Comparative Evaluation of the Efficacy of Two Ectoparasiticides in Preventing the Acquisition of Borrelia burgdorferi by Ixodes scapularis and Ixodes ricinus: A Canine Ex Vivo Model.

In dogs, tick infestation can cause damage ranging from a simple skin irritation to severe diseases and/or paralysis leading to animal death. For example, Ixodes ricinus and I. scapularis are among the tick species incriminated the most in the transmission of Borrelia burgdorferi, the agent of human and canine Lyme borreliosis (LB). In this study, we aimed to compare the efficacy of two products designed for dogs-an oral systemic ectoparasiticide and a topical repellent ectoparasiticide-against the acquisition of B. burgdorferi by adult I. scapularis and I. ricinus using an ex vivo model. Thirty-two beagle dogs were included in a parallel-group-designed, randomized, single-center, negative-controlled efficacy study. The dogs were allocated to three groups based on gender and body weight: a fluralaner (F, Bravecto®) treatment group (n = 8), administered a single oral treatment on day 0 at the recommended dose; a dinotefuran-permethrin-pyriproxyfen (DPP, Vectra® 3D) treatment group (n = 8), topically treated on day 56 at the recommended dose; and an untreated control group (n = 16). Blood and hair were collected from each dog on days 58, 63, 70, 77, and 84. Hair was added to the silicone-based membrane separating two glass chambers forming the feeding unit (FU). Chamber 1 was filled with blood spiked with B. burgdorferi sensu stricto, strain B31 (105 cells/mL). Chamber 2, glued below chamber 1, was seeded with 20 adult I. scapularis or I. ricinus. The FUs (n = 240) were incubated at 37 °C with a humidity >90%. Tick survival, attachment, and feces presence were observed from 1 h up to 72 h after tick seeding. The uptake of B. burgdorferi was determined in ticks using nested polymerase chain reaction (nPCR). The acaricidal efficacy of DPP-treated hair was 100% within 1 h of tick release on every study day for both I. ricinus and I. scapularis. The speed of kill associated with DPP was sufficiently fast to prevent tick attachment and engorgement, and, consequently, to prevent the acquisition of B. burgdorferi. In the F-treated group, the acaricidal efficacy observed at 12 h, throughout the study, was <20% and <28% for I. scapularis and I. ricinus, respectively. Furthermore, tick feces were observed in the FUs, and several female ticks (I. scapularis (n = 55) and I. ricinus (n = 94)) tested positive for B. burgdorferi. The results provide proof of concept for the use of an ex vivo model based on an artificial feeding system to compare two ectoparasiticides against the acquisition of B. burgdorferi by I. ricinus and I. scapularis. In addition, our results demonstrate the superiority of DPP compared to F in the speed of acaricidal activity against ticks, as well as in preventing the acquisition of B. burgdorferi.

Case presentation and management of Lyme disease patients: a 9-year retrospective analysis in France.

Introduction: Lyme borreliosis (LB) is the most common vector disease in temperate countries of the northern hemisphere. It is caused by Borrelia burgdorferi sensu lato complex. Methods: To study the case presentation of LB in France, we contacted about 700 physicians every year between 2003 and 2011. An anonymous questionnaire was established allowing the collection of 3,509 cases. The information collected was imported or directly entered into databases and allowed identifying variables that were validated in a multiple correspondence analysis (MCA). Results: Sixty percent of the cases were confirmed, 10% were probable, 13.5% doubtful, 10.2% asymptomatic seropositive and 6.3% were negative. The clinical manifestations reported were cutaneous (63%), neurological (26%), articular (7%), ocular (1.9%) and cardiac (1.3%). Almost all patients were treated. When focusing more particularly on confirmed cases, our studies confirm that children have a distinct clinical presentation from adults. There is a gender effect on clinical presentation, with females presenting more often with erythema migrans or acrodermatitis chronica atrophicans than males, while males present more often with neurological signs or arthritis than females. Discussion: This is the first time that a comprehensive study of suspected Lyme borreliosis cases has been conducted over several years in France. Although we were not able to follow the clinical course of patients after treatment, these results suggest the interest of refining the questionnaire and of following up a cohort of patients over a sufficiently long period to obtain more information on their fate according to different parameters.

Dirofilaria immitis and Dirofilaria repens in mosquitoes from Corsica Island, France.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are the main causative agents of heartworm disease and subcutaneous dirofilariasis in domestic and wild canids, respectively. Both pathogens have zoonotic potential and are transmitted by mosquitoes. The present study aimed to determine the transmission period, prevalence and diversity of Dirofilaria spp. vectors from endemic areas of Corsica (France). METHODS: A monthly point data model based on average temperature recorded by four meteorological stations during 2017 was used to calculate the Dirofilaria transmission period. From June to September 2017, female mosquitoes (n = 1802) were captured using Biogents® Sentinel 2 traps lured with carbon dioxide and BG-Lure™ or octanol. Mosquitoes were identified to species level, pooled accordingly, and screened using multiplex real-time qPCR to detect D. immitis and D. repens. RESULTS: The monthly point data model showed the possible transmission of Dirofilaria spp. from the third week in May to the last week in October in the studied area. Mosquitoes were identified as Ochlerotatus caspius (n = 1432), Aedes albopictus (n = 199), Culex pipiens sensu lato (n = 165) and Aedes vexans (n = 6) and were grouped into 109 pools (from 1 to 27 specimens, mean 11.4 ± 0.7), of which 16 scored positive for Dirofilaria spp. (i.e., n = 13; estimated infection rate [EIR] = 1.1% for D. immitis and n = 3; EIR = 0.2% for D. repens). Specifically, 6 (i.e., EIR = 3.8%) of 15 pools of Ae. albopictus were positive for D. immitis, 2 of 14 of Cx. pipiens s.l. were positive for D. immitis and D. repens, respectively, and 8 of 77 pools of Oc. caspius were positive for D. immitis (i.e., n = 6; EIR = 0.4%) and D. repens (i.e., 2; EIR = 0.1%). The highest mosquito infection rate was recorded in July (EIR = 2.5%), then in June (EIR = 1.3%) and September (EIR = 0.6%). CONCLUSIONS: The data suggest that both Dirofilaria species are endemic and occur possibly in sympatry in the studied area in Corsica, highlighting the need to implement preventive chemoprophylaxis and vector control strategies to reduce the risk of these filarioids in dog and human populations.

Vectra 3D (dinotefuran, pyriproxyfen and permethrin) prevents acquisition of Borrelia burgdorferi sensu stricto by Ixodes ricinus and Ixodes scapularis ticks in an ex vivo feeding model.

BACKGROUND: We evaluated the efficiency of an ex vivo feeding technique using a silicone membrane-based feeding chamber to (i) assess the anti-feeding and acaricidal efficacy of a spot-on combination of dinotefuran, pyriproxyfen and permethrin (DPP, Vectra® 3D) against adult Ixodes scapularis and Ixodes ricinus ticks, and to (ii) explore its effect on blocking the acquisition of Borrelia burgdorferi sensu stricto. METHODS: Eight purpose-bred dogs were randomly allocated to two equal-size groups based on body weight assessed on day 2. DPP was administered topically, as spot-on, to four dogs on day 0. Hair from the eight dogs was collected individually by brushing the whole body on days 2, 7, 14, 21, 28 and 35. On each day of hair collection, 0.05 g of sampled hair was applied on the membrane corresponding to each feeding unit (FU). Seventy-two FU were each seeded with 30 adults of I. scapularis (n = 24 FU) or I. ricinus ticks (n = 48 FU). Bovine blood spiked with B. burgdorferi sensu stricto (strain B31) was added into each unit and changed every 12 h for 4 days. Tick mortality was assessed 1 h after seeding. One additional hour of incubation was added for live/moribund specimens and reassessed for viability. All remaining live/moribund ticks were left in the feeders and tick engorgement status was recorded at 96 h after seeding, and the uptake of B. burgdorferi s.s. was examined in the collected ticks by applying quantitative real-time PCR. RESULTS: Exposure to DPP-treated hair was 100% effective in blocking B. burgdorferi s.s. acquisition. The anti-feeding efficacy remained stable (100%) against both Ixodes species throughout the study. The acaricidal efficacy of DPP evaluated at 1 and 2 h after exposure was 100% throughout the study for I. ricinus, except the 1-h assessment on day 28 (95.9%) and day 35 (95.3%). The 1-h assessment of acaricidal efficacy was 100% at all time points for I. scapularis. CONCLUSIONS: The ex vivo feeding system developed here demonstrated a protective effect of DPP against the acquisition of B. burgdorferi without exposing the animals to the vectors or to the pathogen.

Examining the Relationship of Clinical and Laboratory Parameters With Infectiousness to Phlebotomus perniciosus and Its Potential Infectivity in Dogs With Overt Clinical Leishmaniasis.

Infected dogs are considered the main domestic animal reservoirs for Leishmania infantum parasite. Infectiousness to competent phlebotomine vectors has been associated with many factors, the main being the severity of the disease exhibited by infected dogs. This study examines the relationship between different clinical parameters and the infectiousness to colonized Phlebotomus perniciosus sand flies having a blood meal on dogs. Data obtained in the present study come from an untreated group of Leishmania sick dogs submitted to xenodiagnosis for the evaluation of a spot on insecticide solution. Seventeen dogs were diagnosed as affected by leishmaniasis through clinical examination, immunofluorescence antibody test (IFAT) serology, and loop-mediated isothermal amplification (LAMP). The disease severity (clinical score) was staged by using a numeric value derived from eight clinical and parasitological parameters. Xenodiagnosis was performed on caged dogs exposed for 1.5 h to sand-fly bites. The following parameters related to sand flies were examined: blood feeding (% of blood engorged females), promastigote detection (% of promastigote-positive sand flies), promastigote burden, and the promastigote stage maturation (potential transmissibility rate). Statistical relationship between the clinical score and entomological parameters was investigated, as well as the possible correlation between each clinical and laboratory parameters and sand fly infection/infectivity. The severity of clinical score may influence the blood feeding by, and the probability of promastigote detection in, sand flies; skin lesions seem to be the main factor that influences the rate of blood feeding. Promastigote burden is related to IFAT titer, skin lesions, and clinical score. All entomological parameters are strongly related among them. This study confirms that both P. perniciosus infection and infectivity are influenced by a dog's clinical condition.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats.

These guidelines are intended to provide an in-depth review of current knowledge and assist the planning and implementation of studies for evaluating the efficacy of parasiticides in reducing transmission of vector-borne pathogens (VBPs) to dogs and cats. At present, the prevention of VBP transmission in companion animals is generally achieved through the administration of products that can repel or rapidly kill arthropods, thus preventing or interrupting feeding before transmission occurs. The present guidelines complement existing guidelines, which focus on efficacy assessment of parasiticides for the treatment, prevention and control of flea and tick infestations, but also give guidance for studies focused on other vectors (i.e. mosquitoes and phlebotomine sand flies). The efficacy of parasiticides in reducing VBP transmission can be evaluated through laboratory or field studies. As such, the present guidelines provide recommendations for these studies, representing a tool for researchers, pharmaceutical companies and authorities involved in the research, development and registration of products with claims for reducing VBP transmission in dogs and cats, respecting the overall principles of the 3Rs (replacement, reduction and refinement). Gaps in our current understanding of VBP transmission times are herein highlighted and the need for further basic research on related topics is briefly discussed.

Potential of Artesunate in the treatment of visceral leishmaniasis in dogs naturally infected by Leishmania infantum: Efficacy evidence from a randomized field trial.

Leishmaniasis is among the world's most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.

Aedes (Stegomyia) aegypti mosquito bite hypersensitivity in a dog: a case report.

BACKGROUND: Mosquitoes are vectors of several pathogens of considerable importance to humans and companion animals, including nematode helminths such as Dirofilaria immitis and Dirofilaria repens that cause heartworm disease and subcutaneous dirofilariosis, respectively. In addition to mosquito-borne pathogen transmission, mosquito bites can cause discomfort and irritation in pets, and even lead to severe hypersensitivity reactions. In the present study, we report an acute local hypersensitivity reaction in a dog following experimental exposure to Aedes (Stegomyia) aegypti. CASE PRESENTATION: A healthy six-year-old male beagle was included in an efficacy study in which dogs (n = 28) were exposed to Ae. aegypti mosquitoes. On Day - 6, the dog was allocated to one of the study groups, consisting of seven dogs to be treated on Day 0 with an imidacloprid/flumethrin collar. After sedation, animals were exposed to approximately 50 females of Ae. aegypti for 60 (± 5) minutes on Days - 6, 1, 7, 14, 21, 28, 55, and 83. On Day - 6, no allergic reaction to the mosquito bites was observed. However, on Day 1, corresponding to the second challenge, the dog demonstrated an acute allergic reaction characterized by swelling of the face (especially in the base of the muzzle and around the eyes), redness of the eyes, and conjunctival edema of the right eye was also observed. The dog was immediately treated with an intramuscular injection of a commercially available antihistamine treatment, Pen-Hista-Strep® containing a suspension of benzylpenicillin, chlorphenamine, dexamethasone, dihydrostreptomycin, and procaine at a dosage of 1 mL per 10 kg. A few hours after treatment, the dog showed noticeable improvement. CONCLUSIONS: This case provides the first evidence of canine acute local hypersensitivity reaction to mosquito bites under laboratory conditions. This observation suggests that invasive mosquito species such as Aedes spp. may affect the health and comfort of our companion animals, especially for pets with outdoor access without individual protective measures against insect bites.

Effect of Dinotefuran, Permethrin, and Pyriproxyfen (Vectra® 3D) on the Foraging and Blood-Feeding Behaviors of Aedes albopictus Using Laboratory Rodent Model.

Dinotefuran-Permethrin-Pyriproxyfen (DPP) is used to kill and repel mosquitoes from dogs. However, the influence of the product on the host-seeking behavior of mosquitoes remains unknown. The interference of DPP with the host selection of unfed female Aedes albopictus was investigated. A total of 18 animals (9 mice and 9 rats) were divided into three groups of six animals each. DU: DPP treated rats (n = 3) with untreated mice (n = 3), UD: DPP treated mice (n = 3) with untreated rats (n = 3) and control UU: untreated mice (n = 3) and untreated rats (n = 3). In each group, the rats and mice were placed 30 cm apart. After sedation, the animals in each group were exposed twice (Day 1 and Day 7 post-treatment) for one hour to 71 ± 3 female mosquitoes. Mosquitoes were categorized after the 2-h post-exposure period as dead or alive. Blood-meal origin was determined from mosquitoes using a newly customized duplex qPCR. The highest values of forage ratio (1.36 ≥ wi ≤ 1.88) and selection index (0.63 ≥ Bi ≤ 0.94) for rat hosts indicates a preference of mosquitoes for this species as compared to mice when co-housed during the exposure. The mosquitoes only seldom fed on mice, even in the untreated group. The anti-feeding effect of DPP was therefore only assessed on rat's hosts. The results showed that DPP, when directly applied on rats, provided a direct protection of 82% and 61% on Day 1 and Day 7, respectively, while when applied on mice hosts (UD), the DPP provided an indirect protection of 21% and 10% on Day 1 and Day 7, respectively. The results showed also that DPP, when applied on rats, provided a direct protection against Ae. albopictus bites. This effect did not result in increased exposure of the untreated host placed in the same cage at a distance of 30 cm.

Interrupted Blood Feeding in Ticks: Causes and Consequences.

Ticks are obligate hematophagous arthropods and act as vectors for a great variety of pathogens, including viruses, bacteria, protozoa, and helminths. Some tick-borne viruses, such as Powassan virus and tick-borne encephalitis virus, are transmissible within 15-60 min after tick attachment. However, a minimum of 3-24 h of tick attachment is necessary to effectively transmit bacterial agents such as Ehrlichia spp., Anaplasma spp., and Rickettsia spp. to a new host. Longer transmission periods were reported for Borrelia spp. and protozoans such as Babesia spp., which require a minimum duration of 24-48 h of tick attachment for maturation and migration of the pathogen. Laboratory observations indicate that the probability of transmission of tick-borne pathogens increases with the duration an infected tick is allowed to remain attached to the host. However, the transmission time may be shortened when partially fed infected ticks detach from their initial host and reattach to a new host, on which they complete their engorgement. For example, early transmission of tick-borne pathogens (e.g., Rickettsia rickettsii, Borrelia burgdorferi, and Brucella canis) and a significantly shorter transmission time were demonstrated in laboratory experiments by interrupted blood feeding. The relevance of such situations under field conditions remains poorly documented. In this review, we explore parameters of, and causes leading to, spontaneous interrupted feeding in nature, as well as the effects of this behavior on the minimum time required for transmission of tick-borne pathogens.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum.

BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10-1 to 1.5 × 10-4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

New Molecular Approach for the Detection of Kinetoplastida Parasites of Medical and Veterinary Interest.

Kinetoplastids are protozoa containing a range of ubiquitous free_living species-pathogens of invertebrates, vertebrates and even some plants. Some of them are causative agents of canine vector-borne diseases. Their diagnosis is often missing in a gold standard. Here, we proposed a molecular approach for the diagnosis and study of Kinetoplastida. The TaqMan qPCR assays target the following genes: 24Sa LSU of Kinetoplastida, 28S LSU of Leishmania/ Trypanosoma spp., 5.8S rRNA of Trypanosoma spp., 18S SSU of Leishmania spp., kinetoplast minicircle DNA (kDNA) of L. donovani complex and kDNA of L. infantum, were designed, validated for their sensitivity (Se) and specificity (Sp) in silico and in vitro using a panel of known DNAs. They were then used to screen 369 blood samples (358 dogs, 2 equids, 9 monkeys). In addition, new 28S LSU primer sets are presented to use for Kinetoplastida's identification by PCR/sequencing. All qPCRs showed consistently high analytical sensitivities and reproducibility. They detect approximately 0.01 parasite/ mL blood for the kDNA based- qPCRs and at least a single cell-equivalent of rDNA for the other systems. Based on the sequencing results, after screening, Se and Sp were: 0. 919 and 0.971, 0.853 and 0.979, 1.00 and 0.987, 0.826 and 0.995 for all of Kinetoplastida, Leishmania/ Trypanosoma, Trypanosoma, Leishmania spp. specific qPCRs, respectively. kDNA based qPCRs were more sensitive and specific (Se: 1.00; Sp: 0.997). PCR/sequencing allowed the detection of Kinetoplastids in animal blood samples such as L. infantum, L. guyanensis, T. congolense, T. evansi and Bodo spp. The molecular approach proposed here is useful for epidemiological studies, fundamental research such as screening for new Kinetoplastida species, diagnosis and therapeutic follow-up. In addition, researchers are free to choose the molecular tools adapted to their aims.

A Suspected Case of Lyme Borreliosis in a Dog from Belgium.

A 5-year-old Jack Russell Spaniel was presented in December 2017 to his veterinarian in Belgium for sudden weakness, reluctance to move, and pain. Blood analysis showed no deviations and serum increased levels of B. burgdorferi s.l. antibodies were detected. The dog recovered a few days after the onset of doxycycline treatment. This case illustrates the possible relationship between tick-borne diseases and orthopedic problems.

Permethrin enhances the agonist activity of dinotefuran on insect cholinergic synaptic transmission and isolated neurons.

Insect resistance mechanisms against pesticides lead to the development and the search of new pesticide combinations in order to delay the resistance. The combination of neonicotinoids with pyrethroids was currently proposed but the mode of action of these compounds at synaptic and extrasynaptic levels needs to be further explored. In the present study, we evaluated the effect of the combination of two insecticides, permethrin and dinotefuran, on cockroach cholinergic synaptic transmission and on isolated cell bodies. We first found that combination of 5 µM permethrin and dinotefuran enhances depolarization of the sixth abdominal ganglion compared to dinotefuran alone, without an inhibition of the spontaneous activity. However, a pretreatment with 1 µM dinotefuran or permethrin before bath application of the mixture inhibits the ganglionic depolarization. Compared to permethrin, 1 µM dinotefuran induces a persistent enhancement of spontaneous activity. Interestingly, at extrasynaptic level, using dorsal unpaired median neurons and Kenyon cells, we found that combination of both 1 µM dinotefuran and permethrin resulted in an increase of the mixture-induced current amplitudes. Pretreatment with 1 µM dinotefuran strongly decreases the currents whereas permethrin induces a time-dependent inhibition. These data demonstrate that the combination of dinotefuran and permethrin enhances the effect of dinotefuran.

Early Babesia canis transmission in dogs within 24 h and 8 h of infestation with infected pre-activated male Dermacentor reticulatus ticks.

BACKGROUND: This study was designed to assess the ability of fed male Dermacentor reticulatus ticks to transmit Babesia canis to dogs after being detached from previous canine or ovine hosts. METHODS: The study was an exploratory, parallel group design conducted in two trials. All the animals were sero-negative for babesiosis prior to enrolment. In a first trial, donor dogs and donor sheep were infested with Babesia canis infected male and uninfected female ticks for 72 h. The ticks were detached and the second group of host dogs were infested for 24 h before tick removal. In a second trial, the experiment was repeated but the donor animals were infested for 88 h and the second group of host dogs were infested for 8 h prior to tick removal. After infestation, the dogs were maintained under clinical surveillance and blood samples were collected for blood smear, IFA and PCR analysis. A dog was considered infected if any of these tests were positive. RESULTS: All of the dogs (6 out of 6) were infected after being exposed to pre-activated male ticks for 24 h. Half of the dogs were infected after being exposed to pre-activated ticks for 8 h: 1 out of 3 dogs infested with ticks removed from sheep and 2 out of 3 dogs infested with ticks removed from dog. All the infected dogs were positive to blood smear, IFA and PCR. Three of these dogs exhibited elevated body temperature (> 39.4 °C). CONCLUSIONS: This study demonstrates the ability of male D. reticulatus to transmit B. canis to dogs. The study also illustrates for the first time that, regardless of the first host on which ticks may attach and start feeding, Babesia canis can be transmitted to dogs within 8 h of infestation. Since no minimal transmission time can be established for all possible natural situations, a strategy of prevention based on anti-attachment or repellency is recommended.

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% of D. immitis, B. malayi and B. pahangi respectively. This study presents the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes. Considering that the results might vary in other mosquito species, further studies are needed to consolidate the obtained preliminary results before applying this tool in entomological surveillance as a fast mass screening method of filariosis vectors in endemic areas.

Blocking the transmission of heartworm (Dirofilaria immitis) to mosquitoes (Aedes aegypti) by weekly exposure for one month to microfilaremic dogs treated once topically with dinotefuran-permethrin-pyriproxyfen.

BACKGROUND: This study assessed the influence of a topical ectoparasiticide (dinotefuran-permethrin-pyriproxyfen, DPP, Vectra®3D, Ceva Animal Health) on the acquisition of heartworm microfilariae by mosquitoes exposed to microfilaremic dogs weekly for 1 month. METHODS: Six beagle dogs (9.2 ± 1.6 kg body weight) infected with Dirofilaria immitis were allocated to two groups of three dogs: an untreated control group and a DPP-treated group. Dogs were treated on Day 0 and exposed under sedation for 1 h to 80 ± 20 unfed Aedes aegypti. Each dog was exposed to mosquitoes released into mosquito-proof containers on Days -7 (pretreatment), 7, 14, 21 and 28. Up to 20 engorged mosquitoes were aspirated from the cage as soon as they were blood-fed. They were dissected and the blood from each midgut was stained for a microfilaria (MF) count. After each exposure, mosquitoes were classified as live, moribund or dead and engorged or nonengorged. The number of dead mosquitoes was recorded daily for 16 days, when the live mosquitoes were dissected to count the infective third-stage larvae (L3). RESULTS: Prior to treatment, 95% of the engorged mosquitoes in both groups had MF. After treatment, engorgement rates for the treated group were 0%, 2.3%, 2.7% and 2.2% for Days 7, 14, 21 and 28, respectively, with anti-feeding efficacy (repellency) of 100%, 98.0%, 95.8% and 97.0%, respectively. A total of 22 mosquitoes fed on treated dogs; most of them were dead within 24 h, and all were dead within 72 h. Only 2 unfed mosquitoes exposed to treated dogs survived the incubation period and no L3 were found in them. A total of 121 of the 132 (91.6%) surviving mosquitoes that had engorged on untreated dogs had an average of 12.3 L3 per mosquito (range, 0-39). CONCLUSIONS: DPP was more than 95% effective in inhibiting blood-feeding and killing both engorged and nonengorged mosquitoes exposed weekly to microfilaremic dogs for 28 days after treatment. Treatment with DPP was completely effective in killing the few mosquitoes that fed on the treated dogs before they lived long enough for the microfilariae to develop to L3 and, consequently, was completely effective in blocking the transmission of L3 to other animals. DPP can break the life cycle of D. immitis and prevent infected dogs and infected mosquitoes from being effective reservoirs and can slow down the spread of heartworms, even those resistant to macrocyclic lactone preventives.

Shifting the paradigm in Dirofilaria immitis prevention: blocking transmission from mosquitoes to dogs using repellents/insecticides and macrocyclic lactone prevention as part of a multimodal approach.

BACKGROUND: This study assessed the influence of a topical ectoparasiticide (dinotefuran-permethrin-pyriproxyfen, DPP, Vectra® 3D, Ceva Animal Health) combined with a macrocyclic lactone (milbemycin oxime, MBO, Interceptor®, Virbac) on transmission of heartworm L3 from mosquitoes to dogs and subsequent development of worms in treated dogs exposed to infected mosquitoes. METHODS: Thirty-two beagle dogs were allocated to four groups of eight: Group 1, untreated controls; Group 2, treated topically with DPP on Day 0; Group 3, treated orally with MBO on Day 51; and Group 4, treated with DPP on Day 0 and MBO on Day 51. Dogs were exposed under sedation for 1 h to Dirofilaria immitis (JYD-34)-infected Aedes aegypti on Days 21 and 28. At the end of each exposure, mosquitoes were classified as live, moribund, or dead and engorged or non-engorged. Live or moribund mosquitoes were incubated for daily survival assessment for 3 days. Mosquitoes were dissected before and after exposure to estimate the number of L3 transmitted to each dog. Dogs were necropsied 148 to 149 days postinfection. RESULTS: A total of 418 mosquitoes fed on the 16 dogs in Groups 1 and 3, while only 6 fed on the 16 DPP-treated dogs in Groups 2 and 4. Mosquito anti-feeding (repellency) effect in Groups 2 and 4 was 98.1 and 99.1%, respectively. The estimated numbers of L3 transmitted to controls, DPP-treated, MBO-treated and DPP + MBO-treated dogs were 76, 2, 78, and 1, respectively. No heartworms were detected in any of the DPP + MBO-treated dogs (100% efficacy), while 8 out of 8 were infected in the control group (range, 21-66 worms per dog), 8 out of 8 were infected in the MBO-treated group (58% efficacy), and 3 out of 8 were infected in the DPP-treated group (96% efficacy). CONCLUSIONS: DPP repelled and killed most mosquitoes that were capable of transmitting heartworm L3 to dogs. The "Double Defense" protocol of DPP + MBO had better efficacy for protecting dogs against heartworm transmission and infection than MBO alone. This added DPP benefit is more pronounced when macrocyclic lactone-resistant strains of heartworms are involved or lack of compliance in macrocyclic lactone administration is known or suspected.

Usefulness of a topical combination of dinotefuran and pyriproxyfen for long-term control of clinical signs of allergic dermatitis in privately-owned cats in Ile-de-France region.

BACKGROUND: The present study assessed the activity of a combination of dinotefuran and pyriproxyfen (Vectra® Felis) for long-term control (3 months) of allergic dermatitis (AD) in privately-owned cats under common household conditions in Ile-de-France region. METHODS: This was an open pre-treatment vs post-treatment study. Twenty-eight client-owned cats with clinical signs of AD were enrolled in the study. They received topical application of the combination of dinotefuran and pyriproxyfen on days 0, 28, 56 and 84. Two parameters (clinical signs and pruritus severity) were used to assess the animals' condition on days 0, 28 and 84. Fleas were counted if they were observed. RESULTS: Of the 28 cats initially enrolled, 26 were presented on day 28 and 20 for the final evaluation on day 84. A significant improvement in clinical signs and pruritus was observed in cats for which fleas and/or flea feces were detected on day 0. Globally, the post-treatment AD clinical scores on days 28 and 84 were different from that of the pre-treatment on day 0, with a reduction of 30% and 71%, respectively. For cats with fleas and/or flea feces, the reduction on days 28 and 84 was 33% and 85%, respectively. The improvement of clinical signs and pruritus was not significant in cats with no visible fleas and no flea feces at the beginning of the trial (n = 8). CONCLUSIONS: The present study indicated that the treatment with a combination of dinotefuran and pyriproxyfen should be considered as useful in controlling fleas on cats without additional environmental treatment and useful for long-term control of clinical signs and pruritus in allergic cats.

Synergy between dinotefuran and fipronil against the cat flea (Ctenocephalides felis): improved onset of action and residual speed of kill in adult cats.

BACKGROUND: The cat flea, Ctenocephalides felis felis (C. felis), is a cosmopolitan hematophagous ectoparasite, and is considered to be the most prevalent flea species in both Europe and the USA. Clinical signs frequently associated with flea bites include pruritus, dermatitis and in severe cases even pyodermatitis and alopecia. Ctenocephalides felis is also a vector for several pathogens and is an intermediate host for the cestode Dipylidium caninum. Treatment of cats with a fast-acting pulicide, that is persistently effective in protecting the animal against re-infestation, is therefore imperative to their health. In addition, a rapid onset of activity ("speed of kill") may also reduce the risks of disease transmission and flea allergic dermatitis. The aim of this study was to evaluate the in vitro insecticidal activity and potential synergism between dinotefuran and fipronil against C. felis. A further aim was to evaluate the onset of activity and residual speed of kill of the combination in vivo on cats artificially infested with C. felis. METHODS: In the first study, the insecticidal activity of dinotefuran and fipronil separately and dinotefuran/fipronil (DF) in combination, at a fixed ratio (2:1), was evaluated using an in vitro coated-vial bioassay. In the second study, the onset of activity against existing flea infestations and residual speed of kill of DF against artificial flea infestations on cats was assessed in vivo. Onset of activity against existing flea infestations was assessed in terms of knock-down effect within 2 h post-treatment and onset of speed of kill assessed at 3 h, 6 h and 12 h post-treatment. Residual speed of kill was evaluated 6 h and 48 h after infestation, over a period of six weeks post-treatment. RESULTS: In vitro results revealed that the DF combination was synergistic and more potent against fleas than either compound alone. The combination also proved effective when tested in vivo. Efficacy was > 97% [geometric mean (GM) and arithmetic mean (AM)] at 3 h after treatment, and ≥ 99.8% (GM and AM) at 6 h and 12 h post-treatment. At 6 h after flea re-infestations, the efficacy of DF remained ≥ 90.8% (GM and AM) for up to 28 days, and at 42 days post-treatment persistent efficacy was still ≥ 54.3% (GM and AM). At 48 h after flea re-infestations, DF remained almost fully effective for up to 28 days, with efficacies ≥ 99.4% (GM and AM) and was persistently ≥ 93.0% (GM and AM) effective for up to 42 days post-treatment. CONCLUSIONS: The combination of dinotefuran and fipronil in a single formulation exhibited strong synergistic insecticidal activity against C. felis in vitro, and also proved effective on artificially infested cats. This activity had a rapid onset that persisted for 6 weeks against re-infestations of C. felis on cats. The rapid curative insecticidal effect was observed as early as 3 h after treatment, and as early as 6 h after re-infestations for up to 6 weeks post-treatment. The insecticidal activity profile of DF makes it an optimal candidate for the protection of cats against flea infestations, and possibly also associated diseases.