Fleas

It would seem that fleas belonging to the genus Ctenocephalides had their origins in Africa (Ménier et al. [4] and Beaucournu et al.[5] recognize 13 species in this genus). Undoubtedly the most important of these, C. felis, has two subspecies, namely C. felis felis and C. felis strongylus. The so-called cat flea C. felis felis feeds on domestic cats and dogs, and has been transported worldwide by humans on their pets, and is seldom if ever found on wild felids [6]. However, its sibling subspecies, C. felis strongylus has largely remained a parasite of small wild felids in Africa [5, 6]. C. felis felis is often more frequently encountered on dogs than the so-called dog flea, C. canis. In addition to the irritation caused by fleas, dogs often develop a condition known as flea allergy dermatitis [7] resulting from repeated exposure to flea bites. Furthermore, C. felis felis is an intermediate host of the tapeworm Dipylidium caninum[1].

The eggs and the three larval stages of dog and cat fleas as well as the pupa are free-living within the sleeping and resting environment of dogs, while the adults are for all practical purposes permanent parasites on their hosts [8]. Under conditions of optimal temperature and humidity, the life cycle from egg-laying until the emergence of adult fleas from pupae, can be completed in 23 days [8].

Ticks

Ticks act as vectors of a wide variety of protozoan, bacterial and viral diseases in domestic livestock and companion animals [3, 9]. There are four stages in the life cycle of ixodid ticks, namely eggs, larvae, nymphs and adults. Once the eggs have hatched, all subsequent stages require a blood meal before they can continue their development.

Several surveys on the ticks infesting dogs have been conducted in various European countries and Dermacentor reticulatus Ixodes ricinus Ixodes hexagonus and Rhipicephalus sanguineus were commonly recovered [10–12]. In Britain and Ireland I. ricinus followed by I. hexagonus were the tick species most prevalent on dogs [13–15], and no R. sanguineus was recovered in any of the British surveys.

In studies conducted in the southern USA, Dermacentor variabilis Ixodes scapularis and R. sanguineus were common parasites of dogs [16, 17], while the only Rhipicephalus species present in South America is R. sanguineus, and it was introduced by humans [18]. In surveys in southern Africa and eastern Australia R. sanguineus was the dominant species [19–23]. It would thus appear that depending on locality, I. ricinus I. scapularis D. reticulatus D. variabilis and R. sanguineus are amongst the tick species most frequently encountered on dogs in both Hemispheres.

Mites

The mite Sarcoptes scabiei is the cause of sarcoptic mange or scabies in a wide range of host species. By preference the mites colonize the less hairy regions of the host’s body. Clinical signs in dogs include a fairly constant pruritus, an erythematous rash, and papules and yellowish crusts that form on the skin surface as well as alopecia [24].

Lice

While infestation with fleas and ticks may be common, infestations with lice are usually rare on dogs, and are often associated with dogs debilitated by disease, age or neglect, on which burdens may become exceedingly large [25].

Active ingredients

Imidacloprid is an ectoparasiticide belonging to the group of chloronicotinyl compounds, and has been registered in European Union (EU) member states as a veterinary medicinal product for use on dogs and cats since 1997. Flumethrin is an ectoparasiticide that belongs to the group of α-cyano-pyrethroids, and has been registered in EU member states for use on companion and food-producing animals since 1986. These chemicals have been combined in a slow release matrix collar formulation incorporating 10% imidacloprid (w/w) and 4.5% flumethrin (w/w). The imidacloprid component of the collar is aimed at the treatment and control of fleas and lice, and the flumethrin component at the treatment and control of ticks and mites. The medicated collar is expected to provide sustained protection over a period of eight months to dogs exposed to ticks, fleas and lice.

The objectives of the present studies are to demonstrate the efficacy of the imidacloprid/flumethrin collar formulation against fleas, ticks, mites and lice on dogs.

General materials and methods

Each study was designed to ascertain the immediate and/or long-term efficacy of a collar containing imidacloprid 10% (w/w) and flumethrin 4.5% (w/w) against a specific ectoparasite or ectoparasites on a group of experimentally infested dogs, compared to an untreated control group of dogs infested with the same ectoparasite or ectoparasites.

In accordance with the protocols for the various studies, custom-bred beagles or mixed-breed dogs that had not been treated with an acaricide, or insecticide, or a compound with an insect-growth regulating activity during the previous 60 to 90 days, were used. The dogs were maintained and handled with due regard for their welfare, and were acclimatized to the kennel environment seven to ten days prior to the commencement of a study. Dogs were individually housed in pens in indoor animal units that conformed to the national standards for floor area, lighting and temperature of the various countries in which the studies were conducted. Water was available ad libitum and an adequate amount of a commercial dog food towards their maintenance was provided daily.

Parent-stock fleas were reared on artificially infested dogs or cats, their eggs collected and incubated with flea rearing medium (dried, powdered beef blood and sand 1:4). The resultant adult fleas were collected and the numbers stipulated in the protocols for the various studies counted and placed in separate containers. These containers were opened on the dogs’ necks and the fleas were allowed to disperse in their hair. Parent-stock ticks were reared on artificially infested dogs or rabbits. Dogs were lightly sedated or restrained by hand and specified numbers of ticks were released onto their backs or necks and allowed sufficient time to disperse into the hair before the animals were released.

Approximately four days before the collars were fitted all animals were infested with a prescribed number of fleas or ticks of the species to be targeted. In some instances a slightly larger number of dogs than specified in the protocol were infested. One or two days after this infestation flea or tick counts were conducted. At this stage surplus animals that harboured the lowest numbers of fleas or ticks were excluded from the remainder of the study. The dogs that qualified for the study were ranked in descending order on their pre-treatment parasite counts, and animal ID’s were used to break ties. Thereafter they were randomly allocated to treatment groups. Each treated and each untreated control group of dogs consisted of at least seven individuals.

One or two days before the collars were fitted all animals in each study were infested with pre-determined numbers of fleas or of ticks or with both fleas and ticks of the species to be targeted. On Day 0 the test collars were fitted to the necks of the dogs allocated to the treatment group, and two days after the collars had been fitted flea and tick counts were done. The collar size was determined by the dog’s weight. Dogs that weighed less than 8 kg were fitted with collars designed for small dogs (35 cm in length) and those weighing more than 8 kg were fitted with collars designed for large dogs (66 cm in length). After the collar had been secured by means of the buckle the loose end was passed through the retaining loop or loops. The excess collar was cut off approximately 2–3 cm from the last retaining loop. At specified time intervals on the day of treatment all animals were carefully observed for adverse signs that could be ascribed to the collars or their active ingredients.

At prescribed intervals thereafter all the dogs in the study were re-infested with fleas or ticks depending on the target species. A day after these infestations flea counts were done and two days after the infestations tick counts were performed. This procedure was repeated at about 28-day intervals for approximately 34 weeks after the collars had initially been fitted.

Fleas were counted by combing the hair with a fine-toothed flea comb to recover fleas present in the animal’s pelage. Several strokes of the comb were made over each body region, each time moving in the same direction following the lie of the hair coat. Fleas showing no movement were considered dead. Tick counts were performed by intensive examination and palpation of the dog’s skin. When a tick was found the hair was parted and visual confirmation of the tick’s attachment status made. Once the ticks discovered by these methods had been removed, the dogs were either combed to ensure that all ticks had been collected, or thoroughly searched until the investigator felt confident that all ticks had been found. Male and female R. sanguineus, D. reticulatus and D. variabilis were counted, but only female I. ricinus and I. scapularis were counted because the males of Ixodes species seldom attach.

The studies were conducted at facilities in Germany, North America and in South Africa and the experimental designs of each were not necessarily identical. For the sake of uniformity, efficacy determinations have thus been allocated to 28-day or 4-week blocks, and any determination of efficacy during a 4-week window was included in the window within which it had been calculated.

Efficacy evaluations are in accord with those published in document EMEA/CVMP/EWP/005/2000-Rev.2 of the European Medicines Agency.

Percent efficacy against fleas was calculated as follows:

Efficacy $\left(\%\right)=100\text{x}\left(\text{mc}–\text{mt}\right)/\text{mc}$, where

mc = geometric mean number of live fleas on the untreated control group of dogs

mt = geometric mean number of live fleas on the treated group of dogs.

Acaricidal efficacy was calculated as follows:

Acaricidal efficacy$\left(\%\right)=100\text{x}\left(\text{Gmc}–\text{Gmt}\right)/\text{Gmc}$

Where Gmc = Geometric mean number of live ticks (categories 1–3) on dogs in the untreated control group at a specific time point.

Gmt = Geometric mean number of live ticks (categories 1–3) on dogs in the treated group at a specific time point

Sustained acaricidal efficacy against subsequent re-infestations, was calculated as follows:

Sustained efficacy $\left(\%\right)=100\text{x}\left(\text{Gmc}–\text{Gmt}\right)/\text{Gmc}$

Where Gmc = Geometric mean number of live ticks (categories 1–3) on dogs in the untreated control group at a specific time point.

Gmt = Geometric mean number of live and dead ticks (categories 1–3 & 6) on dogs in the treated group at a specific time point.

According to the EMEA guidelines, dead engorged ticks (Category 6) should be included with the 3 categories of live ticks as they had succeeded in engorging before being killed.

The use of geometric rather than arithmetic means for the calculation of efficacy in the studies reported here was justified by the statisticians involved in each study. Consequently, unless stated otherwise, all efficacy values have been based on a comparison of transformed geometric means of the individual parasite counts of treated and control animals. Geometric means better reflect the skewed distribution of the numbers of parasites recorded on individual animals within the small groups of animals of necessity included in an experimental study. In fact, in the case of anthelmintic efficacy studies, in which non-normal distributions of parasitic burdens are commonplace, the use of geometric means is practically a prerequisite. However, when large numbers of animals are involved, such as in field trials, both methods of calculating efficacy are acceptable.

Because zero flea and tick counts were recorded in many of the present studies, calculations were based on the geometric means of the individual flea or tick counts (count + 1). One (1) was subsequently subtracted from the result to obtain a meaningful value for the geometric mean of the study groups. Descriptive statistics (mean, minimum, maximum, standard deviation, CV%, geometric mean and median) on flea or tick counts for the various assessment days were also calculated.

Efficacy against adult fleas

Four long-term studies, aimed at determining the efficacy of the imidacloprid/flumetrin collars against C. felis felis, were conducted. Infestation with fleas, the application of collars, the determination of flea numbers and the assessment of efficacy followed the procedures described in the General Materials and Methods and summarized in the Schematic Experimental Design (Table 1).

Onset and speed of efficacy

In order to determine the onset of efficacy of the imidacloprid/flumetrin components of the medicated collars against C. felis felis, dogs were infested with fleas one day before the collars were fitted and efficacy was measured 24 h later. A second study was designed in an attempt to determine the time lapse between collar fitment and the elimination of fleas. In this study the control and the treated group of dogs in one of the long-term studies were infested with fleas on Day 6, after the collars had been fitted and fleas were collected 12 h later on Day 7. Once these collections had been completed the same two groups of dogs were infested on Day 7 and fleas collected 6 h later, and finally the same groups of dogs were again infested with fleas on Day 7 and fleas collected 2 h thereafter. Speed of efficacy could thus be determined at 2 h, 6 h and 12 h intervals after infestation.

In vitro larvicidal efficacy

This study was designed to assess the effect that residues of imidacloprid and flumethrin on blankets that had been in contact with treated dogs, had on the development of flea larvae. The study commenced on Day-7 and continued until Week 35 after the collars had been fitted. Fleecy polyester blankets were fastened to wooden boards, which were then placed on the floor of transport boxes. The untreated control dogs and the collared dogs were confined in these boxes for 3 h on three consecutive days from Days −7 to −5 and again from Days 12 to 14 and thereafter for three consecutive days 3 days prior to each efficacy assessment day. At the end of each 3-day exposure period the blankets were removed and a circular sample, 8.5 cm in diameter, cut from the centre of each blanket. The wooden boards were cleaned with hot water, soap and ethanol and new blankets were attached before the next period of exposure to the control and treated dogs. The blanket samples were placed in Petri dishes and frozen at approximately −20°C for at least 24 h. After removal from the freezer the Petri dishes were allowed to reach room temperature. Thereafter, approximately 50 one-day old flea eggs were placed in the middle of each blanket-sample and about 0.5 g of flea rearing medium (dried, powdered beef blood and sand 1:4) was distributed in a very thin layer over the surface of each sample. The samples were incubated at 26 ± 2°C and 75 ± 8% relative humidity for 27 or 28 days. To facilitate counting of the adult fleas that had emerged, the Petri dishes were frozen at −20°C for at least 2 h. The geometric mean numbers of fleas on the blanket samples exposed to the untreated control dogs compared to the numbers on the blanket samples exposed to collared dogs were used to calculate efficacy.

Flea allergy dermatitis

During the laboratory efficacy studies the dogs were repeatedly infested with a fairly large number of fleas, followed by a 4-week break. This is possibly the ideal way to provoke an allergic reaction. All the dogs in the various studies were observed daily and were clinically examined at pre-scribed times. Signs of flea allergy dermatitis and other conditions were recorded during these observations and examinations.

Efficacy against adult fleas

The sustained efficacy of the imidacloprid/flumethrin collars, without necessarily being flea-repellent in the strict sense of the word, implies that treated dogs will remain attractive to fleas, which will then be killed almost immediately, and certainly before they can lay eggs. Moreover, the existing flea population within a collared dog’s household or surroundings will progressively die-out as the animal will act as a lethal ‘vacuum-cleaner’ over a protracted period of time.

Onset and speed of efficacy

The imidacloprid/flumethrin collars had an immediate lethal effect on an existing one-day old flea population, and also killed fleas within 2, 6 or 12 h after infestation. The results obtained in the onset of efficacy study were based on a single experiment, and the speed of efficacy results were obtained when the collars were 7 days old and the concentration of imidacloprid and flumethrin on the haircoat of the dogs presumably high. Further studies to support these findings are thus indicated. Nevertheless, the above results imply that under field conditions fleas that are present on a dog will be killed within 24 h of a collar being applied and that fleas in any new infestations acquired from the environment are likely to be killed within 2 h after getting onto a collared dog.

In vitro larvicidal efficacy

Flea allergy dermatitis

Considering the multiple infestations with fleas during the long-term studies, it is perhaps surprising that so few dogs in the untreated control groups developed signs of FAD, and then only in two of the six long-term studies. This suggests that other factors may also have been involved in the latter two studies.

Since the clinical signs of FAD are directly related to exposure to flea bites, there are two main strategies in treating animals exhibiting this allergic reaction. The first is to reduce the animal’s exposure to flea bites and the second is to administer appropriate palliative treatment to reduce the pruritus and inflammation. A combination of both strategies usually produces the best results.

Spot-on products containing imidacloprid have been used for several years as an integral component in the treatment of FAD by eliminating the fleas responsible for the allergy. In a field trial conducted at several veterinary clinics in Italy, 1939 flea-infested dogs, of which more than 34% exhibited signs of FAD were presented for treatment [7]. A single treatment with a spot-on formulation of imidacloprid (Advantage®) caused a rapid decline in the percentage of dogs infested with fleas. At the same time there was a marked improvement in the frequency of pruritus associated with FAD until almost complete remission 28 days later [7]. There was also a marked improvement in the extent of alopecia during this time.

The effect of imidacloprid on fleas is rapid and in the present study the imidacloprid/flumetrin collars were 100% effective in killing fleas within 2 h of artificial infestation (Table 2). Thus fleas are likely to be eliminated before they can feed and provoke FAD by the injection of their saliva.

The use of ‘knock down’ insecticides, or those with only a limited time-span of efficacy, requires strict owner-compliance with the prescribed treatment regimen for dogs with FAD. However, the long-term efficacy of the slow-release imidacloprid component of the collars ensures sustained protection against flea bites, thus preventing intermittent exposure to flea saliva which initiates, provokes, sustains or exacerbates FAD.

Ticks

Efficacy against adult ticks

Onset and speed of efficacy

Repellent efficacy

However, 48 h after each infestation all the ticks resulting from each infestation in the study were removed and counted. This was done because the first study was designed in such a manner that not only repellent effectiveness could be detected 6 h after each infestation, but also long-term sustained acaricidal efficacy could be determined 48 h after the same infestations (Tables 7, 8).

In vitro acaricidal efficacy

This study was designed so that the in vitro effect that imidacloprid and flumethrin residues on the hair coat of a collared dog had on the nymphs and adults of I. scapularis could be measured. To this end three female dogs were acclimatized to the study conditions 10 days prior to treatment. Four days before treatment ten hair samples were taken from the same places on each animal, namely the dorsal and ventral surfaces, both lateral sides, and all four legs. The ten samples were first weighed and then combined and well-mixed for each dog separately. The total weight of the ten combined samples always exceeded 5.8 gm. Approximately 1 gm of hair from the combined sample of each dog was added to each of six Petri dishes. Ten unfed I. scapularis nymphs and ten unfed adults were added to the samples in the Petri dishes. These were counted two days later and all were still alive.

The three dogs were randomly allocated to three treatment groups and placed in separate pens. Imidacloprid/flumethrin collars were fitted to two of the dogs and the third remained untreated. One of the treated dogs was randomly chosen for hair-sampling and the other treated dog served as a backup for this dog.

Efficacy against adult ticks

At the commencement of the various studies the therapeutic efficacy of the collars against 1-day old R. sanguineus infestations varied between 32.2% and 46.5%, and against 2-day old infestations it varied between 64% and 84.8% (Table 7). Efficacy against re-infestation with ticks 7 days after the collars had been fitted exceeded 96%, and remained at this level or higher against re-infestations until the termination of one of the studies at 24 weeks, and in two studies terminated at 35 weeks. In the fourth study efficacy decreased to 90.3% by Week 32 and thereafter recovered to 93.2% by Week 35 (Table 7).

The therapeutic efficacy of the medicated collars against a 2-day old infestation of I. ricinus varied between 76.7% and 89.9%, and was 96.4% against a 1-day old infestation of I scapularis (Table 8). Thereafter the sustained acaricidal efficacy against re-infestations of either species remained at 100% for the 34-week duration of the study (Table 8).

The therapeutic efficacy of the collars was 69.7% against a 1-day old infestation of D. reticulatus, and 66.1% against a 2-day old infestation. The sustained acaricidal efficacy of the collars against re-infestation with D. reticulatus was consistently above 97% during the 24-week long study, and but for one transitory decline to 92% at 16 weeks, efficacy exceeded 96% in the 34-week long study (Table 9). The therapeutic efficacy of the collars against a 1-day old established infestation of D. variabilis was 69.1% and the repellent acaricidal efficacy against re-infestation at or above 98.5% during the initial 16 weeks of the study, and thereafter exceeded 90% until the termination of the study at 34 weeks (Table 9).

The collars were particularly effective against re-infestation with both Ixodes species that were targeted. Efficacy of 100% was recorded on every assessment day from Week 1 to Week 34 for I. ricinus and I. scapularis. Although the American tick, D. variabilis appeared to be slightly less sensitive to the flumethrin component of the collars than the European tick, D. reticulatus, efficacy against the former tick never dropped below 90% during the 34 weeks of the study.

As can be deduced from the results summarized in Tables 7, 8 and 9, the flumethrin component of the collars in many instances was not immediately >90% effective against ticks that had been attached to dogs for one or two days at the time-point of collar application (=therapeutic or curative efficacy). This was particularly noticeable for R. sanguineus. However, when infestation took place directly after the collars had been fitted (=preventive efficacy) effectiveness against both R. sanguineus and I. ricinus exceeded 96% (Table 10). Furthermore, two days after the collars had been fitted the concentration of flumethrin on the hair coat of collared dogs was already sufficient to reduce the number of surviving and/or feeding R. sanguineus and I. ricinus 6 h after infestation by 99.2% and 100% respectively (=repellent efficacy) (Table 10).

Onset and speed of efficacy

Efficacy against R. sanguineus and I. ricinus applied immediately after the collars had been fitted exceeded 96%, and efficacy measured at 6 h after re-infestation on Day 2 exceeded 99% (Table 10). This implies that the majority of ticks that get onto a dog on the same day that the collars are fitted will be killed and that nearly every tick that gets onto a dog 2 days after the collars have been fitted will be eliminated within 6 hours (=repellent efficacy).

Repellent efficacy

Repellent or’speed of kill’ efficacy measured 6 h after each infestation from Week 4 to Week 34 exceeded 97% against R. sanguineus and was 100% against I. ricinus for the duration of the study (Table 11). Taken in conjunction with the results obtained in the study in which the onset of efficacy was determined, repellent efficacy was already evident 2 days after the collars had been fitted (Table 10). Thus the vast majority of ticks that get onto a dog would be killed within 6 h from Day 2 after the collars had been fitted until at least 34 weeks thereafter.

A true repellent effect against ticks is evident when the ticks do not even attempt to attach and leave the host spontaneously. The perception of a repellent effect may instead be due to extremely rapid killing of ticks shortly after they have gained access to a host. The EMEA efficacy guidelines for testing of ectoparasiticides on dogs and cats allows for a 24 h time interval to claim a repellent effect, but in the studies described here, this interval was reduced to 6 h. The 6 h interval between infestation and the death of the majority of ticks would certainly interfere with the transmission of tick-borne pathogens, including Ehrlichia spp., which are normally transmitted soon after tick attachment [26].

In vitro acaricidal efficacy

From Day 7 to Week 34 the efficacy of residues of imidacloprid/flumethrin on the hair coat of dogs against I. scapularis nymphs was 98.7% to 100%, and 100% against adult ticks (Table 12). Other in vitro studies using haircoat samples after fitting imidacloprid/flumethrin collars support these results, in that ataxia is evident 4 h after tick/hair contact, and nearly complete mortality is observed after approximately 12 h [27, 28].

Effect of shampooing or water immersion

It is inevitable that dogs with or without medicated collars are going to be washed or shampooed, or swim or go out into the rain. The former two events are usually planned, but the latter two can hardly be prevented by their owners. In all of these the question arises as to whether shampooing or getting wet will affect the efficacy of the medicated collars and whether the collars should be removed before washing or shampooing. The objective of the following study was to evaluate the efficacy of the imidacloprid/flumethrin collars against laboratory infestations of C. felis felis, R. sanguineus and D. variabilis on dogs that were either regularly shampooed or immersed in water.

Thirty-two dogs were allocated to four groups of eight dogs each based on sex and pre-treatment tick counts. Dogs in the first group served as untreated controls and those in the second group were fitted with imidacloprid/flumethrin collars, and both groups were shampooed regularly. Dogs in the third group served as untreated controls and those in the fourth group were fitted with medicated collars, and both of these groups were regularly immersed in water. The animals in all four groups were infested with C. felis felis, R. sanguineus and D. variabilis. The method of infestation, application of collars, determination of flea and tick numbers and assessment of efficacy followed the procedures described in the General Materials and Methods section and summarized in the Schematic Experimental Design (Table 1).

The initial infestations with C. felis felis and R. sanguineus took place on Day-1 and the collars were fitted one day later on Day 0. The initial infestations with D. variabilis only took place on Day 6. From Day 28 and onwards the dogs were infested with R. sanguineus seven days after they were infested with D. variabilis, which was followed one day later by C. felis felis. Flea counts were performed one day after infestation, and tick counts two days after infestation.

On Day 21 and at 28 day intervals thereafter all the dogs in the first two groups were shampooed with a shampoo not containing any active ectoparasiticidal ingredient. They were first wetted by means of a bath-shower and the shampoo was applied and the whole dog thoroughly lathered, followed by a thorough rinsing with approximately 19 litre of water applied over a period of 5 minutes by means of the bath-shower. On Day 21 and at 28-day intervals thereafter the dogs in Groups 3 and 4 were immersed in a tank containing tepid tap water for a minimum of 5 minutes and each animal’s head was thoroughly wetted three times during the procedure. On each day of this procedure the control group was immersed first followed by the collared group without changing the water. The collars were not removed from the dogs in either of the treated groups before shampooing or water immersion. After shampooing or immersion the dogs were allowed to air-dry in their runs. The tank was washed and filled with fresh tepid water before the next immersion procedure.

For the sake of legibility and conformity the infestations with fleas and ticks and concomitant efficacy evaluations have been aligned with each other as far as it is possible for each activity that took place during the same week after treatment.

Mites

Lice