International Program to Monitor Cat Flea Populations for Susceptibility to Imidacloprid.

An international team of scientists and veterinarians was assembled in 1999 to develop a monitoring program to determine the susceptibility of cat fleas, Ctenocephalides felis felis (Bouché) (Siphonaptera: Pulicidae), to imidacloprid. Cat flea eggs were collected, shipped to laboratories, and tested for their susceptibility to imidacloprid. Over 3,000 C. felis populations were collected from 2002 to 2017 from 10 different countries. Of these, 66.3% were collected from cats and 33.7% from dogs. C. f. felis populations (n = 2,200) were bioassayed by exposing cat flea eggs and the emerging larvae to a Diagnostic Dose (DD) of 3 ppm imidacloprid in larval rearing medium. Flea eggs hatched and developed in the untreated controls in 1,837 of the isolates (83.5%) bioassayed. Flea isolates (n = 61) that had ≥5% survival at the DD of 3 ppm were retested with a second DD of 3 ppm. None of them had ≥5% survival to the second dose of 3 ppm. Of the 1,837 valid C. felis isolates tested, there has been no evidence of a decreased susceptibility to imidacloprid over the past 17 yr. The methods outlined in this article should provide an acceptable protocol for testing many of the new active ingredients that have been registered for cat flea control.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition: guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats.

These second edition guidelines, updated from the 2007 version (Marchiondo et al., 2007), are intended to assist the planning and conduct of laboratory and clinical studies to assess the efficacy of ectoparasiticides applied to dogs or cats for the purpose of treating, preventing and controlling flea and tick infestations. Major revisions to this second edition include guidelines on the assessment of systemic flea and tick products, an update of the geographical distribution of the common fleas and ticks species on dogs and cats, determination of flea and tick efficacy based on geometric versus arithmetic means with respect to geographic regulatory agencies, modification of tick categorization in the assessment of efficacy, expanded guidelines on repellency and anti-feeding effects, enhanced practical field study guidance, and considerations on the ranges of flea and ticks for infestations in laboratory studies. The term ectoparasiticide includes insecticidal and acaricidal compounds, as well as insect growth regulators. The range of biological activities from animal treatment that are considered include: repellency and anti-feeding effects, knockdown, speed of kill, immediate and persistent lethal effects, and interference with egg fertility and subsequent development of off-host life cycle stages. Information is provided on the selection of animals, dose determination, dose confirmation and field studies, record keeping, interpretation of results and animal welfare. These guidelines are also intended to assist regulatory authorities involved in the approval and registration of new topical or systemic ectoparasiticides, and to facilitate the worldwide adoption of harmonized procedures.

Understanding attraction stimuli of the cat flea, Ctenocephalides felis, in non-chemical control methods.

Comparisons were conducted of flea catches of four commercially available flea traps in the laboratory and under field conditions, in both rural and urban locations. The results clearly showed the My Flea Trap™, which utilizes an intermittent light to attract fleas, to be far superior in trapping ability to the three continuous light traps; it caught up to 23 times as many fleas as the other traps. Altering the lighting mechanism to provide continuous rather than intermittent light significantly decreased the number of fleas captured. In addition, the use of a green filter significantly increased trapping efficiency, whereas the addition of a heat source had no apparent effect.

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.

Flea blood feeding patterns in cats treated with oral nitenpyram and the topical insecticides imidacloprid, fipronil and selamectin.

A series of studies was conducted to determine the effect of systemically and topically active insecticides on blood consumption by fleas (Ctenocephalides felis). Infestations were conducted by placing fleas into plexi-glass chambers attached to the lateral rib cage of domestic short-hair cats. After pre-defined periods, fleas and flea feces were extracted using vacuum aspiration and spectrophotometrically analyzed for hemoglobin using Drabkin's reagent. To determine how rapidly nitenpyram kills actively feeding fleas, a single oral treatment was administered 24h after infestation. To determine the effect of nitenpyram on blood consumption of newly acquired fleas, cats were infested with fleas 1h post-treatment and fleas and flea feces from both studies were extracted at 15, 30, 60, 120, 240 and 480min post-treatment or post-infestation. To compare the effects of topically versus systemically active insecticides, 20 cats each with 2 chambers attached, were randomly allocated among groups and were infested with fleas 1h after each of 4 nitenpyram treatments, or at 7, 14, 21 and 28 days after a single application of commercial spot-on formulations of fipronil, imidacloprid or selamectin. Infestations were also completed for untreated (control) cats. Twenty-four hours after infestation, fleas and flea feces were removed for host blood quantification. If at any time, flea blood consumption in a treated group did not significantly differ from that of fleas infesting controls, that treatment group was withdrawn from the study. Nitenpyram effects on actively feeding fleas were first observed at 60min post-dosing when 38% of fleas were dead or moribund, and at 240min 100% were dead or moribund. Nitenpyram produced a significant reduction in flea blood consumption (p<0.05), which appeared to cease 15min after infestation. For the treatment comparisons, significantly more (p<0.05) blood was consumed by fleas taken from imidacloprid and fipronil-treated cats than from the nitenpyram or selamectin groups. Only on nitenpyram- or selamectin-treated cats were there significant reductions (p<0.05) in flea blood consumption on days 21 and 28, with significant difference (p>0.05) between these two groups on day 28. In this study systemically acting insecticides such as nitenpyram, and the topically applied but systemically active insecticide selamectin, were more effective in interfering with flea blood feeding than were imidacloprid and fipronil.

Efficacy of fipronil (9.8% w/w) + (S)-methoprene (8.8% w/w) and imidacloprid (8.8% w/w) + permethrin (44% w/w) against Dermacentor variabilis (American dog tick) on dogs.

This study evaluated overall efficacy, percentage of dogs free of live ticks, retention of ticks, and efficacy against retained ticks of fipronil (9.8% w/w) + (S)-methoprene (8.8% w/w) and imidacloprid (8.8% w/w) + permethrin (44% w/w) spot-on topical solutions against Dermacentor variabilis-infested dogs. Tick control was significantly greater (P < .05) on dogs treated with fipronil + (S)-methoprene than on control dogs for the entire 30-day study period. Conversely, the combination product of imidacloprid + permethrin demonstrated activity significantly (P < .05) different from that seen on control dogs only up to day 23. Significantly (P < .05) more dogs treated with fipronil + (S)-methoprene were free of live ticks compared with controls on days 3, 9, and 16. There was never a significant difference (P > .05) between control and imidacloprid + permethrin-treated dogs in numbers of dogs free of live ticks. After the initial 10-minute tick exposure, lower numbers of ticks were retained on the imidacloprid + permethrin-treated dogs than on the fipronil + (S)-methoprene-treated dogs. However, when evaluating the effectiveness of the acaricides on "retained ticks," it appears that while some of the ticks were rapidly knocked down on the imidacloprid + permethrin-treated dogs, efficacy against ticks still retained at 10 minutes was never greater than 50%.

Efficacy of a novel formulation of metaflumizone for the control of fleas (Ctenocephalides felis) on cats.

A novel spot-on formulation containing metaflumizone (ProMeris for Cats, Fort Dodge Animal Health, Overland Park, KS) was evaluated in five laboratory studies to determine the duration of residual efficacy in cats against fleas after a single spot treatment. In each study, eight domestic shorthair cats were randomly allocated to each treatment group and individually housed. One group in each study remained non-treated. In one study, an additional group of eight cats was treated with a placebo formulation. Cats were treated topically with metaflumizone formulation to provide a dose of at least 40mg metaflumizone/kg. Cats were infested with 100 cat fleas (Ctenocephalides felis felis) once per week for approximately 8 weeks. Cats were comb counted 48h after treatment and each infestation to determine the number of viable fleas present. There were no significant differences in flea counts between the non-treated control and the placebo-treated control (P>0.05) other than a 26% reduction at week 1, demonstrating that the formulation excipients had no activity. Metaflumizone treatment resulted in significantly lower flea numbers relative to non-treated controls on all post-treatment count days (P<0.05). Metaflumizone provided >90% control of flea infestations up to 7 weeks following a single treatment.

Evaluation of an imidacloprid (8.8% w/w)--permethrin (44.0% w/w) topical spot-on and a fipronil (9.8% w/w)--(S)-methoprene (8.8% w/w) topical spot-on to repel, prevent attachment, and kill adult Ixodes scapularis and Amblyomma americanum ticks on dogs.

This study evaluated the effectiveness of two topical spot-on formulations -- imidacloprid(8.8% w/w)-permethrin (44.0% w/w) and fipronil (9.8% w/w)-(S)-methoprene (8.8% w/w)--to repel, prevent the attachment of, and kill adult Ixodes scapularis and Amblyomma americanum on dogs. Twelve purpose-bred beagles were distributed into three groups of four dogs each; one group served as untreated controls, and each of the other two groups received one of the test products. Dogs were exposed to 25 adult ticks of each species for 10 minutes on posttreatment days 3, 7, 14, 21, and 28. Unattached or repelled ticks were collected and evaluated for viability, and on-dog tick counts were conducted at 3, 24, and 48 hours after tick exposure. The imidacloprid-permethrin formulation provided significant repellency against I. scapularis for up to 3 weeks after treatment, and both formulations provided good overall control of I. scapularis and A. americanum during the study period.

Evaluation of an imidacloprid (8.8% w/w)--permethrin (44.0% w/w) topical spot-on and a fipronil (9.8% w/w)--(S)-methoprene (8.8% w/w) topical spot-on to repel, prevent attachment, and kill adult Rhipicephalus sanguineus and Dermacentor variabilis ticks on dogs.

This study evaluated the effectiveness of two topical spot-on formulations, imidacloprid (8.8% w/w)--permethrin (44.0% w/w) and fipronil (9.8% w/w)--(S)-methoprene (8.8% w/w), to repel, prevent the attachment of, and kill adult Rhipicephalus sanguineus and Dermacentor variabilis on dogs. Twelve purpose-bred beagles were distributed into three groups of four dogs each; one group served as untreated controls and each of the other two groups received one of the test products. Dogs were exposed to 25 adult ticks of each species for 10 minutes on posttreatment days 3, 7, 14, 21, and 28. Nonattached or repelled ticks were collected and evaluated for viability, and on-dog tick counts were conducted at 3, 24, and 48 hours after tick exposure. The imidacloprid-permethrin formulation provided significant repellency of R. sanguineus and D. variabilis for up to 3 and 4 weeks after treatment, respectively; and provided good overall control for R. sanguineus and D. variabilis during the study period. The fipronil--(S)-methoprene formulation provided good overall tick control during the study period.

Efficacy of imidacloprid (8.8% w/w) plus permethrin (44% w/w) spot-on topical solution against Amblyomma americanum infesting dogs using a natural tick exposure model.

This study evaluated the efficacy of an imidacloprid 8.8% w/w + permethrin 44% w/w spot-on topical solution (K9 Advantix, Bayer Animal Health) against Amblyomma americanum using a natural field exposure model. Sixteen beagles were divided into two groups of eight dogs each. One group of dogs was treated with K9 Advantix and the other group served as untreated controls. On day -1 and at 3, 7, 14, 21 and 28 days after treatment, the dogs were walked for 80 minutes in an A. americanum-infested habitat at the Konza Prairie Biological Station in Northeastern Kansas. Postexposure tick counts (efficacy evaluations) were conducted on each dog at 3 and 48 hours after exposure. At 3 days after treatment, the efficacy of K9 Advantix within 3 hours of natural tick exposure was 88.0% and declined slowly during the study. The 48-hour postexposure efficacy remained above 93.5% throughout the study.

Accurate diagnosis of Giardia spp and proper fecal examination procedures.

A series of investigations evaluated the ability of different testing methods - a swing-head centrifugation technique using two flotation solutions (1.18-specific gravity zinc sulfate and 1.27-specific gravity Sheather's sugar solution), a passive commercial flotation technique, and the SNAP Giardia Test Kit from IDEXX Laboratories - to identify Giardia-positive dogs and recover the eggs of other intestinal parasites. It was determined that the SNAP Giardia test can improve a practice's ability to identify Giardia-infected dogs. Because of its higher specific gravity, the sugar solution was better for recovering heavy parasite eggs, such as Taenia spp, and thus is the flotation solution of choice when conducting routine centrifugation fecal examinations.

Comparative speed of kill of selamectin, imidacloprid, and fipronil-(S)-methoprene spot-on formulations against fleas on cats.

The speed of kill of selamectin, imidacloprid, and fipronil-(S)-methoprene against Ctenocephalides felis infestations on cats for one month following a single treatment was evaluated. Eighty cats were randomly allocated so that there were 20 cats in four different treatment groups. On Days -2, 7, 14, 21, and 28, each cat was infested with 100 adult C. felis from the Kansas 1 flea strain. Following initial application only imidacloprid had caused a significant reduction in adult fleas on treated cats within 6 hours, but by 24 hours all three formulations had killed 96.7% of the fleas. At 7 days post treatment, all three formulations reduced flea populations within 6 and 24 hours by 68.4% and 99.4%, respectively. At 21 and 28 days after treatment, none of the formulations killed significant numbers of fleas as compared to controls within 6 hours of infestation. At 28 days after treatment, selamectin, fipronil-(S)-methoprene, and imidacloprid had killed 99.0%, 86.4%, and 72.6% of the fleas within 48 hours of infestation, respectively. This study demonstrates that the speed of kill of residual flea products on cats decreases throughout the month following application. It also demonstrated that selamectin provided the highest level of residual activity on cats against the Kansas 1 flea strain.

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.

Comparison of common fecal flotation techniques for the recovery of parasite eggs and oocysts.

A variety of procedures are available to detect parasite eggs or oocysts in feces. This study compared the efficacy of simple flotation, a commercial assay, and various centrifugation techniques and three common flotation solutions. Results indicate that centrifugation consistently recovered more eggs than other methods. Proper technique is critical, including ensuring that the specific gravity of the flotation solution is correct and allowing the sample to stand for a sufficient amount of time before examining the coverslip. Because of the zoonotic health risks of many companion animal parasites, veterinarian and their staff should better utilize fecal examinations in their routine diagnostic plan.

Molecular phylogeny of isolates of Ctenocephalides felis and related species based on analysis of ITS1, ITS2 and mitochondrial 16S rDNA sequences and random binding primers.

The phylogenetic relationships among 31 different flea isolates representing seven different species were studied by nucleotide sequence comparison of the internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2) and/or mitochondrial 16S ribosomal RNA gene (mt16S-rDNA) to examine the patterns of variation. Results show that all regions are useful in discriminating among flea species. In Ctenocephalides felis and Tunga penetrans, some differences in these gene regions occurred among different isolates within the same species. In the latter case, the differences are in the mt16S-rDNA region, with one isolate showing 48% divergence in nucleotide sequence. The taxonomic implications of this result are unclear at present. The gene regions revealed differences between C. felis isolates only after DNA sequencing the PCR products. Further differentiation among C. felis isolates was obtained using four different random binding primers (decamers) and primers for mammalian aldolase to amplify narrow differences in the genome. Using these primers we were able to discriminate between different C. felis isolates and determine that some of the genetic variation coincided with minor differences in response to the control agent imidacloprid. However, overall findings do not support the existence of subspecies of C. felis.

Development of a larval bioassay for susceptibility of cat fleas (Siphonaptera: Pulicidae) to imidacloprid.

Strategies for controlling cat fleas, Ctenocephalidesfelisfelis (Bouché), have undergone dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the potential for the development of insecticide resistance increases. A larval bioassay was developed to determine the baseline susceptibility of field-collected strains of cat fleas to imidacloprid. All four laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this bioassay are that smaller numbers of fleas are required because flea eggs are collected for the test. Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating dose, the detection of reduced susceptibility in field strains can be determined with as few as 40 eggs.

Distribution and prevalence of Echinococcus multilocularis in wild predators in Nebraska, Kansas, and Wyoming.

To further determine the distribution and prevalence of Echinococcus multilocularis in the central United States, 245 wild canids (125 red foxes, 120 coyotes) and 33 bobcats were collected from Nebraska, Kansas, and Wyoming and examined for this parasite. Animals examined included 11 red foxes from the western panhandle of Nebraska; 5 red foxes and 30 coyotes from southern Nebraska; 56 red foxes and 1 coyote from northeastern Nebraska; 20 red foxes, 63 coyotes, and 13 bobcats from northern Kansas; 2 red foxes, 26 coyotes, and 20 bobcats from southern Kansas; and 31 red foxes from east-central Wyoming. Of these, 27 of 72 (37.5%) red foxes from Nebraska were positive, including 2 of 11 (18.2%) from the western panhandle and 25 of 56 (44.6%) from the northeastern part of the state. Mean intensity of infection was 282 worms (range, 1-5,150). New distribution records were established for E. multilocularis in western Nebraska as well as for several northeastern counties. These findings support previous estimates that the southernmost front of the parasite's range extends along the southern border of Wyoming, eastward through central Nebraska and central Illinois into Indiana and Ohio.

Effect of 0.29% w/w fipronil spray on adult flea mortality and egg production of three different cat flea, Ctenocephalides felis (Bouché), strains infesting cats.

To evaluate the effect of fipronil spray on adult flea mortality and flea egg production of three different cat flea, Ctenocephalides felis (Bouché) strains, 30 domestic short hair cats were randomly allocated into six groups of five cats each. On day 0, cats in groups 2, 4 and 6 were treated with fipronil at 5-6ml/kg. Cats in groups 1, 3 and 5 served as untreated controls. On days -2, 7, 14, 21, and 28 each cat was infested with 50 adult cat fleas. Groups 1 and 2 were infested with fleas from the Kansas1 Colony (KS1) strain. Groups 3 and 4 were infested with a recently colonized cat flea strain from Florida (R6). Groups 5 and 6 were infested with fleas from the ARC strain. The adulticidal activity of fipronil was determined by flea comb counts 48h after treatment and then 48h after each reinfestation. Any flea eggs produced during the infestations were collected and counted prior to the 48h comb counts. Fipronil spray was > or = 99.5% effective against adults of all three cat flea strains when applied during an active infestation. Fipronil spray provided > or = 98.2 and > or = 99.5% control of adult fleas and egg production, respectively, for all strains through week 2. On days 23 and 30 control of R6 adults and egg production was significantly lower than either the ARC or the KS1 strain. On day 30, control of R6 adults and egg production was 77.3 and 87.3%, respectively. Control of KS1 adults and egg production on day 30 was significantly lower than the ARC strain. Fipronil provided > or = 99.5 and > or = 99.9% control of ARC fleas and egg production, respectively, throughout the entire study. The susceptibility to fipronil for the three strains was also evaluated on filter paper pesticide bioassays. The R6 strain was found to be less susceptible than the KS1 and ARC strains. The LC(95) estimates for the strains were 10.13, 4.77 and 2.62mg/m(2) for the R6, ARC and KS1 strains, respectively.

Control of fleas on dogs and cats and in homes with the combination of oral lufenuron and nitenpyram.

Efficacy of lufenuron (Program, Novartis Animal Health, Greensboro, NC), an insect growth regulator, and nitenpyram (Capstar, Novartis Animal Health), an insecticide for reducing flea populations, was evaluated in 35 flea-infested dogs and cats residing in 18 households in Tampa, Florida. Pets were randomly allocated by household to two treatment groups. Pets in both treatment groups were given lufenuron orally according to label directions on Day 0, then once a month for 3 months. Pets in one group were also given nitenpyram tablets by the owner orally once a day every other day. Pets in the second group were given nitenpyram by their owners as needed but no more frequently than once daily. Flea numbers on pets and in homes were assessed throughout the study by the use of visual body area counts and intermittent-light traps, respectively. The combination of lufenuron and nitenpyram reduced flea populations on pets by at least 97.3% within 7 days and maintained this rate of reduction in flea numbers for the duration of the study, regardless of which regimen was used for administering nitenpyram. For either group, premise flea counts were reduced by up to 89.5% by Day 28 and by as much as 100% by Days 84 to 90.