Efficacy of a combination of fipronil and permethrin in reducing the risk of Dirofilaria immitis transmission to dogs in field conditions

Abstract

Background

Dirofilaria immitis is a life-threatening mosquito-borne parasite affecting dogs. Prevention mainly relies on macrocyclic lactones (MLs), but D. immitis resistance to MLs and lack of compliance may occur. Multimodal strategies combining chemoprophylaxis and vector control with insecticide and repellent products are often recommended. This study evaluated the efficacy of Frontline Tri-Act® (IVP), a topical combination of fipronil and permethrin, in preventing the transmission of D. immitis by repelling mosquitoes in naturally exposed dogs.

Methods

This study was conducted in northern Greece, in an area highly endemic for D. immitis and with seasonal (spring to fall) mosquito season. Dogs were housed in outdoor kennels to allow natural vector exposure throughout the whole study. Twenty-four healthy, heartworm-naive, Beagle dogs were randomly allocated 1:1 to an IVP or an untreated control group. Phase 1 was conducted during the mosquito season, when dogs from the IVP group were treated monthly for seven consecutive months. Phase 2 was conducted after the mosquito season, when all dogs were left untreated and diagnosed for D. immitis infection via SNAP® Leish 4Dx and Knott’s test at scheduled intervals. Mosquito traps were also used to verify D. immitis infection rates, mosquito species composition and presence or absence in relation to Phase 1 and Phase 2.

Results

At the end of the study, 7 in 12 (58.3%) untreated control dogs were heartworm-positive, while the 12 IVP treated dogs remained negative, indicating 100% preventive efficacy (p < 0.001). Several hundred mosquitoes were trapped during Phase 1 and pooled for D. immitis Polymerase Chain Reaction (PCR) detection, 8 pools in 30 (27%) were positive. No treatment-related adverse events were reported.

Conclusions

Frontline Tri-Act® provided 100% protection against D. immitis transmission under natural mosquito challenge. These findings support its inclusion in multimodal strategies for heartworm prevention in endemic areas.

Graphical Abstract

Background

Mosquitoes form a monophyletic family of insects that live in all regions of every continent except Antarctica. They significantly impact humans as vectors of major pathogens, including malaria, filarioses, yellow fever, and dengue [1]. In veterinary medicine, mosquitoes are an important natural vector of several pathogens, including Dirofilaria immitis (Leidy, 1856), commonly known as “canine heartworm”, and D. repens and D. asiatica, the sub-cutaneous filarial worms [2]. There are more than seventy mosquito species recognized as Dirofilaria vectors, the two main species in Europe are Culex pipiens and Aedes albopictus [3, 4].

Dirofilaria immitis, the agent of ‘heartworm disease’ poses an important health threat to domestic and wild canids and is present worldwide across tropical, subtropical, and temperate zones, contributing to significant morbidity and mortality in canine populations [5]. The prevalence of D. immitis in Europe is limited to southern regions, where infection rates exceeding 11% in some domestic canine populations have been reported [6–8]. Around the Mediterranean Sea, the environmental conditions characterized by hot dry summers and mild wet winters, support natural mosquito transmission cycles, increasing infection risk among local canine populations, especially for the ones kept outdoor, where exposure to mosquitoes is permanent [8].

Therapeutic treatments of canine heartworm infection are complex and demanding for pets and their owners. They require long lasting, expensive, risky and potentially difficult to obtain drugs, with exercise restriction and possible severe adverse reactions or fatal outcome [9]. Given therapeutic treatments risks and concerns, reliable preventive medicine is essential to avoid any infection. The American Heartworm Society (AHS) [10] and the European Society of Dirofilariosis and Angiostrongylosis (ESDA) [11] recommend the use of products licensed for the prevention of canine heartworm disease that contain macrocyclic lactones (MLs), e.g., ivermectin, milbemycin oxime, moxidectin, or selamectin in oral, topical or injectable forms [12–14]. Presently, some licensed endectoparasiticide products combining ML, ectoparasiticide, and sometimes other nematicide compounds allow, with monthly administrations, the prevention of heartworm disease and the protection against ectoparasites and other helminths [15–17].

As mosquitoes are obligatory intermediate hosts and vectors for D. immitis, their control may reduce their transmission ability. Therefore, both AHS and ESDA acknowledge the possibility to improve prevention with multimodal approaches addressing both heartworm infection and vectorial transmission [10, 11]. Vector control measures include the elimination of mosquito breeding sites, environmental management, limitation of outdoor access during mosquito activity periods, and the use of environmental insecticides. A multimodal approach against the vector and the parasite becomes even more important with the growing concerns about ML resistance in D. immitis, reported for the first time in 2005 [18] and then clearly demonstrated a decade later in the USA [19, 20]. In the absence of an alternative class of drugs, MLs risk being overused in the domestic dog population and this could lead to D. immitis developing resistance. Australia [21] and some parts of the USA [12] recommend year-round use of ML drugs, which induces a risk of selection pressure that enables drug resistance [22].

One option to prevent the development and expansion of resistance to MLs is to prevent mosquitoes from taking a blood meal from a dog. Licensed products with anti-feeding (repellent) and insecticidal activity applied directly to the dog could achieve this [23]. Currently, few products have demonstrated efficacy against D. immitis transmission through repellency. A topical permethrin formulation combined with dinotefuran, and pyriproxyfen was demonstrated efficacious in preventing mosquito infection following exposure to microfilaremic dogs [24]. This combination was also shown to be efficacious against the transmission of an ML-resistant D. immitis strain (JYD-34) to dogs, when administered alone or in association with a milbemycin oxime (MO) registered product for the prevention of heartworm disease, while this MO product alone provided a lower protection against JYD-34 D. immitis infection [25].

A spot on combination of fipronil 6.76% w/v and permethrin 50.48% w/v (Frontline Tri-Act®, Boehringer Ingelheim Animal Health) has been demonstrated to be 100% efficacious for the prevention of Leishmania transmission to dogs exposed naturally to sandflies in a highly endemic area [26, 27]. It has also demonstrated strong repellency and insecticidal efficacy against three species of mosquitoes: A. albopictus, A. aegypti and C. pipiens [28], competent vectors for D. immitis transmission [29, 30]. Furthermore, the safety and the ectoparasiticide and anthelminthic efficacy of the concurrent use of Frontline Tri-Act® and NexGard Spectra® (Boehringer Ingelheim Animal Health), a combination of afoxolaner and MO, namely, indicated for heartworm prevention, has been demonstrated with a 6-month field study [31].

The study described in this manuscript tested the hypothesis that Frontline Tri-Act® used alone could prevent D. immitis transmission, through its repellent properties. The study was conducted in northeastern Greece (Xanthi), an endemic region for D. immitis [32] and Leishmania infantum [33], and with seasonal (spring to fall) mosquito activity, which made it highly relevant for the proposed study design.

Methods

Study design

This study was designed in compliance with Good Clinical Practices as described in the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products, VICH Guideline 9 [34].

It was a blinded, single-center, randomized, negative-controlled study. It was conducted from May 2024 to April 2025. It was organized in two phases, a first phase during the mosquito season (spring to fall 2024), when the dogs were treated monthly with Frontline Tri-Act®, and a second phase after the end of the mosquito season (fall to spring 2024/2025), when the treatments were stopped and dogs monitored for diagnosis of D. immitis infection.

Animal details, allocation and housing

The 24 included dogs were purpose-bred beagle dogs sourced form a licensed breeder and naive for D. immitis. The dogs had been raised in a flying insect-protected environment, in a non-endemic D. immitis area (central France). The dogs were 5–6 months, 12 males and 12 females (un-neutered) and weighed 3.8 to 8.2 kg at study start. They were tested D. immitis antigen negative with SNAP Heartworm–RT (IDEXX Laboratories) per manufacturer's instructions and confirmed healthy per veterinary examination at the supplier’s location, 9 days before shipment to the study facility. Neither the dogs, nor the environment had been treated with a repellent or insecticidal product (or other active ingredients deemed likely to interfere with the objectives of the study) with any residual activity at least 30 days prior to study start. The 24 dogs were ranked within sex and body weights (measured 3 days before shipment) and subsequently blocked into 12 blocks of two dogs each and randomly allocated to the untreated control group or the Frontline Tri-Act® (IVP) treated group. The allocation was defined before shipment, so that the dogs could be placed in their kennels and treated with the IVP immediately at arrival (see Phase I) to avoid pre-treatment mosquito exposure. The dogs were transported in trucks with insect control measures, and the animals, trucks and drivers were inspected by the Greek authorities at arrival.

On study site, the dogs had a monthly clinical examination and bodyweight measurement on the day (and before) of each treatment and monthly during the second phase. Dogs were vaccinated against common canine diseases using commercial dog vaccines.

Dogs were housed in an outdoor open-air kennel throughout the study, where they stayed night and day for optimum mosquito natural exposure. The kennel was organized into two separate and identical sub-units, each dedicated to one study group and separated by approximately 30 m, so that no physical contacts between study groups were possible. In each sub-unit, dogs were pair-housed within sex in 2.0 × 2.0 × 2.0 m cages. The dogs were exposed to ambient temperatures and natural sunlight; each cage was protected from direct sun exposure and rain. There were two sleeping boxes in each cage, allowing for a covered place to sleep and an elevated sitting position. Toys were provided on a weekly basis. The sleeping boxes were equipped with warm beddings during winter.

Phase I: days 0–196—exposure to mosquitoes and monthly treatments (May 2024 to Nov 2024)

Dogs in the treated group were treated with Frontline Tri-Act® following label instructions, seven times at monthly intervals (on days 0, 28, 57, 84, 112, 140, and 169). Day 0 was the day of arrival at study site.

On days 84 and 197, all dogs were tested for D. immitis antigens with SNAP® Leish 4Dx (IDEXX Laboratories) serology tests, per manufacturer’s instructions. This test also detects antibodies of Leishmania infantum, Anaplasma spp., and Ehrlichia spp.

Two mosquito CDC light traps (one trap per group/sub-unit) were used overnight twice during the peak season, on days 56 and 112. They were placed two meters above the ground at the south side of the kennel to minimize exposure to wind (the traps were used without CO₂). The trapped mosquitoes were collected, counted and stored in ethanol. The species were identified morphologically according to a published method [35], and pools of 5 to 15 mosquitoes were analyzed for D. immitis infection by PCR, using QIAmp DNA Mini kit for DNA extraction targeting D. immitis-specific sequences as published in the literature [36]. Late in Phase 1, starting on day 142, the traps were placed permanently and verified once a week for presence/absence of mosquitoes. A significant drop of mosquitoes was seen on day 177 with only one of the two traps with a mosquito, and no mosquito was trapped afterward. Phase I ended when there were two consecutive light trap collections without any mosquitoes. Application of Frontline Tri-Act® was stopped at this time.

Before and during Phase I, no environmental insecticide was used to control the flying insect population.

Phase II: days 197–360—observation and diagnosis (Nov 2024 to Apr 2025)

The dogs remained in the same enclosures. The environment was sprayed monthly with long-acting residual alpha-cypermethrin insecticide registered against mosquitoes and other dipteran insects (Fendona® Top, BASF), according to the manufacturer instructions, and the dogs were fitted with a deltamethrin collar (Scalibor®, MSD) on day 197 to protect them from sandfly and mosquito exposure (even though exposure was unlikely).

On days 197, 225, 257, 287, 317 and 348 dogs were tested for D. immitis antigens with SNAP® Leish 4Dx per manufacturer's instructions and Knott’s tests [37], designed for microscopic detection of microfilariae using standard parasitological methods with methylene blue staining and formalin fixation.

Efficacy assessment

The efficacy criterion was the incidence of D. immitis infection, determined either by a positive antigen test or Knott’s test at two consecutive sampling timepoints, or concordant positive results from both tests at a single timepoint. Dogs diagnosed as infected were considered positive for the remainder of the study.

Safety assessment

Safety monitoring included daily general health observations throughout both phases and monthly clinical examinations.

Statistical analysis

All statistical analyses were performed using SAS® version 9.4 (SAS Company, 100 Campus Drive Cary, NC 27513–2414, USA). Group differences for infection status were tested using Fisher’s exact tests. The efficacy of Frontline Tri-Act® was measured as percentage reduction in infection incidence compared to control.

Reduction in incidence:

$$=\frac{Incidenceinthecontrolgroup-Incidenceinthetreatedgroup}{\mathit{Incidenceinthecontrolgroup}}\times 100$$

A binomial regression model with a log link function and Pearson scale parameter was used to generate 95% confidence intervals. Statistical significance was set at an alpha level of 0.05.

Results

Dirofilaria immitis infection incidence

Phase I: The D. immitis antigen SNAP tests performed on days 84 and 197 were negative for the 24 dogs.

Phase II: In the negative control group, positive D. immitis antigen and microfilariae Knott’s tests occurred on day 225, when 4/12 dogs (33.3%) tested positive by both methods. Positivity increased to 6/12 (50.0%) on day 257 and reached 7/12 (58.3%) on day 287 when the seven dogs (three females and four males) had tested positive by both methods. No additional dog tested positive until the study ended on day 348.

In the Frontline Tri-Act® group, all twelve dogs remained negative across all assessed timepoints for both D. immitis antigen SNAP and microfilariae Knott’s tests.

The reduction in incidence of D. immitis in the treated group in comparison with the control group was 100% (p < 0.005).

Using the SNAP Leish 4Dx serology test, none of the 24 dogs in both groups tested positive for antibodies to Anaplasma and Ehrlichia. For L. infantum antibodies, none of the Frontline Tri-Act® treated dogs tested positive, while in the negative control group, four in 12 dogs (33%) tested positive (three dogs on day 197, and the fourth dog on day 225).

Mosquito collection and PCR

The two CDC light traps used during Phase I collected 313 mosquitoes on day 57 (July) and 368 mosquitoes on day 112 (September) for a total of 681 mosquitoes. The main collected species, identified through microscopic morphology, were Culex pipiens (510), followed by Aedes caspius (92), Anopheles Hyrcanus (54) and Ae. vexans (25). The numbers of mosquitoes by species were comparable in both light traps, on both collection days (days 57 and 112). The details and mosquito species are provided in Table 1.

Table 1.

Mosquito count and identification in both CDC light traps

Days	Mosquito species	Number
57 (July 2024)	Aedes vexans	5
	Anopheles hyrcanus	15
	Culex pipiens	243
	Total	313
112 (September 2024)	Aedes caspius	42
	Aedes vexans	20
	Anopheles hyrcanus	39
	Culex pipiens	267
	Total	368
Total		681

Pooled PCR screening of ethanol-preserved female mosquitoes (batch sizes ranging from 5 to 15 per pool) detected D. immitis DNA in 3 of 15 pools (20%) on day 57 and 6 of 15 pools (40%) on day 112.

No health abnormalities were observed in any of the dogs, and no adverse event occurred in relation to the investigated product.

Discussion

No adverse events related to Frontline Tri-Act® administration were recorded, and the product was well-tolerated.

The negative D. immitis antigen SNAP tests observed before shipment at the supplier’s facility and during Phase I, on days 84 and 197 for all dogs, confirmed that the 24 study dogs were not infected with D. immitis before study start. These antigen tests, based on immunochromatographic ELISA reaction, are highly accurate to the species level, without risk of cross reactivity with other Dirofilaria species. Antigens are produced by mature females, false positivity is thus possible in the presence of males only, but this is highly unlikely in the canine species.

The seven positive D. immitis antigen SNAP tests and microfilariae Knott’s tests observed in the untreated control group during Phase II on days 225 (4 dogs), 257 (2 dogs) and 287 (1 dogs), confirm that these dogs had been infected during Phase I. In consideration of the seven dogs together, the infections became patent during a 3-month period, between the last negative testing until the last positive testing, i.e., between days 197 and 287. In view of 5–6 month D. immitis pre-patent period in dogs [5], duration necessary for females D. immitis to mature and produce antigens and microfilariae, these data support that the mosquitoes were infective during a period starting in early June and ending in late September. Each time a dog tested positive, the two tests were concurrently positive, which supports the power of the D. immitis diagnoses.

As none of the Frontline Tri-Act® treated dogs had a positive D. immitis test during Phase II, the product was demonstrated efficacious for the prevention of D. immitis transmission under field conditions in naïve purpose-bred dogs, during the full 2024 mosquito season in an endemic Greek region. The high level of D. immitis infection in the untreated control group (58.3%) after only one season of exposure and the positive D. immitis PCR analyses of mosquito pools validate the suitability of the exposure model. It will be interesting to reproduce a comparable study in another D. immitis endemic region, with different D. immitis prevalence and mosquito vector species.

The observed positivity for Leishmania in the untreated control group (33%) confirms the endemic status of the study region (dogs were not tested before arrival at study site, but were born in the winter before the study and were reared in a flying insect-proof environment in a non-endemic area) and supports the suitability of the study model for flying insect-borne pathogens. The negative SNAP test results for Borrelia, Anaplasma, and Ehrlichia for all the dogs reveal that the study model is not adapted to assess protection against tick-borne pathogen transmission, most likely because dogs were not infested by ticks within the kennel environment during the study.

After completion of the study, the seven dogs with a positive diagnosis for D. immitis were retained at study site for another study requiring D. immitis infected dogs. The other 17 dogs were neutered, dewormed, vaccinated and re-homed. Dogs positive for Leishmania were treated using the protocol of miltefosine combined with allopurinol as reported by Solano-Gallego et al., 2009 [38].

Previous epidemiological and molecular studies have confirmed the role of Aedes and Culex species as D. immitis vectors in endemic regions [29, 39]. The observed absence of antigens and microfilaremia in dogs treated with this topical formulation of permethrin and fipronil corroborates earlier findings that repellent and insecticidal products can effectively block vector–host contact, thereby interrupting transmission [31, 40]. This contrasts with the untreated control groups, where progressive detection of infection reached 7 out of 12 dogs (58.3%) for D. immitis and 4 out of 12 dogs (33%) for L. infantum. The reduction of the risk of L. infantum transmission by Phlebotomus sandflies with the present combination of permethrin and fipronil was previously demonstrated using a similar study design [26, 27]. While existing studies have typically evaluated combination products involving systemic macrocyclic lactones, the additional prevention of infection under field challenge conditions using an insecticide and repellent formulation represents a significant improvement. Moreover, the integration of entomologic surveillance, vector PCR, and dual diagnostic confirmation (serology and Knott’s test) strengthens the methodological standard for heartworm prevention trials [38, 41].

Conclusions

The present study demonstrated that monthly administrations of Frontline Tri-Act® to dogs throughout an entire mosquito season in a highly endemic area for D. immitis provided high preventive efficacy against heartworm infection.

Author contributions

L.A., E.T., F.B. and E.P. conceived and designed the study, E.P. and G.S. carried out the study activities and laboratory work. M.M. monitored the experimental phase. L.A., E.T. and F.B. drafted the first versions of the manuscript. The authors read and approved the final manuscript.

Funding

This work was funded by Boehringer Ingelheim Animal Health, 29 Avenue Tony Garnier, 69007 Lyon, France.

Availability of data and materials

Data supporting the main conclusions of this study are included in the manuscript.

Declarations

Ethics approval and consent to participate

Animals were managed with due regard for their well-being, and the study designs and procedures were reviewed and approved by the Ethics Committee of the Aristotle University of Thessaloniki (133804/2024). The trial was approved by the Greek Veterinary Authorities (123387/621 issued on 19 April 2024). The test facility was the Investigator’s property.

Consent for publication

All authors have reviewed this document and consent to publish the data.

Competing interest

LA, ET and FB are employees of Boehringer Ingelheim. No other conflicts of interest were declared.