Molecular screening of piroplasms and Anaplasmataceae agents in Hyalomma dromedarii ticks from camels over different seasons in Egypt.

Piroplasmosis, a disease of domestic and wild animals, is caused by tick-borne protozoa of the genera Babesia and Theileria, while anaplasmosis is caused by tick-borne bacteria of genera Anaplasma. Hyalomma dromedarii is the most dominant tick species infesting camels in Egypt and act as a vector of piroplasms, Anaplasma, Rickettsia and Ehrlichia spp. The available information concerning the detection of these pathogens in H. dromedarii infesting camels is limited. The present study aimed to evaluate the status of these pathogens in H. dromedarii ticks over four seasons of a year, in addition to investigate the infections of piroplasms and Anaplasmataceae besides their genetic diversity starting from June 2021 till April 2022. A total of 275 semi-engorged females of H. dromedarii were collected from different slaughtered camels, Toukh city slaughterhouse then investigated by Polymerase Chain Reaction (PCR) to detect piroplasms (Babesia spp., Theileria spp.) and Anaplasmataceae DNA targeting 18 S rRNA and 16 S rRNA genes, respectively followed by sequencing and phylogenetic analyses. Overall, piroplasms were detected in 38 ticks (13.8%), Babesia spp. was detected in 35 ticks (12.7%), while Theileria spp. was detected in one tick (0.4%). Anaplasmataceae was detected in 57 ticks (20.7%). Mixed infections of piroplasms and Anaplasmataceae were detected in 13 ticks (5%). Single infection either with piroplasms or Anaplasmataceae was detected in 25 (9%) and 44 (16%) ticks, respectively. The highest monthly rate of piroplasms was in April (spring) and Anaplasmataceae was in July (summer). Sequence analysis revealed that Babesia bigemina, Wolbachia spp. and Anaplasma marginale are the most dominant species in the examined tick samples. To the best of our knowledge, this study confirms the presence of B. bigemina, Wolbachia spp. and A. marginale in H. dromedarii in Egypt by sequencing.

Acaricidal activity of Artemisia herba-alba and Melia azedarach oil nanoemulsion against Hyalomma dromedarii and their toxicity on Swiss albino mice.

Biopesticides such as essential oils (EOs) are considered an improvement for integrated pest control as they appear to be less toxic to the environment than chemical acaricides. The current study aimed to evaluate the acaricidal activity of Artemisia herba-alba and Melia azedarach oil loaded nano-emulsion as alternatives for chemical acaricides against the camel tick Hyalomma dromedarii, besides evaluating their toxic effect on Swiss albino mice. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) were used for the characterization of loaded nano-emulsions.The immersion test was used for the bioassay of both loaded nanoemulsions on tick stages (egg, nymph, larva, and adult). Mortality percentages and LC50 values of each tick stage were calculated. Reproductive performance for the survived engorged females after treatment was monitored. The toxicity of both loaded nano-emulsions was evaluated on Swiss albino mice by an oral dose of 1500 mg/kg/day for five successive days. The hematological, biochemical, and histopathological changes were evaluated. TEM characterization revealed spherical droplets for A. herba-alba and M. azedarach oil loaded nano-emulsion with droplet size ranging from 62 to 69 nm and 52-91 nm, respectively. FTIR revealed the absence of extra peaks in the loaded nano-emulsions that confirmed no chemical changes existed by ultrasonication. The LC50 values of A. herba-alba and M. azedarach oil loaded nano-emulsion on embryonated eggs, larvae, engorged nymphs, and unfed adults were 0.3 and 1.1%, 0.7 and 1.7%, 0.3 and 0.4%, 4.4 and 22.2%, respectively. The egg productive index (EPI), egg number, and hatchability percentage were lower in the treated females compared with Butox 5% (deltamethrin) and control. The hematological picture and biochemical analysis revealed insignificant changes in the treatment group compared with the negative control group. The liver of the A. herba-alba and M. azedarach oil loaded nano-emulsion treated group exhibited vacuolar degeneration and infiltration of lymphocytic cells. The kidney of mice treated with A. herba-alba and M. azedarach oil loaded nano-emulsion showed hemolysis and slight degeneration of epithelial cells of tubules. It is concluded that A. herba-alba and M. azedarach oil loaded nano-emulsion have good acaricidal activity against camel tick H. dromedarii.

Myrtus communis Essential Oil; Anti-Parasitic Effects and Induction of the Innate Immune System in Mice with Toxoplasma gondii Infection.

BACKGROUND: Myrtus communis (M. communis) is a wild aromatic plant used for traditional herbal medicine that can be demonstrated in insecticidal, antioxidant, anti-inflammatory, and antimicrobial activity of its essential oils (MCEO). AIM: The present study aimed to evaluate the prophylactic effects of M. communis essential oil (MCEO) against chronic toxoplasmosis induced by the Tehran strain of Toxoplasma gondii in mice. METHODS: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of MCEO. Mice were then orally administrated with MCEO at the doses of 100, 200, and 300 mg/kg/day and also atovaquone 100 mg/kg for 21 days. On the 15th day, the mice were infected with the intraperitoneal inoculation of 20-25 tissue cysts from the Tehran strain of T. gondii. The mean numbers of brain tissue cysts and the mRNA levels of IL-12 and IFN-γ in mice of each tested group were measured. RESULTS: By GC/MS, the major constituents were α-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%), respectively. The results demonstrated that the mean number of T. gondii tissue cysts in experimental groups Ex1 (p < 0.05), Ex2 (p < 0.001) and Ex3 (p < 0.001) was meaningfully reduced in a dose-dependent manner compared with the control group (C2). The mean diameter of tissue cyst was significantly reduced in mice of the experimental groups Ex2 (p < 0.01) and Ex3 (p < 0.001). The results demonstrated that although the mRNA levels of IFN-γ and IL-12 were elevated in all mice of experimental groups, a significant increase (p < 0.001) was observed in tested groups of Ex2 and Ex3 when compared with control groups. CONCLUSION: The findings of the present study demonstrated the potent prophylactic effects of MCEO especially in the doses 200 and 300 mg/kg in mice infected with T. gondii. Although the exceptional anti-Toxoplasma effects of MCEO and other possessions, such as improved innate immunity and low toxicity are positive topics, there is, however, a need for more proof from investigations in this field.

In vitro acaricidal activity of green synthesized nickel oxide nanoparticles against the camel tick, Hyalomma dromedarii (Ixodidae), and its toxicity on Swiss albino mice.

The green synthesized nanoparticles have been determined as a novel pesticide against arthropod pests. This study was designed to evaluate the in vitro acaricidal activity of green synthesized nickel oxide nanoparticles (NiO NPs) using aqueous extract of Melia azedarach ripened fruits against different developmental stages of the camel tick Hyalomma dromedarii in addition to their toxic effect on laboratory animals. The synthesized NiO NPs were characterized by UV-visible (UV-Vis) spectroscopy, Fourier transforms infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). The UV-Vis spectra of the NiO NPs showed an absorption peak at 307 nm. FTIR analysis showed the possible functional groups used for capping and stabilization of NiO NPs with strong bands at 3416.2 and 1626.6 cm-1. The SEM images of the NiO NPs exhibited a size ranging from 21 to 35 nm. The immersion test was used for the in vitro application of the synthesized NiO NPs on the various tick stages (egg, nymph, larva, and adult). Mortality percentages and LC50 values of each tick stage were calculated. The oviposition and hatchability of the engorged females were monitored for the survived tick after treatment. The LC50 values for NiO NPs on embryonated eggs, larvae, and engorged nymphs were 5.00, 7.15, and 1.90 mg/mL, respectively. The egg productive index (EPI), egg number, and hatchability (%) were lower in females treated with the NiO NPs than in control ticks. The toxicity of the NiO NPs on laboratory animals was also investigated using Swiss albino mice by oral dose of 500 mg/kg/day administration for five consecutive days. The hematological, biochemical, and histopathological changes were evaluated. The hematological analysis showed significant increase in the level of white blood cells (WBC) and hemoglobin (Hb). Biochemical analysis showed non-significant decrease in alkaline phosphatase (ALP) and alanine amino transferase (ALT). We concluded that NiO NPs have a significant acaricidal activity as demonstrated on eggs, larvae, engorged nymphs, and fully fed females of H. dromedarii. From a toxicological point of view further in vivo investigations are needed to determine the mechanism of toxic effect of NiO NPs.

Molecular epidemiology of certain vector-borne bacterial microorganisms in domestic animals and their ectoparasites in Egypt.

Vector-borne bacterial diseases (VBBD) are a diverse group of tropical and subtropical zoonotic diseases. This study investigated the possibility of domestic animals to carry certain vector-borne bacterial microorganisms (VBBMs), as well as the presence of these targeted DNAs in their ectoparasites in different localities of Egypt using molecular analyses. For this study, 234 animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) in addition to 115 ectoparasites (95 ticks and 20 lice) were investigated for the molecular detection of Bartonella spp., Borrelia spp., and Rickettsia spp., targeting 16S-23S rRNAITS, 16S rRNA, and gltA genes, respectively. The results indicated that the overall prevalence of VBBD was observed in 17 animals (7.26%), of which 16 (6.84%) were positive for Bartonella spp. and one (0.43%) was positive for Borrelia theileri. All blood samples were negative for the DNA of Rickettsia spp. In addition, the results demonstrated that all ectoparasites were free from VBBDNA. Furthermore, of the animals examined for ectoparasite infestation, 28 (11.97%) and 5 (2.14%) represented Rhipicephalus annulatus ticks and Haematopinus tuberculatus lice, respectively, which infested animals. Analysis of epidemiological factors revealed that gender, age, and ectoparasitic infestation of animals had a significant effect on Bartonella infection, whereas no significant difference between animal species was observed. Hence, we report a potential novel Bartonella sp. from cattle and buffaloes, including a new genotype of Bo. theileri from cattle, in Egypt.

Morphological and molecular description of immature stages of Ornithodoros savignyi (Acari: Argasidae).

This study was designed to provide more details about larva, first nymph, and second nymph of Ornithodoros savignyi using a combination of light microscopy (LM), scanning electron microscopy (SEM), and partial sequence of mitochondrial 16S ribosomal ribonucleic acid (rRNA). The main characteristics of larva are wrinkled integument with many grooves, gnathosoma without camerostome cheeks, hypostome with a pair of large teeth apically, and tarsus without humps. The comparisons between the first and the second nymphs are different shape and distribution of dorsal grooves; a few spots without mammilla on the dorsal surface of the second nymph; 27 and 63-65 pairs of setae on the dorsal surface of the first and second nymphs, respectively; small holes on mammillae that are more dense in the second nymph; basis capitulum with two pairs of small setae in the second nymph; and one pair of sate in the first nymph, hypostome with dental formula 2/2 in the first nymph, and 3/3 in the second nymph. The partial 16S rRNA sequence of the second nymph that was determined as O. savignyi (450 bp) was deposited in GenBank under the accession number KU163242.

Molecular epidemiology of bovine theileriosis and genetic diversity of Theileria annulata in three governorates of Egypt.

Bovine theileriosis is a protozoan disease caused by the intracellular parasite (Theileria spp.) transmitted by ticks and it is considered one of the most significant parasitic diseases, potentially endangering Egyptian cattle herd industry. The present study was conducted for a molecular survey of bovine theileriosis and its associated risk factors (season variations, geographical locations, breeds, age, sex, tick infestation, and acaricide applications) in three Egyptian governorates, Beni-Suef, Al-Faiyum, and Al-Minya for a year extended from December 2021 to November 2022, in addition, genetic diversity of Theileria isolates. A total of 961 cattle were examined for Theileria infection clinically, microscopically, and by Polymerase Chain Reaction (PCR) using 18S rRNA gene for piroplasms DNA detection, Theileria genus-specific primers of the small subunit of rRNA gene, and Theileria annulata specific primers of the Tams-1 gene. The prevalence rate of bovine theileriosis was 9.26%, and 11.86% using Giemsa-stained blood smear and PCR, respectively. All positive samples screened by Theileria genus-specific primers were positive for T. annulata when screened by the specific primers. Based on molecular screening, season, cattle breeds and acaricide applications were considered risk factors for T. annulata infection, while locality, age, sex and tick infestation had insignificant effects with the occurrence of the disease. A potential novel T. annulata haplotype based on the Tam-1 gene was identified with accession numbers OR364144 and OR915851. Therefore, T. annulata was the only Theileria species found and played a significant problem in the cattle population. This study could be the basis for future studies on unexplored regions and different animal species for well-structured prevention and control measures.

Rhipicephalus linnaei (Audouin, 1826) recognised as the "tropical lineage" of the brown dog tick Rhipicephalus sanguineus sensu lato: Neotype designation, redescription, and establishment of morphological and molecular reference.

We re-describe the adult stages of Rhipicephalus linnaei (Audouin, 1826), and characterise its diagnostic molecular traits. A male R. linnaei collected in Esna City, Luxor Governorate, Egypt is designated as the neotype. Rhipicephalus linnaei is re-established as a valid tick name and removed from the synonymy list of Rhipicephalus sanguineus (Latreille, 1806). Rhipicephalus linnaei is most similar to R. sanguineus and Rhipicephalus camicasi Morel, Mouchet & Rodhain, 1976 because they share similar elongated comma-like spiracula that are narrowly visible dorsally, and the dorsal prolongation is narrower than the width of the adjacent festoon. The male of R. camicasi is distinguished from R. linnaei by the non-tapering caudal widening of the spiracula. The male of R. sanguineus is distinguished from R. linnaei by shorter extension that does not taper into a long narrow extension of the spiracula. The genital pore atrium of female R. linnaei is broadly U-shaped, while it is a narrower U-shape in R. sanguineus. The remaining species within the R. sanguineus species complex - Rhipicephalus sulcatus Neumann, 1908, Rhipicephalus turanicus Pomerantsev, 1940, Rhipicephalus guilhoni Morel & Vassilades, 1963, Rhipicephalus secundus Feldman-Muhsam, 1952 and Rhipicephalus afranicus Bakkes, 2020, all exhibit spiracula with the dorsal prolongation as wide as the adjacent festoon. The DNA sequence of R. linnaei is most closely related to R. guilhoni. The phylogenetic analysis of mitogenome (mtDNA) sequences including assembled mtDNA from whole genome sequencing of the neotype supports R. linnaei as a well-defined taxon when compared with DNA sequences of other species of the R. sanguineus species complex, in particular: R. sanguineus, R. turanicus, R. secundus and R. camicasi. Molecularly, R. linnaei belongs to the so-called R. sanguineus s.l. "tropical lineage" distributed globally including the Americas, Africa, Europe, Asia and is the only species from R. sanguineus species complex in Australia.

Acaricidal Efficacy of Biosynthesized Zinc Oxide Nanoparticles Against Hyalomma dromedarii (Acari: Ixodidae) and Their Toxic Effects on Swiss Albino Mice.

PURPOSE: The current study aimed to investigate the efficacy of zinc oxide nanoparticles (ZnO NPs) synthesized by Melia azedarach aqueous extract to control Hyalomma dromedarii tick, and to evaluate their toxic effects on Swiss albino mice. METHODS: ZnO NPs were synthesized using M. azedarach aqueous extract. UV-visible spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive spectroscopy were used to characterize the synthesized NPs. Egg, nymph, larva, and adult immersion tests were used for bioassay of tick stages with the synthesized ZnO NP. A toxicity study was performed on Swiss albino mice after treatment with 1/10 of the oral LD50 of ZnO NPs (8437 mg/kg) for 5 successive days by oral gavage. RESULTS: The LC50 of ZnO NPs on the eggs, larvae, and nymphs was 11.6, 8.03, and 3.9 mg/ml, respectively. The reproductive performance of females treated with ZnO NPs was lower than that of untreated females. The hematological results showed an insignificant increase in the level of white blood cells with normal red blood cells, hemoglobin, in addition to normal platelet count. The biochemical analysis showed an insignificant increased level (P > 0.05) of alkaline phosphatase and alanine aminotransferase. The liver and kidney suffered few histopathological changes after oral administration of ZnO NPs. CONCLUSION: These results suggest that ZnO NPs have good acaricidal activity against eggs, larvae, and engorged nymphs of H. dromedarii. ZnO NPs minimized the number of eggs laid by engorged females and the hatchability of their eggs. ZnO NPs did not affect unfed adults. The toxicity results of the mice revealed insignificant changes in the hemogram, biochemistry, with liver and kidney suffering few histopathological changes. Future studies are needed to assess application routes (topical vs oral). Based on these findings, ZnO NPs may be incorporated in the control of camel tick H. dromedarii.

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 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.

Scanning electron microscopy and morphometrics of nymph and larva of the tick Hyalomma impressum (Acari: Ixodidae).

Nymphs and larvae of ixodid ticks play an important role on the distribution of ticks and transmission of pathogens. They almost infest small mammals and birds which either move from place to place as rodents or migrate across different countries as migratory birds. The morphological descriptions of nymph and larva of the tick Hyalomma impressum were firstly studied in details by scanning electron microscopy and morphometric analysis. The distinguished characters of H. impressum nymph are as follows: dorsal idiosoma (excluding scutum) with 26-27 pairs of setae, posterior margin of scutum is narrowly rounded, posterolateral margins of scutum is straight, cervical grooves on the scutum extend to the midlength, coxa I with two large spurs, coxae (II-IV) with one small spur for each, spiracle is an egg shape with a numerous pores, palpus does not project beyond the hypostome, with nine setae dorsally and six setae ventrally, hypostome has cylindrical shape, dental formula 2/2, teeth number per file is eight in the outer file and seven in the inner file, basis capitulum has triangular shape without setae dorsally and tetragonal shape with three pairs of setae ventrally. The distinctive characters of H. impressum larva are as follows: idiosoma with 13 pairs of setae for each dorsal (including scutal setae) and ventral (excluding coxal setae), scutum with cervical grooves is narrow and shallow extending about one third of the scutal length, posterior margin of scutum is broadly rounded, posterolateral margins of scutum is straight, fold-like indistinctive spurs on coxae II and III, palpus with eight setae dorsally, three setae ventrally and one seta apically, hypostome with dental formula 2/2, teeth number per file (excluding small basal and apical teeth) is seven in the outer file and six in the inner file, basis capitulum without setae dorsally and with three pairs of setae ventrally.

Effect of various levels of dietary Jatropha curcas seed meal on rabbits infested by the adult ticks of Hyalomma marginatum marginatum I. Animal performance, anti-tick feeding and haemogram.

The goal of this study was to investigate the use of Jatropha curcas seed meal (JCSM) in different levels as acaricide in diet of rabbits experimentally infested by Hyalomma marginatum marginatum then determining animal performance, anti-tick feeding and its effects on haemogram of rabbits. Thirty healthy mixed-breed rabbits were randomly divided into five equal groups. The first group was kept as a control fed soya bean meal (20%) as a source of protein. Groups from the second to the fifth fed diets contained 2.5%, 5%, 7.5% and 10% of JCSM instead of soya bean meal as a source of protein, respectively. Feeding and watering were given freely throughout the study. Animal performance for treatment groups were recorded from the 1st week up to the 6th week. Then each group divided into two subgroups, and the ticks were introduced to all of one subgroup and the other kept as control, following them until dropped at the end of the 8th week for all groups of the experiment. Feeding and reproductive performance of the adult tick females were determined. Blood samples were collected and analysed for haematological examination at the 0, 6th and 8th weeks post-treatment from all animals. Result revealed that rabbits received diets containing 5%, 7.5% and 10% had significantly (P < 0.05) lower growth performance than control and 2.5% JCSM groups. Feed intake and body weight gain decreased with increasing the level of JCSM in the diet. The result of anti-tick feeding observed that the highest percentage (60-90%) of rejection was recorded in 10% of JCSM group then the other treated groups (20-30%). Egg mass and reproductive index per female were marked increase (P < 0.001) in groups 2.5%, 5% and 7.5% of JCSM. Macrocytic normochromic anaemia was development after 8 weeks of treatment, which changed to microcytic normochromic anaemia after challenge of ticks in groups received 5%, 7.5% and 10% JCSM. Leukopenia, neutopenia and lymphopenia were noticed (P < 0.05) in all treated groups which more drop in the group received 7.5% JCSM. Also, monocytosis was recorded in 7.5% and 10% JCSM groups. In conclusion, JCSM could be use in the treatment of ectoparasites at level less than 10% in diet. Further investigations should be done to detoxification the Jatropha seed meal to decrease the level of its toxicity.

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.

Therapeutic Potential of Green Synthesized Copper Nanoparticles Alone or Combined with Meglumine Antimoniate (Glucantime®) in Cutaneous Leishmaniasis.

BACKGROUND: In recent years, the focus on nanotechnological methods in medicine, especially in the treatment of microbial infections, has increased rapidly. AIM: The present study aims to evaluate in vitro and in vivo antileishmanial effects of copper nanoparticles (CuNPs) green synthesized by Capparis spinosa fruit extract alone and combined with meglumine antimoniate (MA). METHODS: CuNPs were green synthesized by C. spinosa methanolic extract. The in vitro antileishmanial activity of CuNPs (10-200 µg/mL) or MA alone (10-200 µg/mL), and various concentrations of MA (10-200 µg/mL) along with 20 µg/mL of CuNPs, was assessed against the Leishmania major (MRHO/IR/75/ER) amastigote forms and, then tested on cutaneous leishmaniasis induced in male BALB/c mice by L. major. Moreover, infectivity rate, nitric oxide (NO) production, and cytotoxic effects of CuNPs on J774-A1 cells were evaluated. RESULTS: Scanning electron microscopy showed that the particle size of CuNPs was 17 to 41 nm. The results demonstrated that CuNPs, especially combined with MA, significantly (p < 0.001) inhibited the growth rate of L. major amastigotes and triggered the production of NO (p < 0.05) in a dose-dependent manner. CuNPs also had no significant cytotoxicity in J774 cells. The mean number of parasites was significantly (p < 0.05) reduced in the infected mice treated with CuNPs, especially combined with MA in a dose-dependent response. The mean diameter of the lesions decreased by 43 and 58 mm after the treatment with concentrations of 100 and 200 mg/mL of CuNPs, respectively. CONCLUSION: The findings of the present study demonstrated the high potency and synergistic effect of CuNPs alone and combined with MA in inhibiting the growth of amastigote forms of L. major, as well as recovery and improving cutaneous leishmaniasis (CL) induced by L. major in BALB/c mice. Additionally, supplementary studies, especially in clinical settings, are required.

A report on tick burden and molecular detection of tick-borne pathogens in cattle blood samples collected from four regions in Saudi Arabia.

Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.

Ovicidal and Latent Effects of Pulicaria jaubertii (Asteraceae) Leaf Extracts on Aedes aegypti.

The control of Aedes aegypti with synthetic pesticides may result in adverse effects on wildlife and the environment. Bioactive plant extracts have been proposed as one of the alternatives to chemical pesticides used against mosquitoes. Here, we report on the ovicidal and latent effects of ethanolic, petroleum ether, and chloroform leaf extracts of Pulicaria jaubertii at 25 to 150 ppm each against the life stages of laboratory stain of Ae. aegypti. At 150 ppm, the ethanolic leaf extract resulted in 100% ovicidal activity, followed by petroleum ether extract (74%), and chloroform extract about 7% mortality. The ethanolic extract produced 100% larval and pupal mortality at both 75 and 50 ppm, while the petroleum ether extract produced 76.5 and 58.3%, respectively. The ethanolic extract recorded the highest percentage of adult mortality (72.7%) at the lowest concentration (25 ppm). At 25 and 50 ppm, the ethanolic extract resulted in 62.2 and 85.2% sterility index of Ae. aegypti females, respectively, as compared with the 0.1 and 3.5% sterility index caused by the chloroform extract at the same concentrations. In conclusion, P. jaubertii appears to have potential to be further evaluated as a mosquito control agent. Additional studies are needed on its mode of action, synergism with other products, and efficacy under actual field conditions.

Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia.

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

The efficacy of some wild medicinal plant extracts on the survival and development of third instar larvae of Chrysomyia albiceps (Wied) (Diptera: Calliphoridae).

Four crude wild plants extract of Artemisia herba-alba Asso, Artemisia monosperma Del., Euphorbia aegyptiaca Boiss. and Francoeuria crispa (Forsk.) extracted with four successive solvents; hexane, diethyl ether, ethyl acetate and ethanol were evaluated against the third instar larvae of Chrysomyia albiceps using dipping and thin film techniques. In dipping technique, larvae were immersed in the concentrations of plant extracts for 30 seconds. However, in thin film technique, larvae were exposed to thin layer from each plant extract in the Petri-dishes. Results showed that all extracts had toxic effects on larvae in both two treatments. Hexane and diethyl ether extracts of A. herba-alba and ethyl acetate extract of A. monosperma recorded the highest effect in both two treatments. Ethanol extracts of E. aegyptiaca and A. monosperma were the highest in dipping and thin film treatments, respectively. In dipping treatment, most plant extracts revealed extending effect on pupae especially while in thin film treatment most larvae which succeeded to develop to pupae produced normal flies. Deformed pupae were only recorded with the high concentration of diethyl extracts of A. herba-alba. Hexane, diethyl ether and ethyl acetate extracts of F. crespa only produced low percentages of deformed flies. Histological examination conducted on larvae confirmed that extracts in thin film treatment penetrated to the gut and destroyed its epithelial cells and wall. It concluded that the crude extracts of the four tested plants can be used in controlling of C. albiceps larvae while hexane extracts of E. aegyptiaca, A. herba-alba and A. monosperma are considered the most promising plant preparations against the larvae by using thin film technique.

Species Diversity and Seasonal Distribution of Hard Ticks (Acari: Ixodidae) Infesting Mammalian Hosts in Various Districts of Riyadh Province, Saudi Arabia.

Hard ticks are among the most important blood sucking arthropods that transmit pathogens to humans and animals. This study was designed to determine prevalence, mapping, geographical distribution, and seasonal activity of hard tick species infesting the most common domestic and wild mammals in various districts of Riyadh Province, Saudi Arabia, during the period January to December 2017. In total, 10,832 adult hard ticks were collected from the bodies of 8,435 animals belonging to 18 different mammalian species. The ticks were preserved in 70% alcohol and microscopy was used to identify species. Two genera, Hyalomma and Rhipicephalus, were identified, comprising 10 species of hard ticks, with Hyalomma comprising 68.3% and Rhipicephalus comprising 31.7% of species. The most common species on domestic mammalian hosts was Hyalomma dromedarii (Koch 1844) (39.9%) followed by Rhipicephalus turanicus (Pomerantsev, Matikashvili & Lotosky 1936) (34.9%), whereas on wild mammalian hosts Rhipicephalus sanguineus (Latreille 1806) was by far the most prevalent species (83.0%). However, ticks were most abundant during May through July (36.0%) in the studied areas, and tick intensity and abundance differed among seasons. Our results provide information for human and animal health service managers, as well as governmental authorities, to gain a better understanding of hard ticks infesting mammalian hosts in Riyadh Province, Saudi Arabia, which can help improve prevention and control of tick-borne diseases, especially during outbreaks.