Ticks analysis for molecular detection and phylogenetic evaluation of stray dogs infecting protozoa from Alborz, Iran.

Ticks play an important role in the transmission of parasitic diseases, especially pathogenic protozoa in canine hosts, and it is very important to determine the role and extent of their infection with these pathogens in order to determine important control strategies. This study assessed the molecular prevalence of three protozoan pathogens including Hepatozoon canis, Leishmania spp. and Babesia spp., in ticks using PCR. A total 300 stray dogs were investigated and 691 ticks (171 male, 377 female and 143 nymph) were detected directly from 45 infested dogs. Species, stage of growth, and gender were determined for each tick. DNA extracted from 224 ticks (26 male, 165 female and 33 nymph). The molecular presence of three protozoan pathogens including Hepatozoon spp. (18S rRNA gene), Leishmania infantum (kinetoplastid minicircle DNA) and Babesia spp. (ssrRNA gene) were investigated using PCR method. One species of ticks, Rhipicephalus sanguineus was identified. Two of the target pathogens, Hepatozoon spp. (7/83; 8.43 %) and Babesia spp. (1/83; 1.2 %), were detected by PCR method. Sequence analysis of the ssrRNA gene of detected Babesia spp. showed a close relationship to the deposited strains of Babesia vulpis in the gene bank. To the best of our knowledge, this is the first study to undertake a phylogenetic analysis of H. canis and Babesia spp. in stray dogs in Alborz province, Iran and the first report about molecular detection of Babesia vulpis from tick infesting dogs in Iran. According to the above results, it seems necessary to implement tick control programs in dogs.

An investigation of the acaricidal activity of benzyl alcohol on Rhipicephalus annulatus and Rhipicephalus sanguineus and its synergistic or antagonistic interaction with commonly used acaricides.

The most economically significant ectoparasites in the tropics and subtropics are ixodid ticks, especially Rhipicephalus annulatus and Rhipicephalus sanguineus. Years of extensive use of the readily available acaricides have resulted in widespread resistance development in these ticks, as well as negative environmental consequences. Benzyl alcohol (BA) has been frequently used to treat pediculosis and scabies, and it may be an effective alternative to commonly used acaricides. The main aim of the present study was to evaluate the acaricide activity of BA and its combination with the regularly used chemical acaricides against R. annulatus and R. sanguineus. Different concentrations of BA alone and in combination with deltamethrin, cypermethrin and chlorpyrifos were tested in vitro against adult and larvae of both tick species. The results showed that BA is toxic to R. annulatus and R. sanguineus larvae, with 100% larval mortality at concentrations of ≥50 mL/L, and LC50 and LC90 attained the concentrations of 19.8 and 33.8 mL/L for R. annulatus and 18.8 and 31.8 mL/L for R. sanguineus, respectively. Furthermore, BA in combination with deltamethrin, cypermethrin and chlorpyrifos exhibited synergistic factors of 2.48, 1.26 and 1.68 against R. annulatus larvae and 1.64, 11.1 and 1.14 against R. sanguineus larvae for deltamethrin + BA, cypermethrin + BA and chlorpyrifos + BA, respectively. BA induced 100% mortality in adult R. annulatus at concentrations of ≥250 mL/L with LC50 and LC90 reached the concentrations of 111 and 154 mL/L, respectively. Additionally, BA had ovicidal activity causing complete inhibition of larval hatching at 100 mL/L. The combination of BA with deltamethrin and cypermethrin increased acetylcholinesterase inhibition, whereas the combination of BA with chlorpyrifos decreased glutathione (GSH) activity and malondialdehyde levels. In the field application, the combination of BA 50 mL/L and deltamethrin (DBA) resulted in a significant reduction in the percentage of ticks by 30.9% 28 days post-treatment when compared with groups treated with deltamethrin alone. In conclusion, BA causes mortality in laboratory and field studies alone and in combination with cypermethrin or deltamethrin. BA can be used for control of ticks of different life stages, that is, eggs and larvae, through application to the ground.

Evidence on the prevalence of emerging and re-emerging tick- and flea-borne rickettsial agents in acute encephalitis syndrome endemic areas of northeast Uttar Pradesh, India.

Outbreaks of acute encephalitis syndrome (AES) with unknown aetiology are reported every year in Gorakhpur district, Uttar Pradesh, India, and Orientia tsutsugamushi, the rickettsial pathogen, responsible for scrub typhus has been attributed as the primary cause of AES problem. However, information on the prevalence of other rickettsial infections is lacking. Hence, this study was carried out to assess any occurrence of tick- and flea-borne rickettsial agents in villages reporting AES cases in this district. In total, 825 peridomestic small mammals were trapped, by setting 9254 Sherman traps in four villages with a trap success rate of 8.9%. The Asian house shrew, Suncus murinus, constituted the predominant animal species (56.2%) and contributed to the maximum number (87.37%) of ectoparasites. In total, 1552 ectoparasites comprising two species of ticks and one species each of flea and louse were retrieved from the trapped rodents/shrews. Rhipicephalus sanguineus, the brown dog tick, was the predominant species retrieved from the trapped rodents/shrews, and the overall infestation rate was 1.75 per animal. In total, 4428 ectoparasites comprising five tick species, three louse species and one flea species were collected from 1798 domestic animals screened. Rhipicephalus microplus was the predominant tick species collected from the domestic animals. The cat flea, Ctenocephalides felis, constituted 1.5% of the total ectoparasites. Of all the ectoparasite samples (5980) from domestic animals and rodents, tested as 1211 pools through real-time PCR assays, 64 pools were positive for 23S rRNA gene of rickettsial agents. The PCR-positive samples were subjected to multi-locus sequence typing (MLST). In BLAST and phylogenetic analysis, the ectoparasites were found to harbour Rickettsia asembonensis (n = 9), Rickettsia conorii (n = 3), Rickettsia massiliae (n = 29) and Candidatus Rickettsia senegalensis (n = 1). A total of 22 pools were detected to have multiple rickettsial agents. The prevalence of fleas and high abundance of tick vectors with natural infections of rickettsial agents indicates the risk of transmission of tick- and flea-borne rickettsial diseases in rural villages of Gorakhpur. Further epidemiological studies are required to confirm the transmission of these agents to humans.

Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India.

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

Island hitchhikers: pathogen agents of Madeira and Azores ticks.

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.

Larvicidal and repellent effects of essential oils on the brown dog tick (Rhipicephalus sanguineus Sensu lato) with description of new larval repellent activity test method.

The aim of this research was to investigate the larvicidal and repellent effects of essential oils (EOs) obtained from two Lamiaceae plant species, Origanum minutiflorum O. Schwarz & P.H. Davis and Dorystoechas hastata Boiss. & Heldr. ex Bentham, both endemic to Turkey, on Rhipicephalus sanguineus s.l. Latreille (Acari: Ixodidae). The study also introduces a new test method that can be used to assess the repellent effects against ticks. Both plant EOs exhibited the highest larvicidal activity against brown dog tick larvae after 24 h and LC50 and LC90 values were determined as 0.101% and 0.125% for O. minutiflorum essential oil and 0.937% and 2.1% for D. hastata essential oil, respectively. In this study, we have described a detailed protocol for a novel larval repellent activity test (LRAT) for essential oils and extracts, using simple equipment. The advantages and limitations of LRAT, when compared to other tests commonly used to determine repellent effect against ticks, are also included in this study. The LRAT was developed with modifications of the larval immersion test (LIT) and proves to be a highly efficient and easily observable method. It can be used to test any active substance that may be toxic to humans and animals. According to the LRAT, at the end of 3 h, O. minutiflorum essential oil showed a high repellent effect, varying between 84.14% and 100% at 1% concentration. This result was not statistically different from the DEET, the positive control. When comparing the larvicidal and repellent activities, O. minutiflorum essential oil was found to be more effective than D. hastata essential oil.

Encapsulation of carvacrol and thymol with yeast cell wall and its repellent activity against Amblyomma sculptum and Rhipicephalus sanguineus (Sensu Lato).

The main way to avoid contact with ticks and consequently tick-borne disease is the use of synthetic repellents. The search of new repellent compounds to increase the possibilities of use in strategies controls are necessary. The present study evaluated the repellent activity of two natural terpenes carvacrol and thymol in each one two different formulation (encapsulated and nonencapsulated with yeast cell wall) against the ticks Amblyomma sculptum and Rhipicephalus sanguineus sensu lato nymphs. Nymphs of A. sculptum and R. sanguineus s.l. of a single generation were used. The vertical filter paper repellency assay were performed with different concentration of both terpenes encapsulated and nonencapsulated in yeast cell wall. The repellent concentration 50% (RC50) were calculated to each compound formulation. Both carvacrol and thymol (encapsulated and nonencapsulated), had a repellent activity against A. sculptum and R. sanguineus s.l nymphs. Amblyomma sculptum was more sensitive to nonencapsulated carvacrol (RC50 values: 0.0032 to 0.0082 mg/cm2 after 1 and 15 min) (P < 0.05), while R. sanguineus s.l. was more sensitive to encapsulated carvacrol (RC50 values: 0.00008 to 0.0035 mg/cm2 after 1 and 15 min) (P < 0.05). Among tick species, R. sanguineus s.l. was more sensitive for most compounds than A. sculptum (P < 0.05). Although with distinct repellent activities, carvacrol and thymol encapsulated can be a promising alternative to synthetic repellents against A. sculptum and R. sanguineus s.l.

Occurrence and genotyping of Theileria equi in dogs and associated ticks in Egypt.

Theileriosis is a tick-borne protozoal disease caused by a piroplasm of the genus Theileria. Hard ticks are obligate hematophagous ectoparasites that serve as vectors of Theileria spp. Studies of the presence of theileriosis in Egyptian dogs and associated ticks are scarce. This study was conducted to detect and identify Theileria spp. in dogs and Rhipicephalus sanguineus ticks and to monitor the epidemiological data of this disease. The prevalence rates of Theileria equi infection were 12.02%, 0.73%, 2.93%, and 1.83% by microscopic examination of dog blood, tick hemolymph, tick midgut, and tick salivary smears, respectively. Conversely, the T. equi prevalence in dog blood and associated ticks assessed by PCR was 25.81% and 10.42%, respectively. Epidemiological data about Theileria infection revealed a significant difference in the infection between different seasons and different dog breeds (p value <0.05), whereas host, sex, and age of dogs had no significant effect on the infection. Sequencing of PCR products showed that all PCR positive samples were infected with T. equi. Transmission electron microscopy (TEM) described the different stages of Theileria in the midgut and salivary gland of infected ticks. The current study confirmed that T. equi is not specific to equine hosts, and confirmed that dogs are a susceptible host to T. equi.

First report of acaricidal efficacy from plumbagin on larvae of Rhipicephalus microplus and Rhipicephalus sanguineus resistant to conventional acaricides.

The problem of resistance to acaricides in ticks such as Rhipicephalus microplus and R. sanguineus has motivated the search for control alternatives, such as the use of extracts and secondary metabolites from plants. Plumbagin is a natural product present in plants such as Plumbago zeylanica L., Diospyros kaki, and D. anisandra, of which acaricidal activity has been reported. Therefore, the objective of this study was to evaluate in vitro the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides. Larvae from engorged female ticks, collected from naturally infested dairy cattle and domiciled dogs, in Yucatan, Mexico, were used. The larval packet test and the larval immersion test were performed to detect acaricide susceptibility. Both tick populations were detected as resistant to cypermethrin and amitraz. Then, the modified larval immersion test was used and plumbagin was evaluated at concentrations of 1%, 0.5%, 0.25%, and 0.125% (%w/v), obtaining a mortality of 100% in the four concentrations for both tick species. Subsequently, lower doses of plumbagin were evaluated at concentrations of 0.0625%, 0.03125%, 0.015625% and 0.0078125%, obtaining mortalities of 100 to 36.26% for R. microplus and 100%-5.33% for R. sanguineus. Using Probit analysis, lethal concentrations at 50% (LC50), 99% (LC99) and confidence intervals at 95% (CI95%) were calculated. R. microplus showed a LC50 of 0.011% (CI95%: 0.010-0.011) and LC99 of 0.019% (CI95%: 0.018-0.022). R. sanguineus presented a LC50 of 0.017% (CI95%: 0.015-0.018) and CL99 of 0.031% (CI95%: 0.027-0.036). It was concluded that plumbagin has high acaricidal efficacy against larvae of R. microplus and R. sanguineus resistant to amitraz and cypermethrin. R. microplus larvae were significantly more susceptible to LC50 and LC99 compared to R. sanguineus. This is the first report on the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides.

Efficacy of essential oils of Egletes viscosa and Lippia schaueriana on the reproductive biology of Rhipicephalus sanguineus sensu lato engorged females.

The study analyzed the chemical composition and the acaricide effect of Egletes viscosa Less (macela-da-terra) and Lippia schaueriana Mart. (lipia-da-serra) essential oils (EOs) on Rhipicephalus sanguineus s. l. (Acari: Ixodidae) engorged females. The chemical analysis (GC-MS and GC-FID) identified 27 components in E. viscosa EO and 18 in L. schaueriana EO, which comprise more than 98% of its constituents. The effects of the oils on the reproductive biology of R. sanguineus ticks were assessed by adult immersion test. Both EOs significantly reduced (p < 0.05) the egg production index when the females were exposed to 25 and 50 mg/mL, also affecting the egg viability. During the laying process, the eggs produced by the females exposed to the EO showed several morphological alterations such as dehydrated, darkened, and disaggregated, and these alterations were more severe as the concentrations increased. The mortality percentages were 58.9%, 70.8% and 92.7% when the ticks were exposed to 12.5, 25 and 50 mg/mL of E. viscosa oil, respectively. In the same concentrations, the efficacy of L. schaueriana was 39.3%, 53.4%, and 84.6%. Therefore, it can be concluded that the essential oils of E. viscosa and L. schaueriana have acaricidal effect in females of R. sanguineus s.l ticks.

Characterization of the complete mitochondrial genomes of five hard ticks and phylogenetic implications.

Ticks are blood-sucking ectoparasites with significant medical and veterinary importance, capable of transmitting bacteria, protozoa, fungi, and viruses that cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of five hard tick species and analyzed features of their gene contents and genome organizations. The complete mt genomes of Haemaphysalis verticalis, H. flava, H. longicornis, Rhipicephalus sanguineus and Hyalomma asiaticum were 14855 bp, 14689 bp, 14693 bp, 14715 bp and 14722 bp in size, respectively. Their gene contents and arrangements are the same as those of most species of metastriate Ixodida, but distinct from species of genus Ixodes. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes with two different computational algorithms (Bayesian inference and maximum likelihood) revealed the monophylies of the genera Rhipicephalus, Ixodes and Amblyomma, however, rejected the monophyly of the genus Haemaphysalis. To our knowledge, this is the first report of the complete mt genome of H. verticalis. These datasets provide useful mtDNA markers for further studies of the identification and classification of hard ticks.

Do combinations of fipronil, eugenol and carvacrol have synergistic effects against Rhipicephalus sanguineus?

The tick Rhipicephalus sanguineus is one of the main ectoparasites that affects dogs, causing direct and indirect damage to parasitized animals. Currently, infestation control is mainly carried out by using synthetic acaricidal drugs. However, a decrease in efficacy and an increase in resistance to the main therapeutic protocols against tick infestations have been increasingly reported and confirmed, a factor that has driven research into the potential acaricide activity of natural compounds, including in association with synthetic molecules. The aim of this work was to evaluate whether the combinations of fipronil (FIP) and eugenol (EUG), FIP and carvacrol (CAR), and EUG and CAR would have synergistic effects against immature and unfed adult stages of R. sanguineus through in vitro bioassays. Bioassays were carried out using the larval packet test (FAO 2004) adapted for nymphs and adults. The synergistic activity was explored by combining each solution, based on the estimated LC50, in a 1:1 ratio (FIP: EUG, FIP: CAR and EUG: CAR). CompuSyn software was used to evaluate the various pairwise combinations of FIP, EUG and CAR, checking if there was synergism or antagonism between them. FIP and EUG and FIP and CAR showed combination index (CIn) values above 1.45, indicating antagonism. The synergistic activity between EUG and CAR was verified against all unfed phases of R. sanguineus, since the CIn was below 0.70, a value that indicates synergism. The combination of fipronil with either eugenol or carvacrol presented antagonistic effects against R. sanguineus larvae. On the other hand, carvacrol and eugenol had excellent pharmacological synergism against all tick stages with mortality values in the range of 80 to 100%, including the adult stage, which is less susceptible than immature stages.

Ectoparasites of dogs (Canis familiaris) from northeastern Nigeria: an epidemiological study.

Ectoparasites of dogs represent an important group of parasites. They often suck blood, cause pruritis, and could serve as vectors of many pathogens of veterinary and public health importance. In northeastern Nigeria, there is a lack of data regarding ectoparasites of dogs. Therefore, this study was undertaken to explore the external parasites of dogs and the associated epidemiological risk factors. A total of 1041 dogs (mean age = 8.5 ± 2.1 months) from residential house visit (54.9%) and those attending veterinary clinic (45.1%) were sampled in northeastern Nigeria. Multivariate logistic regression analysis assessed epidemiological risk factors associated with canine ectoparasitic infections. Of the 1041 dogs screened, 92.5% (963/1047) harbored one or more ectoparasites. Rhipicephalus sanguineus (52.4%), Linognathus setosus (7.8%), Ctenocephalides canis (2.3%), and Otodectes cynotis canis (1.4%) were the significantly encountered tick, lice, fleas, and mite species, respectively. Being female dog (OR = 1.8; p = 0.01), cross (OR = 2.2; p = 0.029), and exotic breeds (OR = 2.4; p = 0.02), < 7 months (OR = 3.1; p = 0.04) and 7-12 months (OR = 2.1; p = 0.01) aged dogs as well as rainy season (OR = 1.9; p = 0.02) were strong predictors for tick infestation. Similarly, cross (OR = 7.2; p = 0.001) and local breeds (OR = 2.8; p = 0.0001), dogs aged > 12 months (OR = 2.5; p = 0.03), and dogs used for hunting (OR = 3.2; p = 0.01) and as security guards (OR = 3.8; p = 0.01) were strongly associated with lice infestation. Results from this study revealed a high prevalence of external parasites parasitizing majority of the sampled dogs. Some of these parasites may serve as vectors of zoonotic pathogens posing public health risks.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei.

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally. R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with ß-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 µM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 µM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Exploring the Larvicidal and Repellent Potential of Taurus Cedar (Cedrus libani) Tar against the Brown Dog Tick (Rhipicephalus sanguineus sensu lato).

This study investigated the potential acaricidal and repellent effects of tar obtained from the Lebanon cedar (Cedrus libani A. Rich.) against the brown dog tick species Rhipicephalus sanguineus sensu lato Latreille (Acari: Ixodidae). The goal was to find an alternative, safe, and effective way to eliminate ticks. Tar is traditionally extracted from cedar trees in the Antalya region of Türkiye. The composition of the tar is primarily characterized by a diverse mixture of terpenes, with ß-himachalene (29.16%), α-atlantone (28.7%), ar-turmerone (8.82%), longifolene-(V4) (6.66%), α-himachalene (5.28%), and ß-turmerone (5.12%) emerging as the predominant constituents. The toxic effects of tar on tick larvae were studied through larval immersion tests (LIT), and its repellent activity was evaluated using a new larval repellent activity test (LRAT). The results revealed significant acaricidal effects, with mortality rates of 77.7% and 82.2% for the Konyaalti and Kepez strains of the brown dog tick, respectively, in response to a 1% concentration of tar. LC50 and LC90 values were determined as 0.47% and 1.52% for the Kepez strain and 0.58% and 1.63% for the Konyaalti strain, respectively. When comparing the repellent effect of tar to the widely used synthetic repellent DEET, repellency rates of up to 100% were observed. As a result, this study establishes, for the first time, the larvicidal and repellent effects of C. libani tar on ticks.

Atlas of ticks (Acari: Argasidae, Ixodidae) in Germany: 1st data update.

The first data update of the atlas of ticks in Germany published in 2021 is presented here. This atlas provides maps based on georeferenced tick locations of 21 species endemic in Germany as well as three tick species that are regularly imported to Germany. The data update includes the following numbers of newly georeferenced tick locations: 17 Argas reflexus, 79 Carios vespertilionis, 2 Dermacentor marginatus, 43 Dermacentor reticulatus, 4 Haemaphysalis concinna, 3 Haemaphysalis punctata, 3 Hyalomma rufipes, 3 Ixodes apronophorus, 9 Ixodes arboricola, 1 Ixodes ariadnae, 30 Ixodes canisuga, 3 Ixodes frontalis, 80 Ixodes hexagonus, 3 Ixodes lividus, 497 Ixodes ricinus/inopinatus, 1 Ixodes rugicollis, 17 Ixodes trianguliceps, 14 Ixodes vespertilionis, and 45 Rhipicephalus sanguineus sensu lato. Old and new tick findings were mapped, such as the northernmost occurrence of D. marginatus in Germany observed in 2021, but also the historical records from the first descriptions of I. apronophorus and I. arboricola, which were georeferenced here for the first time. The digital dataset of tick locations available for Germany is supplemented by 854 new tick locations. These records increase the number of tick species mapped in the federal states Bavaria, Brandenburg and Mecklenburg Western Pomerania by five each, those in Berlin and Schleswig-Holstein by four each, those in Hamburg by three, those in Baden-Wuerttemberg, Bremen, Lower Saxony, Northrhine-Westphalia, Rhineland Palatinate and Thuringia by two each, and those in Hesse, Saxony and Saxony-Anhalt by one each. Thus, the first data update of the tick atlas in Germany and the underlying digital dataset significantly improve our knowledge of the distribution of these tick species and helps to investigate the effects of climate change and habitat changes on them.

Effect of Tagetes minuta essential oil on the central nervous system of unfed Rhipicephalus sanguineus sensu lato 'tropical lineage' ticks.

Rhipicephalus sanguineus sensu lato 'tropical lineage' (Acari: Ixodidae) is considered a sanitary concern due to its role as a disease vector. Tick strains resistant to synthetic acaricides have caused difficulties in their control, besides synthetic acaricides are harmful to the environment and to the health of non-target animals. The research of plants with acaricidal and repellent properties has proved to be an efficient alternative in tick control. The genus Tagetes spp. excels for its use as traditional pest control in households and plantations and also for its potential as an acaricide against R. sanguineus under laboratory conditions. The first aim of the present study was to evaluate the effect of different doses of Tagetes minuta essential oil (TMEO) on the central nervous system (synganglion) in unfed R. sanguineus adults. The histological analysis of synganglion exposed to the different concentrations of TMEO and amitraz 12.5% (50% of the recommended dose in the package insert) showed a significant effect with signs of cell damage including volume increase, loss of shape, and vacuolization, in addition to chromatin alterations such as condensation, margination, and fragmentation. TMEO were analyzed by gas chromatography coupled with mass spectrometry showing the presence of 21 compounds that according to their chemical structure are classified as terpenoids. Among them (Z)-ß-ocimene, ocimene, (Z)-tagetone, and verbenone were found in major quantities.

Differences in the reproductive output and larval survival of Rocky Mountain wood ticks (Dermacentor andersoni) and American dog ticks (Dermacentor variabilis) from prairie populations near their northern distributional limits in western Canada.

The effects of temperature and relative humidity (RH) on female reproductive output, egg development and larval survival were determined for Rocky Mountain wood ticks (Dermacentor andersoni) from a prairie population (Chin Lakes, Alberta, Canada) near the northern distribution limit of this species. The responses of D. andersoni eggs and unfed larvae to different temperature (25 or 32 °C) and RH (35, 55, 75, 85 or 95%) regimes were compared to our previously published data (Diyes et al. 2021) for a northern prairie population of American dog ticks (Dermacentor variabilis). Oviposition by D. andersoni females took 21-30 days at 25 °C and 95% RH compared to 10-21 days for D. variabilis. The number of eggs laid by female ticks was strongly dependent on their engorgement weight, and D. andersoni females produced more eggs than D. variabilis females of an equivalent body weight. Eggs of D. andersoni took less time to develop at 32 °C than 25 °C with ≥ 85% RH, and hatched faster than those of D. variabilis. Larval survival times declined as temperature increased and RH decreased, but D. andersoni survived longer at 32 °C and ≤ 75% RH than D. variabilis. The interspecific differences in responses to the same temperature and humidity regimes indicate that D. andersoni is xerophilic, whereas D. variabilis is hydrophilic. Hence, 'prairie' populations of the Rocky Mountain wood tick occur in the drier grassland ecoregions but are absent in Aspen Parklands Ecoregion which is located to the north and east of the distributional range of D. andersoni.

Use of a recombinant positive control in the diagnostic of canine Ehrlichiosis from 16sRNA gen of Ehrlichia canis in Mexico City.

Ehrlichia canis has gained importance over the years as a zoonotic bacterium, nevertheless in Mexico is unknown the extent of the problem in animals and public health. The country had a few studies carried out locally using serology and molecular tests as diagnostic methods. Ehrlichiosis is not considered endemic in the central valley of Mexico, because the climatic conditions in the region have not allowed the vector (Rhipicephalus sanguineus) to establish itself adequately, therefore, diagnosis is not used in clinical practice in this area. A nested PCR (nPCR) offers rapid results with high sensitivity and specificity regardless of cost. The use of a recombinant positive control provides the advantage of timely diagnosis, follow-up treatment and allows the clinician to decide. In this work, the nPCR reported by Wen et al. (J Clin Microbiol 35(7):1852-2185, 1997) was used for the diagnosis of E. canis by modifying the reaction conditions to improve the detection of the test. We constructed a recombinant positive control to nPCR as diagnostic technique for E. canis, also we modified the reaction conditions to improve detection of the test which allowed the diagnosis of E. canis in dogs in the Mexican Republic using 53 samples from dogs with positive serological diagnosis of Ehrlichiosis, some of them from the valley of Mexico. Currently, this nPCR is offered to public at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico at an accessible cost and allows to begin to generate epidemiological information to know distribution of the bacterium.

Metabolomic and transcriptomic responses of ticks during recovery from cold shock reveal mechanisms of survival.

Ticks are blood-feeding ectoparasites but spend most of their life off-host where they may have to tolerate low winter temperatures. Rapid cold hardening (RCH) is a process commonly used by arthropods, including ticks, to improve survival of acute low temperature exposure. However, little is known about the underlying mechanisms in ticks associated with RCH, cold shock and recovery from these stresses. In the present study, we investigated the extent to which RCH influences gene expression and metabolism during recovery from cold stress in Dermacentor variabilis, the American dog tick, using a combined transcriptomics and metabolomics approach. Following recovery from RCH, 1860 genes were differentially expressed in ticks, whereas only 99 genes responded during recovery to direct cold shock. Recovery from RCH resulted in an upregulation of various pathways associated with ion binding, transport, metabolism and cellular structures seen in the response of other arthropods to cold. The accumulation of various metabolites, including several amino acids and betaine, corresponded to transcriptional shifts in the pathways associated with these molecules, suggesting congruent metabolome and transcriptome changes. Ticks, D. variabilis and Amblyomma maculatum, receiving exogenous betaine and valine demonstrated enhanced cold tolerance, suggesting cryoprotective effects of these metabolites. Overall, many of the responses during recovery from cold shock in ticks were similar to those observed in other arthropods, but several adjustments may be distinct from the responses in other currently examined taxa.