Susceptibility of cat fleas (Siphonaptera: Pulicidae) to fipronil and imidacloprid using adult and larval bioassays.

The monitoring of the susceptibility offleas to insecticides has typically been conducted by exposing adults on treated surfaces. Other methods such as topical applications of insecticides to adults and larval bioassays on treated rearing media have been developed. Unfortunately, baseline responses of susceptible strains of cat flea, Ctenocephalides felis (Bouchè), except for imidacloprid, have not been determined for all on-animal therapies and new classes of chemistry now being used. However, the relationship between adult and larval bioassays of fleas has not been previously investigated. The adult and larval bioassays of fipronil and imidacloprid were compared for both field-collected isolates and laboratory strains. Adult topical bioassays of fipronil and imidacloprid to laboratory strains and field-collected isolates demonstrated that LD50s of fipronil and imidacloprid ranged from 0.11 to 0.40 nanograms per flea and 0.02 to 0.18 nanograms per flea, respectively. Resistance ratios for fipronil and imidacloprid ranged from 0.11 to 2.21. Based on the larval bioassay published for imidacloprid, a larval bioassay was established for fipronil and reported in this article. The ranges of the LC50s of fipronil and imidacloprid in the larval rearing media were 0.07-0.16 and 0.11-0.21 ppm, respectively. Resistance ratios for adult and larval bioassays ranged from 0.11 to 2.2 and 0.58 to 1.75, respectively. Both adult and larval bioassays provided similar patterns for fipronil and imidacloprid. Although the adult bioassays permitted a more precise dosage applied, the larval bioassays allowed for testing isolates without the need to maintain on synthetic or natural hosts.

[Climate change - a pioneer for the expansion of canine vector-borne diseases?]. / Der Klimawandel - Wegbereiter für die Ausbreitung vektorübertragener Erkrankungen des Hundes?

Vector-transmitted diseases are one of the major contributors to the global burden of disease in humans and animals. Climate change is consistently held responsible for the spread of parasitic acarid and insect vectors such as ticks, fleas, sand flies and mosquitoes, and their transmitted pathogens (in the case of the dog the so-called canine vector-borne diseases [CVBD]). Currently, there is only insufficient data available to prove whether climate change is a major driving force for vector and disease expansion, but the evidence is growing. Other reasons, such as ecological, demographic and socio-economic factors, e.g. pet travel into and pet import from endemic areas, also play a role in this development. Apart from all the controversial discussion of the factors leading to vector and disease expansion, preventative measures should include dog owners' education as they are responsible for individual parasite protection as well as for the minimisation of adverse risk behaviour, e.g. regarding pet travel. Broad-spectrum vector control should be practised by using parasiticides that repel and kill blood feeders in order to minimize the risk of CVBD-pathogen transmission.

Large-scale monitoring of imidacloprid susceptibility in the cat flea, Ctenocephalides felis.

Although on-animal topical treatment with compounds such as imidacloprid has revolutionized the control of the cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae), the development of insecticide resistance is a continuing threat. As part of a highly co-ordinated and unprecedented resistance monitoring programme for C. felis, 1437 flea isolates were collected by veterinary clinics in Australia, Germany, France, the U.K. and 29 states in the U.S.A. from 2002 to 2009. About 65% of the collections were made from June to October each year and 71% of the collections were from cats. Collections of flea eggs were sent to one of five different laboratories, where they were tested with a diagnostic dose of imidacloprid (3 p.p.m.) applied to larval flea-rearing medium. Of the 1437 collections received, 1064 contained adequate numbers of eggs for testing. Of these isolates, untreated eggs failed to hatch in 22.7% and were not considered valid bioassays. Survival rates >5% and development of adult fleas (a threshold for further testing) occurred in only 22 isolates. They were re-tested with the same diagnostic dose and none produced >5% adult emergence. Complete dose-response bioassays were performed on three of the isolates that had triggered a second test and produced slopes, intercepts and LC(50) values similar to those for existing susceptible laboratory strains. Results confirmed sustained susceptibility of C. felis to imidacloprid, despite its widespread use for over a decade.

Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50% in young dogs: a longitudinal field study.

Canine vector-borne diseases (CVBDs) are highly prevalent and increasing in distribution worldwide. A longitudinal study was conducted in southern Italy to determine the incidence of and protection against CVBD-causing pathogens in dogs treated with a combination of imidacloprid 10% and permethrin 50% (ImPer). One hundred eleven autochthonous young dogs were divided into group A (n=63) and group B (n=48), both groups containing dogs positive and negative for one or more CVBD-causing pathogens. Additionally, 10 naïve male beagles were introduced in each group in May 2008. Group A was treated with ImPer on day 0 and every 21+/-2 days whereas group B was left untreated. Blood and skin samples were collected at baseline (March-April 2008) and at the first, second and third follow-up times (July and October 2008 and April 2009). Bone marrow was sampled at baseline and at the third follow-up. Serological, cytological and molecular tests were performed to detect Anaplasma platys, Babesia spp., Bartonella spp., Dirofilaria immitis, Ehrlichia canis, Hepatozoon canis and Leishmania infantum. Ectoparasites (fleas, ticks, and sand flies) were monitored throughout the study. The baseline prevalence of CVBDs was 39.6% with 44 dogs positive for at least one pathogen. A. platys (27.5%) and Babesia spp. (15.6%) were the most prevalent species and co-infections with up to two pathogens were detected in 16 (14.7%) individuals. At the end of the evaluation period, there was a 90.7% reduction in overall CVBD incidence density rate (IDR) in group A, as following: 100% reduction in L. infantum; 94.6% in E. canis; 94.4% in Babesia spp.; and 81.8% in A. platys. Initially positive treated dogs showed significantly lower pathogen prevalence at the third follow-up than untreated ones. At the end of the evaluation period, 8 of the 10 untreated beagles were infected with at least one pathogen whereas one of the treated beagles was A. platys positive at a single time point (second follow-up). Overall efficacy against ticks was 97.9%. In October 2009, samples were collected from the remaining 83 dogs (44 from group A and 39 from group B) to investigate the annual incidence of CVBDs in the same, at this time untreated, dog population. A high year incidence for tick-borne diseases (78.1%) and for L. infantum (13.6%) was detected in dogs from group A, seven months after the treatment had been withdrawn. The results demonstrate that ImPer preventive treatment against arthropods protects autochthonous and naïve beagle dogs against CVBD-causing pathogens.

Seasonal dynamics of the brown dog tick, Rhipicephalus sanguineus, on a confined dog population in Italy.

This study evaluated the seasonal dynamics of Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) on naturally infested dogs in a private shelter in southern Italy. From March to May 2008, 39 autochthonous mixed-breed young dogs and 10 beagles were enrolled in the study. From March 2008 until March 2009, every 21 +/- 2 days, 11 body sites of each dog were checked for ticks. At each follow-up, the number of ticks, their developmental stage, sex and location on the dog's body were recorded. Adult ticks were found throughout the year, but immatures were absent in January and February. The adult tick population increased from July to August, whereas the load of immatures increased in early July and peaked in September, which suggests that R. sanguineus develops one generation per year in this area. The mean number of immature ticks per infested dog was higher than that of adults from March to October 2008. Ears, interdigital areas and armpits were the most frequent attachment sites of adult ticks. At the last follow-up, a total of 2266 ticks were collected and identified as R. sanguineus. The results suggest that R. sanguineus develops one generation per year in the study area, but that it infests dogs in all seasons. This information should be taken into account when planning control programmes against this tick species and the pathogens it transmits.

Transmission of feline calicivirus via the cat flea (Ctenocephalides felis).

In this study, a possible role of the cat flea (Ctenocephalides felis) in transmitting feline calicivirus (FCV) was examined. Fleas were fed via artificial membranes with FCV-spiked bovine blood, free of anti-FCV antibodies. Flea feces were collected daily for 10 days and incubated at room temperature. Infectivity of the feces was tested in vitro using Crandell-Reese Feline Kidney (CRFK) cells. FCV remained infectious for 8 days. These flea feces were also used to oronasally inoculate four specific pathogen-free (SPF) kittens. All kittens were successfully infected as demonstrated by virus isolation from pharyngeal swabs and seroconversion. Two of the cats showed, in addition, clinical signs. Besides the infection of cats with flea feces containing FCV, four SPF kittens were exposed to fleas that were fed with FCV-spiked bovine blood. One of the kittens was successfully infected via this route as demonstrated by virus isolation from pharyngeal swabs and virus isolation. The results of this study show that fleas can spread infectious virus through their feces or by stitch and must be considered a source of infection for uninfected cats.

Investigations into the prevention of neonatal Ancylostoma caninum infections in puppies by application of imidacloprid 10% plus moxidectin 2.5% topical solution to the pregnant dog.

The aim of the investigation was to examine whether a single topical administration of a combination of imidacloprid and moxidectin to pregnant dogs could prevent neonatal infections with reactivated Ancylostoma caninum larvae. Three pregnant beagles, infected with A. caninum, were treated topically with the combination on day 56 of pregnancy. Three further dogs served as untreated controls. Treatment appeared to prevent neonatal infections in the puppies completely. Neither intestinal stages nor somatic larvae were found in two examined puppies per litter. All puppies and dams of the treatment group remained coproscopically negative. No side-effects in dams or puppies were observed. Two of three untreated dams showed a patent infection after parturition. Necropsy of two puppies of each negative control litter revealed seven intestinal and five somatic A. caninum stages in total. One litter of the untreated dams showed a patent infection 33 days after parturition. In the other two litters, no representative sample sizes could be collected.

Comparative efficacy of imidacloprid, selamectin, fipronil-(S)-methoprene, and metaflumizone against cats experimentally infested with Ctenocephalides felis.

Four active ingredients--imidacloprid selamectin, fipronil-(S)-methoprene, and metaflumizone--were tested to assess the speed of flea kill against existing flea infestations and subsequent reinfestations. Thirty flea-infested cats were allocated to four treatment groups and one untreated control group. Flea counts were performed 6, 18, and 48 hours after treatment (day 0) and 2, 4, and 24 hours after weekly flea reinfestations. Cats were also reinfested with fleas after the 6- and 18-hour counts on day 0 and after the 2- and 4-hour counts on subsequent count days. Imidacloprid provided significantly greater flea kill at diverse time points. At the 24-hour counts, all compounds showed expected and similar high efficacies. On study day 34, imidacloprid showed the highest efficacy at 24 hours after reinfestation, with 90.8% flea reduction compared with 55.7% to 67.4% in the other treatment groups. A single topical application of imidacloprid provided a high efficacy in the early elimination of adult fleas, limiting the risk of pathogen transmission and flea allergy dermatitis.

Monitoring of basophil sensitization to antigens of the cat flea (Ctenocephalides felis felis): a new tool for the diagnosis of feline flea bite hypersensitivity?

Suitability of blood basophils for in vitro diagnosis of flea allergy dermatitis (FAD) or flea bite hypersensitivity was studied in cats. A functional in vitro test (FIT) for sensitized type I allergic effector cells was used to evaluate the degree and kinetics of in vivo basophil sensitization against flea antigens in cats under long-term flea exposure. FIT results were compared with intradermal (IDT) and serological testing. Before, during, and after weekly repeated exposure to Ctenocephalides felis; 14 cats were repetitively FIT-assessed for general and flea-specific sensitization. In three cats, flea-specific sensitization was seen before and throughout flea exposure. Five cats, although generally sensitized, never developed a flea-specific sensitization. Six cats initially FIT-negative became sensitized for flea antigen during flea infestation. Induction, upregulation, and binding of C. felis-specific sensitizing antibodies to basophils during flea challenge may explain the developing sensitization in these cats. Strong discrepancies between the levels of flea-specific circulating IgE and basophil sensitization contrasted comparable results for basophil and mast cell sensitization using FIT and IDT, respectively. Hence, the FIT might provide an immunological supplement to the clinical diagnosis of FAD in cats by elucidating the state of basophil-sensitization to flea antigens. And it may be a comfortable alternative to IDT.

Investigations on the life cycle and morphology of Tunga penetrans in Brazil.

In the present study, the life cycle of Tunga penetrans was established in Wistar rats in the laboratory, and the morphology of the resulting developmental stages was studied by means of light and scanning electron microscopy. It was seen that the females enter at a nonfertilized stage through the skin of their hosts. Only there the copulation occurs, while females and males brought together in a Petri dish showed no interest in each other. In any way -- fertilized or not -- the females start about 6 days after penetration and hypertrophy with the ejection of eggs. While fertilized eggs proceed to development, the unfertilized ones remain arrested. The eggs are ovoid and measure about 600 x 320 mum. The larvae hatch from the eggs 1-6 days (mean 3-4) after ejection. Formation of larvae 2 took at least another day, while 4 up to 10 days more were needed until this larva starts pupation (mean 5-7 days). The formation of the adult fleas inside the puparium occurred within 9-15 days (with a maximum hatch at day 12). Adult female fleas having reached the skin of a host start blood sucking within 5 min and prepare to enter the skin. After 24 h, the flea stacked already with two thirds of its body inside the skin. After 40 h, the penetration was completed, and feeding and hypertrophical enlargement started, which was completed on day 6, when eggs became ejected. When studying the morphology of the fleas obtained from different hosts, slight variations were seen, which, however, are not significant for a species separation but may be an indication of the presence of different strains/races or the beginning of such a formation.

Acaricidal and repellent properties of permethrin, its role in reducing transmission of vector-borne pathogens.

Vector-borne pathogens causing canine vector-borne diseases (CVBD) are recognized as being of increasing importance in small animal clinics. The pathogens and their vectors (eg, fleas, ticks, mosquitoes and sand flies) have a global distribution. Their prevalence may vary depending on region, climate, hygiene and many other factors; however the risk of infection for companion animals in our highly developed and supposedly regulated environments will never be zero. Even in highly developed markets, with high socioeconomic standards, ectoparasites are still a threat to both pets and humans. One has to understand the complexity of arthropod biology and esp. the complex mechanisms of host seeking, attachment and skin penetration and finally feeding, to differentiate the various acaricides and their therapeutic as well as prophylactic properties. Prevention of arthropod bites is mainly by prevention of attachment and thus any engorgement if possible. To achieve this, acaricides with repellent properties, such as the synthetic pyrethroid permethrin are ideal compounds to reach this goal.

Experimental quantification of the feline leukaemia virus in the cat flea (Ctenocephalides felis) and its faeces.

Cat fleas (Ctenocephalides felis) were fed via artificial membranes and infected with the feline leukaemia virus (FeLV) from cell cultures. After removing the fleas from the blood source, the quantity of virus in the flea and its faeces was measured over a defined period of time. The virus was detectable in the fleas for up to 30 h at room temperature and up to 115 h at 4 degrees C. In the faeces, the amount of virus decreased much more slowly--after 2 weeks half of the initial amount of virus could still be detected. Thus the faeces might be a source of further infections, e.g. for the flea larvae or the cat itself.

Efficacy of imidacloprid, imidacloprid/permethrin and phoxim for flea control in the Mustelidae (ferrets, mink).

Farmed mink (Mustela vison), a close relative of the domestic ferret (Mustela putorius furo), naturally infested with the squirrel flea (Ceratophyllus sciurorum) were included in a study to investigate three compounds for flea control. The test products were imidacloprid in a 10% (w/v) solution, an imidacloprid 10% (w/v)/permethrin 50% (w/v) solution, and phoxim; all three are well-known compounds for the control of different ectoparasites in a wide range of animals. Two groups of mink received 0.1 ml per animal of the imidacloprid or the imidacloprid/permethrin combination at days 0 and 28, respectively. Two groups of mink were sprayed with 25 ml of a 0.1% phoxim solution at day 0 and either 1x25 ml or 2x25 ml, respectively, of a 0.05% phoxim solution at day 28. One group of mink served as an untreated control. At assessment on day 56 the efficacy was 91.9% in the imidacloprid group, 89.3% in the imidacloprid/permethrin group, 92.2% in the phoxim 1x25-ml group and 99.3% in the phoxim 2x25 ml group, respectively. In the untreated control group an average of 757 fleas per mink nesting material was recorded.

Field trial of the efficacy of a combination of imidacloprid and permethrin against Tunga penetrans (sand flea, jigger flea) in dogs in Brazil.

In a field trial in Brazil 17 dogs penetrated by females of the jigger flea, Tunga penetrans, were topically treated with a combination of 10% imidacloprid and 50% permethrin (Advantix), while 17 dogs remained untreated. The follow-up controls on days 7, 14, 21 and 28 post-treatment clearly showed that, beginning from day 7, the flea load in treated dogs decreased, so that most of the dogs became free of tungiasis lesions, while in the untreated group the flea load remained high. Since the dogs distribute the flea eggs throughout the village, leading to a high incidence of tungiasis in humans, treatment of dogs probably also decreases the number of cases of tungiasis in the latter.

Efficacy of imidacloprid/permethrin and fipronil/(S)-methoprene combinations against Haemaphysalis longicornis ticks evaluated under in vitro and in vivo conditions.

Haemaphysalis longicornis is one of the most important ticks infesting a wide range of mammals including dogs in Japan. H. longicornis is recorded to be a vector of, for example, Babesia gibsoni. It was the aim of the study presented here to evaluate the efficacy of imidacloprid/permethrin and fipronil/(S)-methoprene against larval, nymphal and adult stages of H. longicornis under in vitro as well as in vivo conditions. In the in vitro part of the study, ticks showed avoidance behaviour to imidacloprid/permethrin-treated filter papers. The onset of acaricidal efficacy in the imidacloprid/permethrin group was recorded earlier than in the fipronil/(S)-methoprene group. In the in vivo experiment three beagles per group were treated with either imidacloprid/permethrin, fipronil/(S)-methoprene or left untreated. Each dog was infested with 30 adult female H. longicornis. Ticks were place on a shaved area of skin of the treated dogs and behaviour of the ticks was recorded as before. After 3 h all ticks were removed and placed in Petri dishes. Ticks were further examined until day 4 post-treatment (p.t.). All ticks recovered from the untreated dogs survived. At 4 h p.t. (1 h post-removal) 40 of the 90 ticks exposed to the imidacloprid/permethrin treatment and 25 of the 90 ticks in the fipronil/(S)-methoprene-treated group were found dead. At day 1 p.t., 61 ticks in the imidacloprid/permethrin- and 81 ticks in the fipronil/(S)-methoprene-treated group were recorded dead. At the final examination day 4 p.t., all 90 ticks were found dead in the imidacloprid/permethrin group, while five ticks remained alive in the fipronil/(S)-methoprene group.

Determining a diagnostic dose for imidacloprid susceptibility testing of field-collected isolates of cat fleas (Siphonaptera: Pulicidae).

The susceptibility of four laboratory strains of cat fleas, Ctenocephalides felis (Bouche), to imidacloprid was determined by three different laboratories, by using a standardized bioassay protocol. The probit lines generated by the different laboratories were very similar, with LC50 values ranging from 0.32 to 0.81 ppm. Based on these data, a diagnostic dose (DD) of 3 ppm imidacloprid in larval rearing media was provisionally identified for detecting shifts in tolerance, possibly as a consequence of incipient imidacloprid resistance. None of the larvae from the susceptible laboratory strains survived the DD. Eighteen field-collected isolates were evaluated for their susceptibility to imidacloprid and to validate a DD of 3 ppm. Probit lines from 18 field-collected isolates were very similar, with LC50 values ranging from 0.14 to 1.52 ppm. When exposed to the DD, between 3 and 10% of the exposed larvae emerged as adults from only three of the 18 isolates. All other field isolates gave 100% mortality at the DD. Under the criteria established (>5% survivorship at 3 ppm), two isolates would be established on mammalian hosts and more extensive tests conducted to exclude or confirm the presence of resistance. The DD of 3 ppm is robust enough to eliminate most of the susceptible isolates collected until today, yet low enough to identify possible isolates for further testing.

Infestation of Werneckiella equi on Icelandic horses, characteristics of predilection sites and lice dermatitis.

Lice infestations on horses caused by the lice Werneckiella (Damalinia) equi and Haematopinus equi are observed worldwide. In this study, the distribution and clinical manifestations of lice on Icelandic horses were examined. Thirty-eight out of 93 animals (40.86%) were identified as infested with W. equi. Sixty-eight animals (73.12%) presented dermatological lesions associated with lice infestation, while only 32 of these animals presented lice. Six animals had no clinical signs although of being lice-positive, and 19 animals (20.43%) showed neither lice nor clinical manifestations. Lice burdens varied from animal to animal, and clinical manifestations occurred at all levels of infestation. Focal alopecia was the main clinical sign (83.78%) on lice-positive horses, while scaling and crusts occurred in 10.81% and 9.68% of the cases, respectively. Clinical signs present in the head and the neck/mane area were found to be an indication of lice infestation in horses.

Dermatological and parasitological evaluation of infestations with chewing lice (Werneckiella equi) on horses and treatment using imidacloprid.

Lice infestations in horses caused by the chewing louse Werneckiella (Damalinia) equi are observed worldwide. In the present study, the efficacy of 10% imidacloprid was tested on horses naturally infested with lice. Two groups of animals received a double application of 4 ml and 8 ml Advantage 10% spot-on on day 0 and 28 either. Horses, presenting dermatological signs with negative lice counts, were also included in this investigation. 40.86% of the horses presented positive lice counts and 84.21% of these animals showed clinical dermatologic signs. 65.45% of the lice-negative horses also showed clinical manifestations. Two days after treatment, lice counts dropped in both the treatment groups and on day 56, all animals were free of alive lice, and dermatological lesions decreased significantly (P<0.001) in both the lice-positive and the negative animals. No correlation (P>0.050) between lice burden and clinical signs was detected.

Molecular biological investigations of Brazilian Tunga sp. isolates from man, dogs, cats, pigs and rats.

Twenty-four isolates of Tunga were collected from afflicted humans, dogs, cats, pigs and rats in Brazil. To investigate genetic diversity, a hypervariable section of mitochondrial 16S rDNA was amplified using PCR and subsequently sequenced. In order to compare results with another species of the genus Tunga, three isolates of the recently described Tunga trimamillata were also checked. Whereas eleven isolates (five from cats, three from dogs and three from humans) were of identical sequence, thirteen isolates collected from dogs, humans, pigs and rats showed differences in sequence up to 49%, so that the existence of one or more new species of Tunga may be presumed.

Natural infestation of the chewing lice (Werneckiella equi) on horses and treatment with imidacloprid and phoxim.

Infestation with the chewing louse (Werneckiella (Damalinia) equi) can be found on horses world-wide. Louse infestations, including clinical signs of louse-derived dermatitis, are known from Icelandic horses. A clinical field investigation was conducted in Iceland using horses with natural louse infestations to evaluate the efficacy of imidacloprid in a 10% solution in comparison with phoxim in a 0.05% solution. A total of 27 horses received a single imidacloprid treatment using 16 ml of the 10% solution along the mane and on the dorso-lateral trunk. A further 43 horses were treated twice, 14 days apart, with phoxim, using 2 x 50 ml solution applied along the mane and the dorso-lateral trunk. At the final evaluation on day 28, complete control of the lice was obtained for the imidacloprid treated horses and only a single moribund louse was found on two horses treated with phoxim.