Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

Isolation and molecular characterization of lumpy skin disease virus from hard ticks, Rhipicephalus (Boophilus) annulatus in Egypt.

BACKGROUND: Lumpy skin disease (LSD), a disease of cattle and buffaloes, has recently become widely prevalent in Egypt. The aim of this study was to ascertain the potential role of Rhipicephalus (Boophilus) annulatus ticks in the transmission of this disease. Samples collected from suspected lumpy skin disease virus (LSDV) infected cows that had previously been vaccinated with the Romanian sheep pox virus (SPPV) in various Egyptian governorates were obtained between May to November over two consecutive years, namely 2018 and 2019. Ticks were morphologically identified and the partial cytochrome oxidase subunit I gene (COI) were sequenced, revealing that they were closely related to R. (Boophilus) annulatus. The G-protein-coupled chemokine receptor (GPCR) gene of the LSDV was used to test hard ticks. RESULTS: Two positive samples from Kafr El-Sheikh province and one positive sample from Al-Behera province were reported. BLAST analysis revealed that the positive samples were closely related to the Kazakhstani Kubash/KAZ/16 strain (accession number MN642592). Phylogenetic analysis revealed that the GPCR gene of the LSDV recently circulating in Egypt belongs to a global cluster of field LSDV with a nucleotide identity of 98-100%. LSDV isolation was successfully performed four days after inoculation using 9 to 11-day-old embryonated chicken eggs showing characteristic focal white pock lesions dispersed on the choriallantoic membrane after three blind passages. Intracytoplasmic inclusion bodies, cell rupture, vacuoles in cells, and virus particles ovoid in shape were demonstrated by electron microscopy. CONCLUSION: In this study the role of hard ticks in the transmission of the LSDV to susceptible animals in Egypt was revealed and confirmed by various methods.

Predicting the potential distribution of the cattle fever tick Rhipicephalus annulatus (Acari: Ixodidae) using ecological niche modeling.

Rhipicephalus annulatus is a tick species of veterinary importance due to its potential to transmit babesiosis to cattle. This species has a Holarctic distribution with some Afrotropical records and is one-host species of veterinary importance. This study was carried out from September 2021 to February 2022 at 6 Egyptian collection sites, and a total of 1150 cattle were scanned randomly to collect ticks. A total of 1095 tick specimens were collected and identified as R. annulatus using taxonomic keys. Males were found on all parts of the cattle except the head and around the eyes, but females were found on all parts; in addition, the highest number of specimens was gathered from the udder, (neck and chest), and belly. Maximum entropy (MaxEnt) modeling was used to predict the potential global distribution of R. annulatus. The MaxEnt model performed better than random with an average test area under the curve (AUC) value of 0.96, and model predictions were significantly better than random and gave (AUC) ratios above the null expectations in the partial receiver operating characteristic (pROC) analyses (P < 0.001). Based on correlation analyses, a set of 9 variables was selected for species from 15 bioclimatic and 5 normalized difference vegetation index (NDVI) variables. The study showed that the current distribution of R. annulatus is estimated to occur across Asia, Africa, Europe, South America, and North America. Annual mean temperature (Bio1) and median NDVI had the highest effect on the distribution of this species. The environmentally suitable habitat for R. annulatus sharply increased with increasing annual mean temperature (Bio1). These results can be used for making effective control planning decisions in areas suitable to this vector of many diseases worldwide.

Synergistic larvicidal and repellent effects of essential oils of three Origanum species on Rhipicephalus annulatus tick.

Ticks are of great economic importance worldwide, both because they represent major obstacles to livestock productivity and because of their ability to transmit diseases to humans and animals. Although synthetic acaricides are the most common method for tick control, their overuse has led to the development of resistance as well as unacceptable residual levels in animal products and in the environment in general. There is therefore an urgent need to identify alternative treatments. Among such alternative approaches for tick control is plant essential oil (EO) therapy. In the present study, we investigated the synergistic effect of EOs of three oregano species-Origanum onites, O. majorana and O. minutiflorum-against Rhipicephalus annulatus larvae. Gas chromatography-mass spectrometry profiles of the three EOs revealed that carvacrol was their major component, with a concentration of 86.2% in O. majorana, 79.1% in O. minutiflorum and 77.4% in O. onites. The results of larvicidal assays revealed that the doses that lead to the death of 50% of the ticks (LC50) were 22.99, 25.08 and 27.06 µL/mL for O. majorana, O. minutiflorum and O. onites EOs, respectively, whereas the doses that lead to the death of 99% (LC99) were 41.26, 43.62 and 48.96 µL/mL. In addition, the LC50 and LC99 of the three oils combined was lower (viz., 4.01 and 6.97 µL/mL) than that of each oil alone. The tested EOs were also able to repel larvae of R. annulatus to varying degrees, with O. onites oil exhibiting the greatest repellent effect, as shown by the lowest RC50 dose, followed by O. minutiflorum and O. majorana. Interestingly, this means that the oil that was least effective in killing the larvae was the most effective in repelling them. The calculated synergistic factor of any combination was higher than 1 which means that combinations have a synergistic effect. In conclusion, the combination of all three oils showed higher toxic and repellent activities than either oil separately or combinations of any two oils, suggesting synergistic effects with low doses. Further studies including field trials and the establishment of the mode of action and side effects are urgently needed to expand on these findings, and other tick stages such as adults should also be tested.

Role of antioxidant activity of essential oils in their acaricidal activities against Rhipicephalus annulatus.

Essential oils of Origanum majorana and Satureja thymbra as well as carvacrol are natural products that are known to have potent antioxidant activities. The current study was designed to investigate the role of the antioxidant properties of these natural products in their acaricidal activities against Rhipicephalus annulatus larvae. The synergistic and/or antagonistic effects of the addition of vitamins E and C and hydrogen peroxide (H2O2) to these natural products were also evaluated. Larval packet tests were used to evaluate the acaricidal activities against the larvae of R. annulatus. The antioxidant effectiveness of these products was determined by a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. The addition of vitamin E at 100 mg/mL to O. majorana and S. thymbra decreased the concentrations required to achieve the death of half of the larvae (LC50) to 0.44 and 0.47%, respectively. The combination of O. majorana and S. thymbra attained the LC50 at 1.54% which was decreased to 0.69% after addition of vitamin E. Also, the addition of vitamin E to carvacrol reduced the LC50 to 0.27%. The total antioxidant activity of these natural products increased significantly in presence of vitamin E. The addition of H2O2 inhibited the acaricidal activity of all tested materials, especially at low concentrations. All treatments induced an increase in lipid peroxidation, whereas carvacrol-treated larvae revealed the lowest values for the superoxide dismutase. Glutathione peroxidase and catalase activity decreased in larvae treated with S. thymbra combined with vitamin E. In conclusion, the addition of vitamins E and C increased the acaricidal activities of the tested compounds, whereas the addition of H2O2 decreased these activities. The antioxidant activities of essential oils and their active components may play an important role in mediating their acaricidal activities.

Hard ticks (Acari: Ixodidae) infesting domestic animals in Egypt: diagnostic characters and a taxonomic key to the collected species.

Ticks are important vectors of emerging health problems in humans and animals. We conducted several field surveillances to investigate the fauna of hard ticks on domestic animals in seven governorates of Egypt during 2018-2019. A total of 3265 individual tick specimens were collected and identified to the species level; the specimens belong to 11 species within three genera (Amblyomma, Hyalomma, and Rhipicephalus). Tick infestation was highest in dromedary camels (70%), followed by dogs (52.5%), cattle (50%), buffaloes (38%), and horses (12%). Ten species were collected from dromedary camels, 2 from cattle, and one species from dogs, buffaloes, and horses. Diagnostic characters with high-quality photographic images of the 11 tick species were provided. New measurements of the body regions such as spiracles and body grooves were also obtained to provide further details on morphological variations among the diverse tick species. This study provided detailed descriptions of the genital apertures of five Hyalomma species. Morphological variations of male Hyalomma impeltatum, and genital apertures of female Hyalomma dromedarii and Hyalomma excavatum were also examined in this study. Finally, we provided additional details on local geographic distributions, hosts, endemicity status, and disease relationships of the eleven tick species.

Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts.

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.

Toxicity, repellency and anti-cholinesterase activities of thymol-eucalyptus combinations against phenotypically resistant Rhipicephalus annulatus ticks.

The current study was designed to compare the acaricidal activity of pure thymol (T) crystals, eucalyptus oil (E), and their combinations (T + E) at concentrations ranging from 0.625 to 10% against phenotypically deltamethrin-resistant Rhipicephalus annulatus ticks. The adulticidal, larvicidal, ovicidal, and repellent activities of the tested compounds and their possible mechanism of action were studied. Thymol showed 93.3% adulticidal activity at 10% concentration, whereas at 2.5% it achieved 100% larvicidal activity. Eucalyptus oil had a significant effect on tick adults at concentrations > 5%, and at 10% concentration it showed 96.7% adulticidal activity and stopped the egg hatching. On deltamethrin-resistant larvae, the median lethal concentration (LD50) was 0.61, 7.5, and 0.61% for T, E, and T + E combinations, respectively, and repellent activities were 85.7, 62.5, and 100% at the 10% concentration used. Combinations of T + E did not induce additional effects as adulticidal and larvicidal in a comparison with T treatments. The tick's acetylcholinesterase significantly decreased as a result of all treatments including deltamethrin, whereas lipid peroxidation significantly increased compared with the control untreated ticks. The glutathione content significantly decreased as a result of all treatments except for treatments with 10% thymol and its combination with eucalyptus oil. Moreover, 10% eucalyptus oil significantly decreased the tick's glutathione peroxidase and catalase activities reflecting the attenuation of antioxidant defense in the ticks. In conclusion, thymol and eucalyptus oil showed excellent activities against deltamethrin-resistant R. annulatus isolates. Their action may be mediated via acetylcholinesterase inhibition. The effect of thymol-eucalyptus combinations recorded higher activities as an ovicidal and as a repellent.

Rearing of Rhipicephalus annulatus ticks on rabbits for the biological transmission of Anaplasma marginale.

Background and Aim: Anaplasma marginale is an obligate intraerythrocytic rickettsial parasite that infects cattle in tropical and subtropical regions. There is no evidence that A. marginale inoculation can be used to culture Rhipicephalus annulatus in rabbits. This study aimed to determine the molting of R. annulatus larvae, nymphs, and adults on rabbits as well as nymphs and adults of R. annulatus on calves with or without A. marginale. Transstadial, horizontal, and transovarial transmissions of A. marginale in R. annulatus reared on rabbits and calves were evaluated. Materials and Methods: Engorged female ticks were collected from field samples of A. marginale-infected and non-infected cattle. We divided the eight rabbits into two groups: A and B. Group A rabbits were infected with A. marginale through parenteral inoculation, whereas Group B rabbits were kept as a control. The "clean rabbits" in Group B were observed for tick rearing without A. marginale. Polymerase chain reaction was used to screen A. marginale in rabbits and stages of tick. The complete life cycle of R. annulatus with or without A. marginale was observed on rabbits. Results: A 6.5-day longer life cycle was observed in ticks harboring A. marginale than in ticks without A. marginale. To observe transstadial transmission, transstadial, horizontal, and transovarial transmissions of A. marginale in R. annulatus ticks were experimentally observed in one clean calf fed separately with infected nymphs and female adult ticks. Conclusion: We experimentally observed transovarian, transstadial, and transovarial transmission of A. marginale in R. annulatus ticks as a biological vector reared on calves and rabbits. We used rabbits as a model animal for rearing R. annulatus ticks and culture of A. marginale.

Assessment of cattle tick infestation: Molecular insights into Rhipicephalus annulatus and the efficacy of garlic oil and nanoemulsion as acaricidal agents.

Ticks, particularly Rhipicephalus annulatus, pose significant threats to livestock, causing economic losses and transmitting various infectious diseases. This study aimed to evaluate the potential acaricidal properties of garlic oil and its nanoemulsion against ticks infesting cattle, Rhipicephalus annulatus through the evaluation of mortality rate and morphological changes of the treated ticks. The study also included prevalence, risk factors, and molecular confirmation of tick species. Genetic characterization confirmed the identity of R. annulatus. Our results revealed a high prevalence of R. annulatus (46.9%) with a higher risk in male cattle (50%) than females (44.9%) and a nonsignificant high infection (49.1%) in animals ≤ 1 year old. The acaricidal efficiency of garlic oil and its nanoemulsion was concentration and time-dependent. The high concentration of garlic oil (20 mg/L) induced complete mortality within 48 hours. The nanoemulsion formulation enhanced efficacy, particularly at 5 mg/L, which exhibited rapid and substantial acaricidal activity. Scanning electron microscopy revealed morphological alterations induced by garlic oil and its nanoemulsion, including changes to the anterior capitulum, dorsal, and ventral cuticles. The study contributes to the exploration of effective, safe, and eco-friendly alternatives for tick control. Further research is warranted to validate their efficacy under diverse conditions and assess practical strategies.

Efficacy of D-Limonene Nanoemulsion Against Rhipicephalus annulatus and Rhipicephalus sanguineus Ticks.

PURPOSE: Ticks infestation has a negative impact against human and animal health through blood sucking, transmission of blood-borne diseases and also caused economic losses. METHODS: In the present study the adulticidal, ovicidal and larvicidal activity of D-limonene nanoemulsion (DLN) were evaluated against two tick species; Rhipicephalus annulatus and Rhipicephalus sanguineus. Nanoemulsion form of D-limonene was prepared, and its characteristics were evaluated using a UV spectrophotometer and zeta droplet size measurement. Acetylcholinesterase activity was determined. RESULTS: The results revealed significant adulticidal effect with low LC50 and LC90 for D-limonene pure form (DL) against both adult tick spp. (R. annulatus and R. sanguineus) ((0.958 and 1.559%) and (2.26 and 3.51%), respectively). DLN LC50 and LC90 values were ((1.277 and 2.396) and (3.97 and 7.28), respectively) against R. annulatus and R. sanguineus, respectively. DL and DLN showed significant ovicidal effect against R. sanguineus at high concentrations (10 and 5%). In larval packet test, LC50 and LC90 values of DL were ((1.53 and 2.22%) and (6.81 and 12.07%), respectively) against R. annulatus and R. sanguineus, respectively, while LC50 and LC90 values of DLN were ((6.48 and 11.26%) and (7.82 and 13.59%), respectively) against R. annulatus and R. sanguineus, respectively. Significant acetylcholinesterase inhibition percentage was detected for both ticks spp. which treated by DL and DLN. CONCLUSION: Pure DL is more effective than DLN form against R. annulatus and R. sanguineus.

Potential of Marava arachidis, a Newly Recorded Earwig Species in Egypt as a Biological Control Agent of Rhipicephalus annulatus Tick in Laboratory.

In Egypt, only five species of Dermaptera (earwigs) have been reported. Based on both the morphological and molecular data of the earwig samples collected from a bakery in Beni-Suef, Egypt, we identified the species as Marava arachidis (Spongiphoridae), a cosmopolitan species with no prior records in Egypt. The current study was designed to analyze its predation capability on newly emerged eggs and larvae of the Rhipicephalus annulatus tick. A laboratory functional response study was set up by applying a predation test with various predator-prey ratios as treatments. This experiment was applied using the undefined mix of collected earwigs and the laboratory-collected eggs and the larvae of R. annulatus. The laboratory results showed that the mean number of predated tick eggs was 18.64 ± 11.18 in 24 h under the highest predator-prey ratio (1:10) examined, accompanied by 12.04 ± 4.38 broken but unconsumed eggs. Moreover, M. arachidis predated an average of 12.32 ± 9.07 tick larvae per day. In contrast, the mean dead larvae increased to 38.4 ± 2.30 per day with the highest predator density (1:10). The number of eggs and larvae consumed increased with the predator density. A linear relationship was detected between earwig density and the consumption rates of tick eggs (R2 = 0.99; p = 0.0001) and larvae (R2 = 0.96; p = 0.003). In conclusion, M. arachidis was first recorded in Egypt. This earwig has predation capability on R. annulatus tick eggs and larvae.

Morphological and molecular studies on tick species in Ismailia Governorate in Egypt and Al Gabal Al Akhdar in Libya.

Background: Ticks are obligate blood-sucking ectoparasites of vertebrates that have an impact on both domestic and wild animals, especially in tropical and subtropical areas. Aim: The objective of this study is to compare the prevalence and the tick species in both Al Gabal Al Akhder regions in northeastern Libya and Ismailia, Egypt. Methods: Tick specimens collected from predilection sites on the hosts were identified by morphological (light microscopy) and molecular methods. Results: In Ismailia, Egypt, 23.9% of the 230 (examined cattle and buffaloes) were infested with one species of hard ticks, Rhipicephalus annulatus. In the Libyan province of Al Gabal Al Akhdar, the prevalence of tick infestation in cattle, sheep, and goats, was 47% and 59%, respectively. R. annulatus is the identified tick species for cattle, Hyalomma marginatum, and Rhipicephalus bursa, are the identified tick species of the infested sheep and goats. Conclusion: The 16S rDNA gene sequencing and phylogenetic analysis of sample species from Egypt and Libya proved instrumental in overcoming the difficulties associated with morphological identification techniques.

Molecular Epidemiological Investigation of Piroplasms and Anaplasmataceae Bacteria in Egyptian Domestic Animals and Associated Ticks.

Piroplasmosis and anaplasmosis are serious tick-borne diseases (TBDs) that are concerning for the public and animal health. This study aimed to detect the molecular prevalence and epidemiological risk factors of Piroplasma and Anaplasma species in animal hosts and their associated ticks in Egypt. A total of 234 blood samples and 95 adult ticks were collected from animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) from six provinces of Egypt (AL-Faiyum, AL-Giza, Beni-Suef, Al-Minufia, Al-Beheira, and Matruh). Blood and tick samples were investigated by polymerase chain reaction coupled with sequencing targeting 18S and 16S RNA genes for Piroplasma and anaplasmataceae, respectively. Statistical analysis was conducted on the potential epidemiological factors. Of the 234 animals examined, 54 (23.08%) were positive for pathogens DNA distributed among the six provinces, where 10 (4.27%) were positive for Piroplasma, 44 (18.80%) for anaplasmataceae, and 5 (2.14%) were co-infected. Co-infections were observed only in cattle as Theileria annulata and Anaplasma marginale plus Babesia bigemina, A. marginale plus B. bigemina, and T. annulata plus B. bigemina. Piroplasmosis was recorded in cattle, with significant differences between their prevalence in their tick infestation factors. Animal species, age, and tick infestation were the potential risk factors for anaplasmosis. All ticks were free from piroplasms, but they revealed high prevalence rates of 72.63% (69/95) with anaplasmataceae. We identified T. annulata, B. bigemina, and A. marginale in cattle; A. platys in buffaloes; A. marginale and A. ovis in sheep; for the first time, A. ovis in goats; and Ehrlichia sp. in Rhipicephalus annulatus ticks. Our findings confirm the significant prevalence of piroplasmosis and anaplasmosis among subclinical and carrier animals in Egypt, highlighting the importance of the government developing policies to improve animal and public health security.

Molecular characterization of some equine vector-borne diseases and associated arthropods in Egypt.

Equine vector-borne diseases (EVBDs) are emerging and re-emerging diseases, and most of them are zoonotic. This study aimed to investigate EVBDs in equines and associated arthropods (ticks and flies) from Egypt using molecular analyses, in addition to a preliminary characterization of associated ticks and flies by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and molecular techniques. In this study, 335 blood samples were obtained from equines that appeared to be in good health (320 horses and 15 donkeys) in Cairo and Beni Suef provinces, Egypt. From the same animals, 166 arthropods (105 sucking flies and 61 ticks) were collected. Ticks and flies were preliminary characterized by the MALDI-TOF and molecular tools. Quantitative PCR (qPCR) and standard PCR coupled with sequencing were performed on the DNA of equines, ticks, and flies to screen multiple pathogens. The MALDI-TOF and molecular characterization of arthropods revealed that louse fly (Hippobosca equina) and cattle tick (Rhipicephalus annulatus) infesting equines. Anaplasma platys-like (1.6%), Anaplasma marginale (1.6%), Candidatus Ehrlichia rustica (6.6%), a new Ehrlichia sp. (4.9%), and Borrelia theileri (3.3%) were identified in R. annulatus. Anaplasma sp. and Borrelia sp. DNAs were only detected in H. equina by qPCR. A. marginale, Anaplasma ovis, and Theileria ovis recorded the same low infection rate (0.6%) in donkeys, while horses were found to be infected with Theileria equi and a new Theileria sp. Africa with recorded prevalence rates of 1.2% and 2.7%, respectively. In conclusion, different pathogens were first detected such as A. platys-like, Candidatus E. rustica, and a new Ehrlichia sp. in R. annulatus; A. marginale, A. ovis, and T. ovis in donkeys; and a new Theileria sp. "Africa" in horses.

Molecular detection of pathogens in the ova and unfed larvae of Rhipicephalus annulatus and Haemaphysalis bispinosa ticks infesting domestic cattle of south India.

This study aimed to investigate the presence of pathogens in the engorged ticks infesting domestic cattle, their ova, and unfed larvae. The engorged female ticks infesting domestic cattle of Wayanad district of Kerala, south India were collected and kept for oviposition. The dead females after the complete oviposition, their egg masses, and unfed larvae were screened for the presence of various pathogens by specific PCRs. The presence of Babesia bigemina, Anaplasma marginale, A. phagocytophilum, and Rickettsia spp. similar to R. raoultii was confirmed in Rhipicephalus annulatus ticks, their egg masses, and unfed larvae. Theileria orientalis was detected in Rh. annulatus females, but not in their egg masses or progenies. The presence of A. phagocytophilum and Rickettsia spp. similar to R. raoultii was confirmed in Haemaphysalis bispinosa ticks, their egg masses, and unfed larvae too. The presence of coinfections of B. bigemina with A. phagocytophilum and A. marginale were detected in Rh. annulatus ticks and their progenies.

Control of Rhipicephalus annulatus resistant to deltamethrin by spraying infested cattle with synergistic eucalyptus essential oil-thymol-deltamethrin combination.

The current study investigated the synergistic effect of combinations containing deltamethrin (D), Eucalyptus essential oil (E), and the thyme essential oil component thymol (T), against a field population of Rhipicephalus annulatus in Egypt that was characterized to be resistant to D. Solutions of T, E, or TE at concentrations of 1.25-5% were combined with 5% deltamethrin at different dilutions (0.25-2 mL/L). Results of the adult immersion test used to estimate the in vitro acaricidal activity of these combinations at 5% yielded LC50 values for D, E-D, T-D, and TE-D of 3.87 mL/L, 3.89 mL/L, 0.14 mL/L, and 0.05 mL/L, respectively. Biochemical analyses using whole-body homogenate of ticks from the in vitro tests revealed that the lowest acetylcholinesterase and glutathione peroxidase activity, and the maximum lipid peroxidation were recorded in ticks treated with 5% TE-D. Glutathione content significantly decreased (p ≤ 0.05) in all treated ticks. Three groups, each containing five cross breed cattle naturally infested with R. annulatus from the same area where resistance to D was detected, were sprayed twice at two-week intervals using 1 mL/L of 5% solutions of D, T-D, or TE-D. Overall efficacy of the D, T-D, and TE-D sprays by day 30 post-treatment was 21.6, 88.3, and 95 %, respectively. Ticks collected from infested cattle three days after treatment with the D spray deposited egg masses that were able to hatch, deposited small masses of eggs unable to hatch when exposed to the T-D spray, and laid few eggs that didn't hatch when sprayed with the TE-D combination. Values for liver and kidney function parameters were comparable in cattle before and after treatment with the combination sprays tested. The TE-D spray overcame the insensitivity to D of this R. annulatus population in Egypt, which also highlighted the significant synergistic effect of thymol on the acaricidal activity of deltamethrin observed in vitro. Acaricidal activity of the TE-D combination apparently has deleterious effects on multiple tick systems involving inhibition of acetylcholinesterase, increased lipid peroxidation, and oxidative stress. These findings document that combinations of natural and synthetic products can be part of integrated management solutions to the problem with widespread resistance to pyrethroids like deltamethrin in populations of cattle ticks, including R. annulatus, around the world.

Effect of polyphenols extracted from Punica granatum and Acacia saligna plants on glutathione S-transferase of the cattle tick Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae).

Ticks are hematophageal ectoparasites that transport major pathogens around the world. Glutathione S-transferases (GST) are involved in resistance to acaricide and redox balancing during the life cycle of the tick. The inhibition of tick GST enzymes by certain phenolic compounds, such as phenolic acids and tannins, can be a promising approach to tick control. The objective of this study was to evaluate the effects of Punica granatum red peel and Acacia saligna leaf extracts on Rhipicephalus (Boophilus) annulatus GST activity in order to reduce the resistance of cattle to acaricide. The results showed that P. granatum ethanol extract (70%) contained the highest total phenol content (350 ± 1.2 µM GAE g-1), the highest condensed tannin content (270 ± 1.3 µM CE g-1) and the highest hydrolysable tannin content (70 ± 5.0 µM TAE g-1). Adult immersion test with a dosage of 100 mg ml-1 of A. saligna ethanol extracts had a significant mortality of 50% and 75% after 24 h and 96 h, respectively (p < 0.01). A simple and reproducible procedure was established to purify the whole R. annulatus GST (wRaGST) while a full-length cDNA of GST was cloned from a cDNA library of the local Egyptian cattle tick R. (B.) annulatus (rRaGST). Aqueous extracts of P. granatum inhibited both wRaGST and rRaGST with values of IC50 = 0.114 and 0.07 µg ml-1, respectively, compared to A. saligna extracts (IC50 values = 2.08 and 1.35 µg ml, respectively). These inhibitory effects were attributed to the presence of a high tannin concentration (≥ 80%). HPLC analysis indicated the presence of gallic acid and catechin in both extracts, in addition to the rutin, which was only observed in A. saligna extracts. The addition of a tannin inhibitor, polyethylene glycol, suggested the existence of other phenolic compounds in combination with catechins responsible for inhibiting the activity of these extracts. Non-competitive behaviour of catechins may be helpful in preventing, or at least delaying, the development of chemical acaricide resistance in R. annulatus.

Raw pacific biosciences and illumina sequencing reads and assembled genome data for the cattle ticks Rhipicephalus microplus and Rhipicephalus annulatus.

Ticks from the genus Rhipicephalus have enormous global economic impact as ectoparasites of cattle. Rhipicephalus microplus and Rhipicephalus annulatus are known to harbor infectious pathogens such as Babesia bovis, Babesia bigemina, and Anaplasma marginale. Having reference quality genomes of these ticks would advance research to identify druggable targets for chemical entities with acaricidal activity and refine anti-tick vaccine approaches. We sequenced and assembled the genomes of R. microplus and R. annulatus, using Pacific Biosciences and HiSeq 4000 technologies on very high molecular weight genomic DNA. We used 22 and 29 SMRT cells on the Pacific Biosciences Sequel for R. microplus and R. annulatus, respectively, and 3 lanes of the Illumina HiSeq 4000 platform for each tick. The PacBio sequence yields for R. microplus and R. annulatus were 21.0 and 27.9 million subreads, respectively, which were assembled with Canu v. 1.7. The final Canu assemblies consisted of 92,167 and 57,796 contigs with an average contig length of 39,249 and 69,055 bp for R. microplus and R. annulatus, respectively. Annotated genome quality was assessed by BUSCO analysis to provide quantitative measures for each assembled genome. Over 82% and 92% of the 1066 member BUSCO gene set was found in the assembled genomes of R. microplus and R. annulatus, respectively. For R. microplus, only 189 of the 1066 BUSCO genes were missing and only 140 were present in a fragmented condition. For R. annulatus, only 75 of the BUSCO genes were missing and only 109 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.

Detection of single nucleotide polymorphism in the para-sodium channel gene of Rhipicephalus annulatus populations from Egypt resistant to deltamethrin.

Rhipicephalus annulatus field populations collected from small cattle farms in Beni-Suef province in Egypt were evaluated for deltamethrin resistance by toxicological in vitro bioassays (adult immersion test-AIT and larval packet test-LPT). Moreover, a quantitative PCR high resolution melting (PCR-HRM) technique was used to detect nucleotide substitutions in the voltage-gated sodium channel (Na-channel) gene. By the in vitro bioassays, the examined ticks were phenotypically categorized as deltamethrin susceptible (populations El-Wasta - A, and El-Hakamna - C) or resistant (populations El-Wasta - B, El-Hakamna - D, EL-Halabia - E, and Kom-abokhalad - F). Based on LPT findings, the phenotypic resistant populations were found to have a resistance ratio between 6.5 - 10.8. The PCR-HRM genotyping of the ticks showed variable melting curves among the populations in domain II of the Na-channel gene. Analysis of the curves showed the presence of wild type, mutant homozygous, and mutant heterozygous tick individuals. By sequencing the PCR amplified fragments, the C190A mutation was the only detected nucleotide polymorphism of domain II among the phenotypically resistant populations, which was present in 39.5 % (34/86) of the ticks tested. On the other hand, the phenotypically susceptible populations A and C did not show C190A mutant homozygous (RR) individuals. Meanwhile, in domain III all of the examined populations revealed melting curves like the wild type. Furthermore, the sequence analysis of these populations confirmed the absence of SNPs in domain III. The C190A single point mutation was detected for the first time in domain II of the Na-channel gene of deltamethrin-resistant R. annulatus in Egypt using PCR-HRM. Screening for efficacy of chemical compounds used by farmers to control ticks on cattle should be considered as part of animal health programs to manage the emerging resistance to acaricides in R. annulatus populations.