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Abstract Background The poultry red mite Dermanyssus gallinae (De Geer, 1778) is a major ectoparasite of poultry. Infestations are found in most laying hen farms in Europe, and breeder flocks have also been reported to be affected. Mite infestation has detrimental effects on animal welfare, it causes significant economic losses, and, additionally, D. gallinae is often considered as a vector for pathogens. Despite suspicion of a close relationship between the poultry red mite and Salmonella enterica enterica serovar Gallinarum biovar Gallinarum (serovar Gallinarum), the causative agent of fowl typhoid disease (FT), there has been no definitive proof of mite-mediated transmission. Therefore, an investigation was conducted to determine if D. gallinae-mediated transmission of serovar Gallinarum could be demonstrated among four different hen groups. Methods Two groups of 8 hens (A and B) were experimentally infected with serovar Gallinarum in two isolators. After 7 days, when birds showed signs of FT, about 25,000 mites were introduced. After 3 days, mites were harvested and used to infest two other hen groups of 8 (C and D), in two separate isolators. The health status of hens was constantly monitored; detection and quantification of serovar Gallinarum were performed by PCR and qPCR from mites and organs of dead hens. The maximum likelihood estimation of the infection rate and mite vectorial capacity were calculated. Results Clinical disease was observed in groups infected with serovar Gallinarum (A and B) and in hens of groups C and D infested with mites harvested from the isolators containing groups A and B. In all four groups, serovar Gallinarum was detected from liver, spleen, ovary, and cecum of hens, thus confirming the diagnosis of FT. Mite analysis demonstrated the presence of the pathogen, with an estimated infection rate ranging between 13.72 and 55.21 infected per thousand mites. Vectorial capacity was estimated to be 73.79. Conclusions Mites harvested from birds infected with serovar Gallinarum were shown to carry the mite, and then to transfer serovar Gallinarum to isolated groups of pathogen-free birds that subsequently showed signs of FT. Mite vectorial capacity was high, demonstrating that D. gallinae should be considered an effective vector of FT.

Abstract Background The poultry red mite Dermanyssus gallinae negatively impacts bird welfare and health, and interferes with egg production and quality, while emerging acaricide resistance limits control options. Fluralaner, a novel miticide for administration in drinking water, is approved for control of D. gallinae infestations. Mite sensitivity testing is relevant to gauge field isolate susceptibility to available treatments. Methods Thirteen D. gallinae isolates collected during 2014 through 2016 from farms in Germany, France, Spain and Brazil, and a 2001 laboratory-maintained isolate were used for acaricide contact sensitivity testing. Tested compounds were cypermethrin, deltamethrin, phoxim, propoxur, and the recently available acaricides, spinosad and fluralaner. In each study, at least one isolate was exposed to increasing concentrations of at least one acaricide. In one study, additional testing determined the sensitivity of the 2001 isolate to fluralaner using a mite-feeding test, and of fluralaner, phoxim and spinosad using an immersion test. At least two replicates were used for each dilution. Vehicle and untreated controls were also included. Results Based on 90% mortality (LC90) values, the laboratory isolate was susceptible to fluralaner (15.6–62.5 parts per million, ppm), phoxim (< 500 ppm), propoxur (< 125 ppm), and deltamethrin (500–1000 ppm). All field isolates remained sensitive to fluralaner concentrations ≤ 125 ppm. Spinosad LC90 values for laboratory and field isolates ranged between 2000–4000 ppm. For phoxim, relative to the laboratory isolate, there was reduced sensitivity of two German isolates (LC90 up to 4000 ppm) and two French isolates (> 4000 ppm). An isolate from Spain demonstrated reduced sensitivity to phoxim, propoxur and deltamethrin; an isolate from Brazil showed reduced sensitivity to propoxur and cypermethrin. Mite LC90 when exposed to fluralaner by blood feeding was < 0.1 ppm. Conclusions Contact sensitivity testing indicated apparent resistance to at least one of phoxim, deltamethrin, cypermethrin and propoxur in 13 field isolates from Europe and Brazil. All isolates were highly susceptible to fluralaner. Fluralaner was approximately 1000 times more active by feeding than by contact. Fluralaner’s distinct mode of action and efficacy against isolates largely refractory to those acaricides, makes it a promising option for the control of D. gallinae infestations of poultry.

Abstract Background Dermanyssus gallinae, the poultry red mite, is a growing threat to chickens in poultry farms. This nocturnal hematophagous ectoparasite has a rapid rate of proliferation with a negative impact on the birds’ health, welfare and productivity resulting in severe economic consequences for poultry farmers. A study was performed with fluralaner, a novel systemic ectoparasiticide, to evaluate its effect on mite vitality and reproduction after oral administration to laying hens. Methods Sixteen healthy hens were randomly allocated to two study groups (n = 8). One group was orally treated with fluralaner by gavage at a dose of 0.5 mg/kg bodyweight twice 7 days apart. The negative control group received no treatment. Hens in each group were repeatedly infested with approximately 200 unfed adult D. gallinae at 1, 5, 8, 12, 15, 19, 22 and 26 days after the initial administration. After infestation and feeding for 2.5 h, 25 engorged mites per hen were collected and incubated in tubes. Mites were assessed for vitality (dead/live) at 4, 8, 12, and 24 h after each infestation. Tubes containing eggs and/or living mites were incubated another 8 days for assessment of mite reproductive capacity. Results Fluralaner demonstrated a fast speed of kill in mites within 4 h post-infestation for 12 days after treatment initiation. An efficacy (mite mortality) of 98.7–100% was achieved. At 15 days after treatment initiation, 100% efficacy was achieved within 24 h post-infestation, and no mite oviposition occurred during this period. Nineteen days after treatment initiation, the mites’ ability to generate nymphs was reduced by 90.8%, which decreased to

Abstract Background Northern fowl mites (Ornithonyssus sylviarum) are obligate hematophagous ectoparasites of both feral birds and poultry, particularly chicken layers and breeders. They complete their entire life-cycle on infested birds while feeding on blood. Infestations of O. sylviarum are difficult to control and resistance to some chemical classes of acaricides is a growing concern. The contact susceptibility of O. sylviarum to a new active ingredient, fluralaner, was evaluated, as well as other compounds representative of the main chemical classes commonly used to control poultry mite infestations in Europe and the USA. Methods Six acaricides (fluralaner, spinosad, phoxim, propoxur, permethrin, deltamethrin) were dissolved and serially diluted in butanol:olive oil (1:1) to obtain test solutions used for impregnation of filter paper packets. A carrier-only control was included. Thirty adult northern fowl mites, freshly collected from untreated host chickens, were inserted into each packet for continuous compound exposure. Mite mortality was assessed after incubation of the test packets for 48 h at 75% relative humidity and a temperature of 22 °C. Results Adult mite LC50 /LC99 values were 2.95/8.09 ppm for fluralaner, 1587/3123 ppm for spinosad, 420/750 ppm for phoxim and 86/181 ppm for propoxur. Permethrin and deltamethrin LC values could not be calculated due to lack of mortality observed even at 1000 ppm. Conclusions Northern fowl mites were highly sensitive to fluralaner after contact exposure. They were moderately sensitive to phoxim and propoxur, and less sensitive to spinosad. Furthermore, the tested mite population appeared to be resistant to the pyrethroids, permethrin and deltamethrin, despite not being exposed to acaricides for at least 10 years.

Abstract Background Fluralaner is a novel isoxazoline eliciting both acaricidal and insecticidal activity through potent blockage of GABA- and glutamate-gated chloride channels. The aim of the study was to investigate the susceptibility of juvenile stages of common tick species exposed to fluralaner through either contact (Rhipicephalus sanguineus) or contact and feeding routes (Ornithodoros moubata). Methods Fluralaner acaricidal activity through both contact and feeding exposure was measured in vitro using two separate testing protocols. Acaricidal contact activity against Rhipicephalus sanguineus life stages was assessed using three minute immersion in fluralaner concentrations between 50 and 0.05 μg/mL (larvae) or between 1000 and 0.2 μg/mL (nymphs and adults). Contact and feeding activity against Ornithodoros moubata nymphs was assessed using fluralaner concentrations between 1000 to 10−4 μg/mL (contact test) and 0.1 to 10−10 μg/mL (feeding test). Activity was assessed 48 hours after exposure and all tests included vehicle and untreated negative control groups. Results Fluralaner lethal concentrations (LC50, LC90/95) were defined as concentrations with either 50%, 90% or 95% killing effect in the tested sample population. After contact exposure of R. sanguineus life stages lethal concentrations were (μg/mL): larvae - LC50 0.7, LC90 2.4; nymphs - LC50 1.4, LC90 2.6; and adults - LC50 278, LC90 1973. After exposure of O. moubata nymphs to fluralaner lethal concentrations were (μg/mL): contact exposure - LC50 720, LC95 1133; and feeding exposure- LC50 0.00007, LC95 0.09. Conclusions Fluralaner demonstrates potent in vitro acaricidal activity against all life stages of the brown dog tick, R.sanguineus. The testing of fluralaner contact and feeding routes using O. moubata nymphs demonstrates a high acaricidal activity in both exposure routes.

Abstract Background Fluralaner, a novel isoxazoline, has both acaricidal and insecticidal activity through potent blockage of GABA- and L-glutamate-gated chloride channels. This study investigated the in vitro and in vivo effects of fluralaner exposure on flea (Ctenocephalides felis) reproduction. Methods Blood spiked with sub-insecticidal fluralaner concentrations (between 0.09 and 50.0 ng/mL) was fed to fleas for 10 days using a membrane system. Cessation of reproduction in exposed fleas was assessed using flea survival, egg hatchability, and control of oviposition, pupae, and flea emergence. Fluralaner efficacy for in vivo Ctenocephalides (C.) felis control on dogs was assessed using a simulated flea-infested home environment. During a pre-treatment period, dogs were infested twice on days -28 and -21 with 100 adult unfed fleas to establish a thriving population by day 0 of the study. On day 0, one group of dogs was treated with fluralaner (Bravecto™; n = 10), while another group served as negative control (n = 10). Following treatment, dogs were infested three times with 50 fleas on days 22, 50 and 78 to simulate new infestations. Live flea counts were conducted weekly on all dogs for 12 weeks starting 1 day before treatment. Results Fluralaner potently inhibited flea reproduction capacity in vitro. Oviposition ceased completely at concentrations as low as 25.0 ng/mL. While no ovicidal effect was observed, fluralaner exerted a larvicidal effect at exceptionally low concentrations (6.25 ng/mL). In the simulated flea-infested home environment, flea-control efficacy on fluralaner-treated dogs was >99% at every time point measured for 12 weeks. No adverse events were observed in fluralaner-treated dogs. Conclusions Fluralaner completely controls egg laying, larval development and flea reproduction even at sub-insecticidal concentrations. Oral treatment of dogs with fluralaner is highly effective for eliminating fleas in a simulated flea-infested home environment.