Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons.

Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

Prevention of transmission of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by Ixodes spp. ticks to dogs treated with the Seresto® collar (imidacloprid 10% + flumethrin 4.5%).

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Comparing response of buff-tailed bumblebees and red mason bees to application of a thiacloprid-prochloraz mixture under semi-field conditions.

Recent studies have reported interspecific differences in how bee species respond to various stressors. Evaluating the exposure and responses of different bee species to plant protection products is considered an essential part of their risk assessment. This study was conducted to assess the impacts of thiacloprid-prochloraz mixture on buff-tailed bumblebees (Bombus terrestris) and red mason bees (Osmia bicornis) in a worst-case scenario under semi-field conditions. Bumblebee colonies or solitary bee trap nests were confined in tunnels with flowering oilseed rape. The recommended maximum application rates of 72 g thiacloprid/ha and 675 g prochloraz/ha were applied as a tank mixture during bee flight in full flowering oilseed rape. Several parameters such as flight and foraging activity, population parameters, and exposure level were investigated. Our results show adverse effects of the combination of thiacloprid and prochloraz on the reproductive performance of red mason bees. The number of cocoons produced by O. bicornis was significantly reduced in the treatment compared to the control group. Regarding bumblebees, we found no effects of the thiacloprid-prochloraz mixture on any observed parameters of colony development. The maximum detected concentrations of both active substances three days after application were higher in O. bicornis pollen mass compared to B. terrestris stored pollen. We conclude that this worst-case scenario of thiacloprid-prochloraz exposure poses a high risk to solitary bees and thus the use of such mixture should be restricted.

Genotoxic effects of imidacloprid in human lymphoblastoid TK6 cells.

Among neonicotinoid insecticides, the fastest growing class of insecticides worldwide over the past decade, imidacloprid (IMI) is the most widely used one. The effects of IMI on human health, especially on genetic toxicity have gradually aroused more attention. In this study, a combined in vitro approach employing the thymidine kinase (TK) gene mutation assay, the comet assay and the micronucleus test was taken to assess the genotoxicity of IMI. The mechanism behind IMI was also explored by measuring reactive oxygen species (ROS) in the human lymphoblastoid TK6 cells. The cells were treated with 0.01, 0.1, 1, 5, and 10 µg/mL IMI, and ROS generation was measured by the use of 2,7,-dichlorofluorescin diacetate (DCFH-DA) assay. IMI significantly increased the micronucleus (MN) frequency, TK mutations and DNA damage with a dose-effect relationship, and the lowest effective concentration in those tests was 0.1 µg/mL. However, no obvious change of intracellular ROS was observed for any concentrations. The results indicate that IMI has potential genotoxic effects on TK6 cells, but ROS does not seem to be involved as a mechanism of genotoxicity under the experimental conditions.

Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs.

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

Co-encapsulation of imidacloprid and lambda-cyhalothrin using biocompatible nanocarriers: Characterization and application.

A well-known strategy for managing pest resistance is application of mixture of pesticides. Conventionally formulated pesticides have several environmental incompatibilities. The use of biocompatible and biodegradable nanocarriers in formulating pesticides could improve environmental protection. In this study, a mixture of imidacloprid and lambda-cyhalothrin was co-encapsulated for the first time using liposomes as nanocarrier to simultaneously deliver these insecticides. Ethanol injection was used to produce self-assembled liposomes. The formed nanoliposomes were coated with different concentrations of chitosan. Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscope (SEM) and FT-IR spectroscopy. The encapsulation efficiencies of lambda-cyhalothrin and imidacloprid were about 93% and 51%, respectively. The insecticide carrying liposomes had a size and surface charge of 57 nm and +0.6 mV, respectively. The size and surface charge of the particles produced were increased to 69 nm and +31 mV after being coated with chitosan (0.1%, W/V). In this study, residual activity of technical grade imidacloprid, lambda-cyhalothrin and their mixture and the effect of adjuvants used in commercial and nano formulations of these insecticides on Myzus persicae Sulzer was investigated. The insecticidal effects and duration of residual activity of nano-formulations was correlated with concentration of chitosan in final formulation. In accordance with the life cycle of M. persicae, using the mixture of imidacloprid and lambda-cyhalothrin improves the residual effect over their use alone. The use of lipid nanocarriers makes the improvement even further and can be a better alternative to conventional combination of these insecticides due to their more environmental friendliness.

Dermatitis caused by autochthonous Cercopithifilaria bainae from a dog in Florida, USA: clinical, histological and parasitological diagnosis and treatment.

BACKGROUND: Cercopithifilaria bainae is a tick-vectored filarioid nematode associated with erythematous dermatitis in dogs. It has not been reported previously in the United States. HYPOTHESIS/OBJECTIVE: To describe clinical, histological and parasitological diagnosis and treatment of C. bainae in a dog. ANIMALS: An 11-month-old golden retriever/standard poodle mixed breed dog from Florida (USA). METHODS AND MATERIALS: The dog had no travel history within or outside the United States, was presented with a one month history of annular erythematous plaques on the head and ulcers on the medial canthi. Lesions were unresponsive to antibiotic treatment. RESULTS: Histopathological evaluation of skin biopsies revealed an eosinophilic to lymphohistiocytic perivascular dermatitis with multiple microgranulomas and rare 5-10 µm diameter microfilariae within microgranulomas. Microfilarial morphology was consistent with C. bainae. PCR and sequencing of 18S rRNA and mitochondrial cytochrome oxidase subunit I genes confirmed the nematodes as C. bainae. The dog was treated with a commercial spot-on containing imidacloprid and moxidectin, and clinical resolution occurred. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first report of C. bainae in a dog in the United States and the first description of dermatological lesions caused primarily by C. bainae.

Imidacloprid induced alterations in oxidative stress, biochemical, genotoxic, and immunotoxic biomarkers in non-mammalian model organism Galleria mellonella L. (Lepidoptera: Pyralidae).

Imidacloprid (IMI), a neonicotinoid insecticide, is widely used to control pests in agriculture. We investigated the changes in antioxidant enzyme activities, lipid peroxidation levels, biochemical effects, genotoxic effect, and immunotoxic effect of sublethal doses (0.25, 0.50, 0.75, and 1.00 µg) of IMI at different time periods (24, 48, 72, and 96 h) on a model organism, Galleria mellonella L. The results indicated that there were dose-dependent increases in both antioxidant enzyme activities (SOD and CAT) and MDA levels. Protein content was not affected by IMI at 24th and 48th, whereas it was decreased by the highest dose of IMI (1.00 µg) at 72nd and 96th h. Lipid and carbohydrate contents were reduced with increasing doses of IMI. Micronucleus frequency significantly increased in all IMI doses. All IMI doses caused a significant decrease in THC at 24th, 48th, and 72nd h. Our results can help to illustrate the effects of IMI in target organisms and indirectly may aid to discover potential risk of it on nontarget organisms. Future studies, at molecular levels, will be helpful in understanding the mechanism of action of IMI on these biomarkers.

CUTANEOUS DEMODICOSIS AND UV-INDUCED SKIN NEOPLASIA IN TWO GOELDI'S MONKEYS (CALLIMICO GOELDII).

Two nonrelated Goeldi's monkeys (Callimico goeldii) from the same enclosure developed multifocal alopecia with hyperkeratotic to ulcerative skin lesions on the lower abdomen and inner thighs. Necropsy samples of the first animal showed hyperplastic dermatitis together with in situ carcinoma and intralesional Demodex organisms. The second monkey developed similar lesions 2.5 yr later. Skin scrapings and biopsies also revealed Demodex mites within hyperplastic dermatitis. Long-term treatment with ivermectin, imidacloprid-moxidectin, and sarolaner resolved the demodicosis but skin lesions progressed to actinic keratosis and carcinoma. Both cutaneous neoplasia and demodicosis are rarely described in New World monkeys and these are the first reported cases in Goeldi's monkeys. Since the animals had access to ultraviolet (UV) light, as recommended for indoor-housed callitrichids, the skin tumors were likely UV-induced and the mites have settled particularly within impaired regions. Thus, apparent demodicosis can indicate cutaneous immunosuppression and might alert caretakers to adjust the UV regime.

The effects of imidacloprid combined with endosulfan on IgE-mediated mouse bone marrow-derived mast cell degranulation and anaphylaxis.

Low levels of endosulfan are known to stimulate mast cells to release allergic mediators, while imidacloprid can inhibit IgE-mediated mast cell degranulation. However, little information about the effects of both pesticides together on mast cell degranulation is available. To measure the effects, IgE-activated mouse bone marrow-derived mast cells (BMMCs) were treated with imidacloprid and endosulfan, individually, and simultaneously at equi-molar concentrations in tenfold steps ranging from 10-4 to 10-11 M, followed by measuring several allergy-related parameters expressed in BMMCs: the mediator production and influx of Ca2+, the phosphorylation content of NF-κB in the FcεRI signaling pathway. Then, the effects of the mixtures on IgE-induced passive systemic anaphylaxis (PSA) of BALB/c was detectded. This study clearly showed that the application of equi-molar mixtures of both pesticides with 10-4-10-5 M significantly inhibited the IgE-mediated mouse bone marrow-derived mast cells degranulation in vitro and 10-4 M of them decreased IgE-mediated PSA in vivo, as the application of imidacloprid at the same concentration alone did. Morever endosulfan alone had no remarkable stimulatory effects on any of the factors measured. In conclusion, simultaneous application of equi-molar concentrations of both pesticides generally showed highly similar responses compared to the responses to imidacloprid alone, suggesting that the effects of the mixture could be solely attributed to the effects of imidacloprid.

Toxic effects of imidacloprid combined with arsenic: Oxidative stress in rat liver.

Imidacloprid (IMI), a newer neonicotinoid insecticide, induces oxidative insult to hepatocytes due to the formation of reactive metabolites during hepatic metabolism. The present study aimed to determine the potentiating effect of arsenic (As) on IMI-induced hepatic damage in Wistar rats. Rats, randomly divided into eight groups with six in each, were subjected to daily oral administration for 28 days. Group I served as control; group II received IMI at the dose rate of 16.9 mg/kg body weight; groups III, IV, and V received As at the dose rate of 50, 100, and 150 ppb, respectively, in drinking water; groups VI, VII, and VIII received both IMI (16.9 mg/kg) and As in drinking water at the rate of 50, 100, and 150 ppb, respectively. Repeated oral administration of IMI or As resulted in significant ( p < 0.05) elevation of plasma phosphatases, transferases, hepatic malondialdehyde, and advanced oxidation protein product levels, but significantly ( p < 0.05) decreased levels of total proteins, thiols, and activities of antioxidant enzymes that indicate oxidation-induced hepatotoxicity. These findings were further corroborated by histological alterations in hepatic tissue of IMI or As-administered rats. The coadministration of both IMI and As in rats produced more severe alterations in these parameters in hepatic tissue. Reduced antioxidant indices and increased hepatic damage biomarkers with pronounced histopathological alterations in hepatic tissue after combined exposure to toxicants indicate potentiating toxic effect of As on IMI-induced hepatotoxicity.

Effect of long term exposure to sublethal concentration of imidacloprid on some biochemical and haematological parameters of Grass carp and Goldfish.

During the present research, C. idella and C. auratus fish were exposed to 2 ppm concentration of imidacloprid for 28 and 24 days, respectively, and the effect on biochemical and haematological parameters was investigated. During the study of biochemical parameters, there occurred significant increase (P<0.05) in the serum levels of ALT and creatinine of imidacloprid exposed groups of both species of fish. The level of serum albumin of imidacloprid exposed groups of both fish species was significantly lower as compared to control group (P<0.05). Serum globulin level in imidacloprid exposed group of C. idella was insignificantly lower as compared to control group, however the serum globulin level of C. auratus was significantly lower than the control group (P < 0.05). The level of total proteins in serum of imidacloprid exposed groups of both fish species was insignificantly lower as compared to control groups (P>0.05). During the study of haematological parameters, TLC of C. idella was insignificantly (P>0.05) higher than control group but the TLC of C. auratus was significantly (P<0.05) higher than control. There was also observed increasing trend in the percentage of neutrophils and lymphocytes of imidacloprid exposed group of each fish species. The platelets count of imidacloprid exposed group of each fish species was significantly (P<0.05) lower than control group. The haemoglobin concentration of imidacloprid exposed group of C. idella was significantly lower than control group (P<0.05). In case of C. auratus, the haemoglobin level of imidacloprid exposed group was insignificantly lower than control group (P>0.05). From the finding of the present research it was concluded that 28 days exposure of C. idella and 24 days exposure of C. auratus to 2 ppm concentration of imidacloprid does not cause mortality however the exposure causes alteration in the normal level of biochemical and haematological parameters.

Effect of wall type, delayed mortality and mosquito age on the residual efficacy of a clothianidin-based indoor residual spray formulation (SumiShield™ 50WG) in southern Mozambique.

Indoor residual spraying (IRS) is one of the main malaria vector control strategies in Mozambique alongside the distribution of insecticide treated nets. As part of the national insecticide resistance management strategy, Mozambique introduced SumiShield™ 50WG, a third generation IRS product, in 2018. Its residual efficacy was assessed in southern Mozambique during the 2018-2019 malaria season. Using a susceptible Anopheles arabiensis strain, residual efficacy was assessed on two different wall surfaces, cement and mud-plastered walls, using standard WHO (World Health Organization) cone bioassay tests at three different heights. Female mosquitoes of two age groups (2-5 and 13-26 day old) were exposed for 30 minutes, after which mortality was observed 24h, 48h, 72h, and 96h and 120h post-exposure to assess (delayed) mortality. Lethal times (LT) 90, LT50 and LT10 were estimated using Bayesian models. Mortality 24h post exposure was consistently below 80%, the current WHO threshold value for effective IRS, in both young and old mosquitoes, regardless of wall surface type. Considering delayed mortality, residual efficacies (mosquito mortality equal or greater than 80%) ranged from 1.5 to ≥12.5 months, with the duration depending on mortality time post exposure, wall type and mosquito age. Looking at mortality 72h after exposure, residual efficacy was between 6.5 and 9.5 months, depending on wall type and mosquito age. The LT50 and LT10 (i.e. 90% of the mosquitoes survive exposure to the insecticides) values were consistently higher for older mosquitoes (except for LT10 values for 48h and 72h post-exposure mortality) and ranged from 0.9 to 5.8 months and 0.2 to 7.8 months for LT50 and LT10, respectively. The present study highlights the need for assessing mosquito mortality beyond the currently recommended 24h post exposure. Failure to do so may lead to underestimation of the residual efficacy of IRS products, as delayed mortality will lead to a further reduction in mosquito vector populations and potentially negatively impact disease transmission. Monitoring residual efficacy on relevant wall surfaces, including old mosquitoes that are ultimately responsible for malaria transmission, and assessing delayed mortalities are critical to provide accurate and actionable data to guide vector control programmes.

The clinical toxicity of imidacloprid self-poisoning following the introduction of newer formulations.

BACKGROUND: Self-poisoning with imidacloprid has been previously shown to have low toxicity in humans. Since 2007 newer formulations of Imidacloprid with unknown solvents have been introduced and the potential clinical consequences of these products have not been described. METHODS: Clinical and demographic data were prospectively collected from admissions following oral ingestion of imidacloprid from seven hospitals in Sri Lanka. Data was collected from 2002 to 2007 in an already published study. We compared this data on poisonings collected from 2010 to 2016 following the introduction of new formulations of imidacloprid. RESULTS: From 2002-2007, there were 56 patients with ingestion to imidacloprid compared to 67 patients post 2010 The median time to presentation prior to 2007 was 4 h (IQR 2.3-6.0 hrs) and post 2010 was only 2.0 hr (IQR 1.5 to 3.1 hrs). The median amount ingested was 15 ml (IQR 10.0-50.0mls) prior to 2007 and 27.5mls (IQR 5.0-71.8mls) post 2010. In both studies most patients developed non-specific symptoms including nausea, vomiting, epigastric pain and headache. However, prior to 2007 only 1.9% of the cohort required mechanical ventilation due to respiratory failure and there were no reported deaths. In contrast, post 2010; deaths occurred in 3.0% of the cohort and 6.0% required mechanical ventilation for respiratory failure. The cause of mortality was due to one case of cardiorespiratory failure and the other due to a prolonged admission complicated with lobar pneumonia leading to decompensated liver failure on the background of undiagnosed liver cirrhosis. CONCLUSION: Although acute exposure to imidacloprid is usually associated with mild non-specific symptoms, since the introduction of new formulations of imidacloprid, the toxic profile has changed with reported cases of death as well as an increase in cases requiring mechanical ventilation. The change in toxicity could be due to the solvents used in the newer formulations but also due to higher dose of imidacloprid described in our latter cohort. Further research into these solvents needs to be done and continued toxicovigilance is required.

Aging-related changes in the sensitivity of behavioral effects of the neonicotinoid pesticide clothianidin in male mice.

Neonicotinoids, which act as agonists of the nicotinic acetylcholine receptors of insects, are widely used pesticides worldwide. Although epidemiological studies revealed that the detection amounts of neonicotinoids in urine are higher in the elderly population than other age-groups, there is no available information regarding the risks of neonicotinoids to older mammals. This study was aimed to investigate aging-related differences in the behavioral effects of the neonicotinoid pesticide clothianidin (CLO). We acutely administered a sub-NOAEL level (5 mg/kg) of CLO to adult (12-week-old) and aging (90-week-old) mice and conducted four behavioral tests focusing on the emotional behavior. In addition, we measured the concentrations of CLO and its metabolites in blood, brain and urine. There were age-related changes in most parameters in all behavioral tests, and CLO significantly decreased the locomotor activity in the open field test and elevated plus-maze test in the aging group, but not in the adult group. The concentrations of most CLO and its metabolites were significantly higher in the blood and brain and were slightly lower in the urine in the aging group compared to the adult group. These findings should contribute to our understanding of age-related differences in the adverse effects of neonicotinoids in mammals.

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.

An integrated approach, based on mass spectrometry, for the assessment of imidacloprid metabolism and penetration into mouse brain and fetus after oral treatment.

Imidacloprid is an insecticide belonging to neonicotinoids, a class of agonists of the nicotinic acetylcholine receptors that shows higher affinities in insects compared to mammals. However, recent evidence show that neonicotinoids can bind to the mammalian receptors, leading to detrimental responses in cultured neurons. We developed an analytical strategy which uses mass spectrometry with multiple reaction monitoring (targeted approach) and high-resolution acquisitions (untargeted approach), which were applied to quantify imidacloprid and to identify its metabolites in biological tissues after oral treatments of mice. Mouse dams were treated with doses from 0.118 mg/kg bw day up to 41 mg/kg day between gestational days 6-9. Results showed quantifiable levels of imidacloprid in plasma (from 30.48 to 5705 ng/mL) and brain (from 20.48 to 5852 ng/g) of treated mice, proving the passage through the mammalian blood-brain barrier with a high correspondence between doses and measured concentrations. Untargeted analyses allowed the identification of eight metabolites including imidacloprid-olefin, hydroxy-imidacloprid dihydroxy-imidacloprid, imidacloprid-nitrosimine, desnitro-imidacloprid, 6-chloronicotinic acid, 5-(methylsulfanyl)pyridine-2-carboxylic acid and N-imidazolidin-2-ylidenenitramide in plasma and brain. Moreover, analysis of embryonic tissues after oral treatment of mouse dams showed detectable levels of imidacloprid (816.6 ng/g after a dose of 4.1 mg/Kg bw day and 5646 ng/g after a dose of 41 mg/Kg bw day) and its metabolites, proving the permeability of the placenta barrier. Although many studies have been reported on the neurotoxicity of neonicotinoids, our study paves the way for a risk assessment in neurodevelopmental toxicity, demostrating the capability of imidacloprid and its metabolites to pass the biological barriers.

Simultaneous quantification of imidacloprid and its metabolites in tissues of mice upon chronic low-dose administration of imidacloprid.

This study aimed to (i) develop a sensitive method for simultaneous detection and quantification of imidacloprid (IMI) and seven of its metabolites in tissue specimens, and to (ii) determine the biodistribution of the IMI compounds in tissues of C57BL/6J male mice; after exposure to 0.6 mg/kg bw/day of IMI (10% of no observable adverse effect level of IMI) through a powdered diet for 24 weeks. We successfully developed a method which was accurate (recoveries were ≥ 70% for most compounds), sensitive (LODs ≤ 0.47 ng/mL and LOQs ≤ 1.43 ng/mL were recorded for all detected compounds, R2 ≥ 0.99) and precise (RSDs ≤ 20%) for routine analysis of IMI and seven of its metabolites in blood and various tissue matrices. After bio-distributional analysis, IMI and five of its metabolites were detected in mice. Brain, testis, lung, kidney, inguinal white adipose tissue and gonadal white adipose tissue mainly accumulated IMI, blood and mesenteric white adipose tissue mainly accumulated IMI-olefin; liver mainly accumulated desnitro-IMI; pancreas predominately accumulated 4-hydroxy-IMI. The desnitro-dehydro-IMI and the desnitro-IMI metabolites recorded tissue-blood concentration ratios ≥ 1.0 for testis, brain, lung and kidney. The cumulative levels of the six detected IMI compounds (Σ6 IMI compounds) were found in the decreasing order: blood > testis > brain > kidney > lung > iWAT > gWAT > mWAT > liver > pancreas. Altogether, this study provided essential data needed for effective mechanistic elucidation of compound-specific adverse outcomes associated with chronic exposures to IMI in mammalian species.