Laboratory assessment of the anti-feeding effect for up to 12 months of a slow release deltamethrin collar (Scalibor®) against the sand fly Phlebotomus perniciosus in dogs

Leishmaniasis consists of a wide group of diseases, caused by different Leishmania species. Leishmaniasis caused by Leishmania infantum, a disease primarily of dogs and humans occurs after susceptible hosts are exposed to the feeding behavior of L. infantum-infected feeding sand flies. While many infections subclinical, in susceptible individuals clinical signs, attributed to protozoon-induced deposition of immune complexes, may develop soon after infection, or may be insidious in onset following an incubation period of months to years [1–3]. Poor body condition, lymphadenopathy and exfoliative dermatitis represent the most common clinical manifestations in dogs, followed by a range of signs including, ocular disease, epistaxis and lethargy, with a potentially fatal outcome [1–4]. Immunosuppressed individuals are at greatest risk of developing clinical leishmaniasis. Traditionally prevalent in warmer latitudes including southern Europe, northern Africa, Middle East and central Asia, South and Central America and sporadically in the USA, Leishmania is endemic in locations which favor the sand fly life-cycle [2, 4–7]. Non-arthropod modes of transmission have also been reported [1]. The northward geographical spread of L. infantum in Europe has been linked to climate change-induced increases in ambient temperatures and to increased travel of pets between endemic and non-endemic areas [2, 4, 8, 9]. Sand flies feeding on infected dogs, whether or not those dogs are presenting clinical signs, ingest the protozoon and then transmit infection to the host of their next blood meal, be it a dog, a human or another animal species. Prevention of sand fly feeding on dogs is therefore an essential feature of disease control that helps limit the occurrence of infection in dogs and in people.

Numerous publications have demonstrated that the pyrethroid deltamethrin, impregnated into a collar and worn by dogs during the sand fly exposure season, reduces the incidence of Leishmania seroconversion under natural challenge conditions [10–15]. While these field reports, as well as two small-scale laboratory-challenge studies [16, 17] suggest that the anti-feeding activity of this collar is season-long, there is a need for well-controlled studies to investigate the specific duration of the period over which the collar prevents sand fly feeding, and the degree to which feeding is prevented. A one-year study was therefore designed with the objective of determining the anti-feeding and insecticidal efficacy against Phlebotomus perniciosus, the most commonly occurring sand fly in the Mediterranean basin [5], provided by a single application of the deltamethrin collar to dogs.

This was a randomized, assessor-blinded, single-site study in which the efficacy of the deltamethrin collar (Scalibor® protector band, Intervet International B.V., The Netherlands) in preventing engorgement of P. perniciosus in Beagles was compared to a placebo control group at the Charles River Laboratory in County Mayo, Ireland. Study personnel carrying out general health observations, clinical assessments, and assessments of P. perniciosus exposure, engorgement, and counting were masked to the treatments.

There was a 97% reduction in arithmetic mean numbers of engorged P. perniciosus in the deltamethrin group, relative to the control group, at the first P. perniciosus challenge assessment, seven days post-application of the collars (Table 1, Fig. 1). The deltamethrin collar then provided sustained anti-feeding efficacy of ≥ 94% throughout the study. At each post-application assessment, the mean counts of engorged P. perniciosus in the deltamethrin group were statistically significantly lower than those in the control group (ANOVA or Kruskall-Wallis H-test: P ≤ 0.0041) (Table 1, Fig. 1). The maximum number of engorged P. perniciosus collected from any dog with a deltamethrin collar was four, while in the control group the minimum to maximum number of engorged sand flies ranged between 12–54, thereby validating the challenge methodology (Table 1).

Day	Control			Deltamethrin collar			Comparison		% Efficacy AM (GM)
	AM ± SD	GM	Range	AM ± SD	GM	Range	χ 2 -value b F-valuec	P-value
7a	22.4 ± 5.0	22.0	16–29	0.6 ± 0.5	0.5	0–1	χ2 = 8.2206, df = 1	0.0041	97 (98)
28	24.8 ± 9.7	23.2	14–41	0.6 ± 0.7	0.5	0–2	χ2 = 11.5315, df = 1	0.0007	97 (98)
56	33.9 ± 12.1	32.1	20–52	1.3 ± 1.8	0.8	0–5	F(1, 14) = 113.92	<0.0001	96 (98)
84	31.5 ± 11.8	29.6	14–53	1.4 ± 1.1	1.1	0–3	F(1, 14) = 135.63	<0.0001	96 (96)
112	25.4 ± 10.0	23.7	14–42	1.1 ± 1.4	0.8	0–4	F(1, 14) = 111.83	<0.0001	96 (97)
140	31.6 ± 13.0	29.0	12–48	1.5 ± 1.4	1.1	0–3	χ2 = 11.4286, df = 1	0.0007	95 (96)
168	38.1 ± 8.7	37.3	27–53	0.6 ± 0.7	0.5	0–2	χ2 = 11.5315, df = 1	0.0007	98 (99)
196	40.0 ± 10.2	38.8	25–52	1.6 ± 1.4	1.3	0–4	χ2 = 11.3442, df = 1	0.0008	96 (97)
224	37.1 ± 6.3	36.7	28–46	1.8 ± 1.0	1.5	0–3	χ2 = 11.3947, df = 1	0.0007	95 (96)
252	36.4 ± 6.7	35.8	24–44	1.1 ± 0.8	1.0	0–2	χ2 = 11.4456, df = 1	0.0007	97 (97)
280	39.3 ± 5.9	38.9	31–51	1.6 ± 1.6	1.2	0–4	χ2 = 11.3947, df = 1	0.0007	96 (97)
308	41.4 ± 8.1	40.6	27–54	2.0 ± 1.2	1.7	0–4	χ2 = 11.4627, df = 1	0.0007	95 (96)
336	39.3 ± 8.9	38.3	25–52	1.9 ± 1.5	1.5	0–4	χ2 = 11.3442, df = 1	0.0008	95 (96)
364	35.4 ± 6.1	34.9	27–42	2.3 ± 1.3	2.0	0–4	χ2 = 11.3947, df = 1	0.0007	94 (94)

Abbreviations: AM arithmetic mean, SD standard deviation, GM geometric mean, df degrees of freedom

^aDay 7 data for one control and three deltamethrin collar dogs were not included because their collars were damaged

^bKruskal-Wallis test

^cANOVA

All dogs in both groups gained weight during the study. There were no treatment-related adverse events.

This is the first well-controlled laboratory study to directly demonstrate that the deltamethrin collar provides a strong and sustained anti-feeding effect on sand flies for up to one year. Therefore, a deltamethrin collar maintained on a dog for one year could prevent or reduce the risk of transmission of L. infantum for this extended period. These results help to explain the reported reductions in L. infantum infections from two-season field studies in which dogs received collars in consecutive years and were exposed to natural challenge [10–15].

In a study in northern Tunisia involving 80 Leishmania-free dogs, none of the 42 dogs treated with the deltamethrin collar seroconverted to Leishmania, while 15.8% of the control dogs seroconverted [10]. In another study, collars were applied to dogs in a town located in a Leishmania endemic region in southern Italy, while dogs in four neighbouring towns did not receive collars [11]. After the 1997 and 1998 seasons, 2.7 and 3.5% of collared dogs seroconverted, respectively, compared to a seroconversion rate of 5.4 and 25.8% of control dogs. This translated to protection rates provided by the collar of 50%, under conditions of apparent relatively low challenge, to 86% when challenge was more intense. In a 2008 report of a study in a coastal region of north-western Italy, just three of 119 (2.5%) collared dogs seroconverted, based on a Leishmania immunofluorescent antibody assay, compared with 15% of untreated control dogs [12]. A study in southern Italy estimated the anti-Leishmania protection rates provided by the collar, relative to un-collared control dogs, to be 72.3 and 41.2% in 2003 and 2004, respectively [13]. Of the seroconverting dogs in that study, clinical signs of canine leishmaniosis were significantly more frequent and rapidly progressive in control than in collared dogs. A study conducted in Brazil between January 2014 and November 2015 compared Leishmania infection rates in seronegative dogs from two neighboring endemic areas; dogs in one area received deltamethrin collars, those in the other area did not [15]. Despite the many limitations involved in an extended large-scale field study involving roaming domestic dogs, the results from serology testing showed a tendency for the deltamethrin collar to provide population-based protection against infection with Leishmania.

In another field study involving a twenty-year follow-up of canine leishmaniosis incidence in military working dogs, the reduction of the risk of leishmaniosis infection provided by deltamethrin-impregnated collars was demonstrated [14]. Usage of the deltamethrin collar started in 2002, and by comparing the incidence of leishmaniosis in the military kennels from 1993 to 2001, with the incidence observed in the period 2002 to 2012, the authors demonstrated a leishmanial protection of collared dogs of 93.8%.

Sand flies are active from dusk until dawn, with a reported peak of activity from 11 pm to 2 am [5]. The exposure period of one hour in our study was possibly shorter than exposures under field conditions. However, sedation of the study dogs would have made them more susceptible to becoming hosts for sand fly bites, as shown by the engorgement rates of flies exposed to control dogs. Although in the control dogs in our study those rates were lower than those reported from some other similar experiments, they were sufficient to validate the experimental methodology [18, 19]. Additionally, our results are consistent with, and extend those of a 1997 report of a shorter study from southern France [16]. In that study, the efficacy of the deltamethrin collar against sand fly bites in dogs sedated for two-hour exposure durations was 96% over 34 weeks.

Deltamethrin, like most pyrethroids, provides spatial-repellency and/or contact irritancy to insects [20, 21], and this effect is postulated to explain why the deltamethrin collar in our study provided a strong (≥ 94%) anti-feeding efficacy, with lower insecticidal efficacy (0–77%). Although the freezing of flies immediately following removal from the exposure chambers may have resulted in an underestimation of the insecticidal activity of the collar, the insecticidal activity we found generally aligns with an earlier study finding of a fly mortality between 21–60% over an 8-month exposure period of collared dogs to sand fly challenge [16]. We therefore postulate that the strong anti-feeding effect of the collar is due to avoidance of the sand flies to make sufficiently long contact with the protected dogs to engage in feeding behavior.

A number of studies have demonstrated that low volume insecticide “spot-on” and sprays applied to dogs can also be useful in the prevention of sand fly engorgement on dogs [19, 22–25]. Importantly, these products need to be applied once or twice monthly, meaning that owners must be conscientious in adhering to treatment recommendations. Additionally, optimal protection of on-dog, short-term measures requires accurate determination of the beginning and end of the sand fly season, which has been shown to vary by location, and from year to year due to changing climatic factors [5]. The extruded collar formulation used in this study provides extended deltamethrin release and is a key factor in the observed 12 month duration of anti-feeding efficacy. This extended duration of protection that could allow just once yearly treatment can be particularly relevant in geographies, for instance in areas of South America, where phlebotomine flies present an almost year-long risk of transmission of L. infantum [26, 27]. Additionally, recent evaluation of long-acting ectoparasiticides has shown that increasing the duration of protection leads to increased owner compliance and therefore more months of protection during the year [28]. Thus, once-yearly application of the deltamethrin collar to dogs reduces the risk of infection with Leishmania, relieves the pressure on dog owners for compliance to a repeated treatment scheme, and avoids the need for defining the beginning and end of the sand fly season.

In dogs receiving the slow release deltamethrin collar, sand fly anti-feeding efficacy was ≥ 94%, relative to unprotected control dogs for one full year following treatment. Thus, one collar applied to a dog for one year can prevent or reduce sand fly transmission of Leishmania for this entire time.