG NEG POS POS CON NEG

No positive biopsy

Average % of POS biopsiesc

Average number of biopsies not CONd

Dogs with at least one POS biopsye

79%

3.667

75% (6/8)

0%

2.5

0% (0/7 or 0/8)

p valuef

Not applicable

0.007

a NEG = negative, POS = positive, CON = contaminated. Four skin biopsy sites were selected because they had either the highest number of attached female ticks or the most notable inflammation when a single tick was attached. For dogs that had less than 4 tick attachment sites, the attachment sites most frequently selected on the control dogs were selected. b Treatment groups had 8 dogs each. Control group 1 dogs were not treated. Groups 2, 3 and 4 dogs were treated once topically with fipronil, amitraz and (S)-methoprene on Day 0, 7 or 14, respectively, i.e., 28, 21 and 14 days before each dog was infested with 25 male and 25 female Ixodes scapularis on Day 28. c For each group, the average percent of positive biopsies amongst those animals that had at least one positive biopsy. d For each group, the average number of biopsies not contaminated (4 biopsies per dog). e Proportion of dogs with at least one positive biopsy amongst dogs with at least one biopsy not contaminated (numbers in parentheses). f Two-sided p-value for Fisher’s Exact test comparing expected proportion of dogs with at least one positive biopsy in treatment group 1 with expected proportions of dogs with at least one positive biopsy in each treatment group (analysis conducted using SAS Version 9.1.3.).

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vent the transmission of any infectious agent carried by this tick species. The protection of all 24 treated dogs from infection by A. phagocytophilum is particularly significant because this pathogen can be found in the salivary glands of unfed I. scapularis and is therefore more likely to be transmitted faster than the pathogens that remain confined to the midgut until host attachment, such as B. burgdorferi (de Silva and Fikrig, 1995; Kidd and Breitschwerdt, 2003). 5. Conclusions In this study, field-collected I. scapularis ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 control dogs with A. phagocytophilum, whilst a single topical application of the new spot on formulation combining fipronil, amitraz and (S)-methoprene protected all treated dogs from infection with both agents for at least 28 days. To the authors’ knowledge, this study is the first time a single treatment with an ectoparasiticide has been shown to provide at least 28 days of complete protection of dogs against the transmission of both B. burgdorferi and A. phagocytophilum from carrier ticks. Conflict of interest The work reported herein was funded by Merial Limited, GA, USA. Six authors (CFB, STC, JPI, SLY, LGC and MGP) are current employees of Merial, and assisted with study design, data analysis and review of the manuscript, however there were no conflicting interests that may have biased the work reported in this paper. Acknowledgments The authors gratefully acknowledge the expert contributions of all technical staff from the Center for Vector-Borne Disease, the University of Rhode Island, TRS Labs, Inc., and Merial Limited in the execution and data analysis for this study. Drs. A. Glaser, Y.F. Chang and B. Wagner and their staff at the Animal Health Diagnostic Center of the Cornell University College of Veterinary Medicine were responsible for the execution and reporting of the k-ELISA tests and for the PCR and cultures of the skin biopsies. References Alleman, A., Chandrashekar, M., Beall, M., Cyr, K., Barbet, A., Lundgren, A., Sorenson, H., Wamsley, H., Wong, S., 2006. Experimental inoculation of dogs with a human isolate (NY18) of Anaplasma phagocytophilum and demonstration of persistent infection following doxycycline therapy. J. Vet. Intern. Med. 20, 763. Alleman, A.R., Wamsley, H.L., 2008. An update on anaplasmosis in dogs. Vet. Med. 103, 212–220. Anon., 1985. International Guiding Principles for Biomedical Research Involving Animals. Council for International Organizations of Medical Sciences (CIOMS), World Health Organization, Accessed 03 November 2010 at: http://www.cioms.ch/publications/guidelines/ frame guidelines.htm. Artsob, H., Maloney, R., Conboy, G., Horney, B., 2000. Identification of Ixodes scapularis in Newfoundland, Canada. Can. Commun. Dis. Rep. 16–26, 133. Baker, C.F., Hunter III, J.S., McCall, J.W., Young, D.R., Hair, J.A., Everett, W.R., Yoon, S.S., Irwin, J.P., Young, S.L., Cramer, L.G., Pollmeier, M.G., Prullage,

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