Phylogeny and ultrastructure of Elthusa raynaudii (Isopoda, Cymothoidae) firstly recorded from the invasive silver cheeked toadfish (Lagocephalus sceleratus) (Gmelin 1789) in eastern Mediterranean Sea coast.

With the opening of the Suez Canal in 1869, many changes have occurred in the Mediterranean Sea ecosystem so became a home to many invasive Lessepsian marine species that have migrated from the Red Sea. About 500 marine species including pufferfish have immigrated and rapidly established a population in the Mediterranean Sea causing significant impact on its ecosystem and fisheries sector. The parasitic fauna of these pufferfish has scarcely been studied in the Mediterranean Sea and also in their native habitat. During this surveillance study on the invasive pufferfish species from the Egyptian Mediterranean coast, the female cymothoid isopod Elthusa raynaudii was detected from the branchial cavity and also in the buccal cavity of 23.9% of the examined Lagocephalus sceleratus. The isolated isopod species was firstly identified and described through electron microscopy and molecular phylogeny based on the sequences of mitochondrial 16S rRNA gene. Additionally, the description of eggs, embryonic stage, and manca of E. raynaudii was firstly provided. The pathological impact on the infested fish tissues was investigated and revealed curling and loss of secondary gill lamellae in addition to mucous exudates in between the gill filaments and granuloma formation in the gill arch. The study provided the first report of L. sceleratus as a new host for the isopod E. raynaudii collected from the Egyptian Mediterranean coast as a new locality record. The role of the Lessepsian invasive pufferfish in transmitting parasites to the native fish species was discussed.

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.

Morphological, ultrastructural, and molecular characterization of Livoneca redmanii (Leach, 1818) (Isopoda: Cymothoidae) infestation among Solea solea.

In the present investigation, we identified a cymothoid isopod species, Livoneca redmanii (Isopoda, Cymothoidae), in the branchial cavity, skin, and muscle pouch of the common sole (Solea solea) (Soleidae, Pleuronectiformes) from Qarun Lake, Egypt. The parasite was identified by morphometric and ultrastructural analysis, and molecular typing of the 16S rRNA gene. Out of 625 S. solea specimens collected, 8.16% were infested with the isopod. For the first time, the ultrastructural features of L. redmanii in different growth stages were described, revealing the fine characteristics of the body appendages, the eyes, the antennae, and the antennules of the female, male, and juvenile isopod, as well as the external layers of the ova. Additionally, the detailed structures of the mouth parts, the sensory microtriches, and the male organs (penis, appendex masculine, and the genital furrow) were elucidated. A histological examination of the infested gill revealed damaged gill lamellae, leukocyte infiltration, and epithelial hyperplasia. Besides, microscopic examination of the skin showed focal erosion and periodic acid stain-positive foci in the dermis. Phylogenetic analysis revealed the close relationship between the L. redmanii isolated in this study and those recovered from mugiliid fry in the Mediterranean Sea in 2017. Based on our findings, we recommend using hatchery-reared fry instead of wild fry for supplying lakes and fish farms. Also, a strict biosecurity program must be implemented to avoid the transmission and spread of these disease-causing parasites.

Screening and phylogenetic characterization of tick-borne pathogens in a population of dogs and associated ticks in Egypt.

BACKGROUND: The incidence or recurrence of tick-borne diseases (TBDs) in animals and humans is increasing rapidly worldwide, but there is insufficient information about TBDs infecting dogs in Egypt. Thus, the present study was conducted to screen and genetically identify tick-borne pathogens (TBPs) in dogs and associated ticks by microscopic examination and polymerase chain reaction (PCR). METHODS: In Cairo and Giza governorates, 208 blood samples were collected from dogs of different breeds, ages, and sex. In addition, 1266 dog-associated ticks were collected (546 ticks were used to prepare hemolymph smears, and 720 ticks were kept in 70% ethanol until PCR analysis). PCR was applied to 124 dog blood samples and 144 tick pools prepared from 720 ticks. RESULTS: All ticks collected from dogs were Rhipicephalus sanguineus (s.l.). Microscopic examination revealed that TBP prevalence among dogs was 23.56% (49/208), including Anaplasma and Ehrlichia with 11.1% (23/208) and Babesia canis with 8.2% (17/208). Hepatozoon canis was not detected in blood smears. Co-infections with two pathogens were visible in 4.33% (9/208) of examined dogs. The prevalence of TBPs in hemolymph smears was 45.97% (251/546) including 35.89% (196/546) for H. canis, 8.1% (44/546) for B. canis, and 2.01% (11/546) for Anaplasmataceae (A. phagocytophilum, A. marginale, A. platys, and E. canis). The overall molecular prevalence rate of TBPs was 25.81% and 29.17% in the blood of examined dogs and in ticks, respectively. The molecular prevalence of Anaplasmataceae family, Babesia canis, and H. canis in dog blood samples was 19.35%, 6.45%, and 0.0%, respectively, while in ticks, it was 20.83%, 5.55%, and 2.8%, respectively. A sequential analysis identified six different species of TBPs, namely B. canis vogeli, Hepatozoon canis, A. phagocytophilum, A. marginale, A. platys, and E. canis. The obtained sequences were submitted to GenBank and assigned accession numbers. CONCLUSIONS: The present study detected a wide range of TBPs (B. canis, H. canis, A. platys, A. phagocytophilum, A. marginale, and E. canis) that are considered a threat to domestic animals and humans in Egypt. Hepatozoon canis and A. marginale were reported in dogs and associated ticks for the first time in Egypt.

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.

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.

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.

Additional morphometric and phylogenetic studies on Mothocya melanosticta (Isopoda: Cymothoidae) parasitizing the Red Sea Nemipterus randalli fish in Egypt.

Cymothoidae, Leach, 1818 (Crustacea: Isopoda) are ectoparasites of marine, brackish and freshwater fishes that are reported to induce deleterious tissue impacts on the infested fish hosts. The present study aimed for the first-time screening of Mothocya melanosticta collected from the red sea fish Nemipterus randalli in Egypt. Surveillance study was conducted for isopod infestation among the Red Sea fish N. randalli revealing a total infestation rate of 40.96% with the species identified as M. melanosticta. The parasite species was isolated from the branchial cavity and gills. Morphometric description was estimated using dissecting stereo-microscope, and scanning electron microscope (SEM) provided new additional features for the isolated species including the microtrich sensillum in the body cuticle and the fine structure of the mouth parts and body appendages. Mitochondrial COI gene of M. melanosticta female isolated in the present study from N. randalli was detected for the first time in Egypt and recorded in the GenBank (MK168807). The study showed that the detected isopod species represents one monophyletic group closely affiliated to the genospecies of M. melanosticta, and can be distinguished clearly from other isopods genospecies. Based on the genetic distance values, lower level of genetic divergence was indicated within the genospecies of the present M. melanosticta isolated from Egypt and the same species isolated from India. The present investigation recorded N. randalli fish as a new host for the isopod M. melanosticta in Egypt and provided additional morphological features through SEM as well as molecular characterization of this isopod species for the first time.

First Report of Cattle Tick Rhipicephalus (Boophilus) annulatus in Egypt Resistant to Ivermectin.

Tick control is mainly dependent on the application of acaricides, but resistance has developed to almost all classes of acaricides, including macrolactones. Therefore, we aimed to investigate ivermectin resistance among tick populations in middle Egypt. The larval immersion test was conducted using a commercial formulation of ivermectin (1%). Different concentrations of the immersion solution (0.0000625% (625 × 10-7%), 0.000125% (125 × 10-6%), 0.0005% (5 × 10-4%), 0.001% (1 × 10-3%), 0.0025% (2.5 × 10-3%), 0.005% (5 × 10-3), and 0.01% (1 × 10-2%)) were prepared by diluting a commercial ivermectin (1%) with distilled water containing 1% (v/v) ethanol and 2% (v/v) TritonX-100. Field populations of Rhipicephalus annulatus were collected from five different localities in Beni-Suef province, Egypt. Adult engorged female ticks were collected and assessed for oviposition and egg fertility. Eggs were collected, and hatched larvae were then used in the experiment. Application of acaricides was conducted on 10-day-old larvae. There was a significant difference in the LC50 (50% lethal concentration) among the examined localities on the log dose-response plot, where, the LC50 of tick populations from two localities (Emin elaros and Aldiabia) was higher than the other localities (Alhalabia, Alkom, and Beshnna). Besides, tick populations from Emin elaros and Aldiabia showed higher LC90 values with lower slope values compared to those from Alhalabia, Alkom, and Beshnna. According to these values (LC50, LC90, and slope values), as well as a history of acaricide failure to ticks in these areas, R. annulatus developed resistance to ivermectin. This study documents the first report of field populations of R. annulatus resistant to ivermectin in Egypt.

In vitro acaricidal effect of Melia azedarach and Artemisia herba-alba extracts on Hyalomma dromedarii (Acari: Ixodidae): embryonated eggs and engorged nymphs.

The present study aimed to evaluate the in vitro efficacy of four medicinal plant extracts: petroleum ether and ethyl alcohol extracts of the ripen fruits of Melia azedarach and whole aerial parts of Artemisia herba-alba against the two inactive stages of the camel tick Hyalomma dromedarii, embryonated eggs and engorged nymphs in comparison to reference acaricide Butox®5.0 (Deltamethrin). Egg and nymphal immersion tests at four concentrations with three replicates were used. The deformity in larvae hatched from treated eggs and adults moulted from treated nymphs were observed and photographed by light microscope (LM) and scanning electron microscope (SEM). The results showed that M. azedarach and A. herba-alba extracts revealed higher significant toxic effects on embryonated eggs and engorged nymphs comparing with the reference acaricide (Butox®5.0) and control. In egg emmersion test, the LC50 of petroleum ether extracts of M. azedarach and A. herba-alba was 3.14 and 3.91%, respectively and LC50 of the respective ethyl alcohol extracts was 1.77 and 2.45%. In nymphal immersion test, LC50 of petroleum ether extracts of M. azedarach and A. herba-alba was 0.26 and 1%, respectively, and LC50 of the respective ethyl alcohol extracts was 4.17 and 8.7%. Abnormalities were observed by LM and SEM in the larvae hatched from the treated eggs as incomplete development of legs and mouth parts as well as shrinkage mainly in legs and mouthparts of adults emerged from treated nymphs. In conclusion, all extracts and petroleum ether extracts of the two plants have great potential to be developed as a novel acaricidal for controlling eggs and nymphs of H. dromedarii, respectively.

Molecular detection of Rhipicephalus (Boophilus) annulatus resistance against deltamethrin in middle Egypt.

This study was designed to investigate the susceptibility/resistance of Rhipicephalus (Boophilus) annulatus to commercial deltamethrin 5%. The work was based on in vitro bioassays; adult immersion test (AIT) and larval packet test (LPT). Fully engorged female ticks were collected from 11 locations in Beni-Suef Governorate, Egypt (Alfashin, Eminelaros, Ehnasia, Smusta, Belifia, Aldiabia, Alhalabia, Beshnna, Alkom, Nasr and Beba). Two concentrations of commercial deltamethrin 5% (200 and 400 ppm) were tested by AIT on R. (B.) annulatus. The mortality of ticks at 400 ppm was higher than 200 ppm which was not reached to 100%. Ehnasia tick population was the least susceptible to deltamethrin while Alfashin population was the most susceptible one. Different deltamethrin concentrations (0.000625, 0.00125, 0.0025, 0.005, 0.010 and 0.020) were used in LPT to evaluate the larval mortality. The highest LC50 appeared in Ehanasia tick population (0.01) with 95% confidence interval (0.005 to 0.01) meanwhile, the lowest LC50 appeared in Alfashin (0.0006) with 95%CI (0.0005 to 0.0007). Thus, it is possible to deduce that one population was considered susceptible; seven populations were resistant level I and three populations were resistant level II. Additionally, PCR revealed that the resistant alleles (R) appeared in two localities (Ehnasia and Aldiabia). It was concluded that at least two tick populations of the investigated locations were of possible resistant to deltamethrin.

Immunological evaluation of some antigens of Lucilia sericata larvae.

The present study aimed to select an antigen of Lucilia sericata larvae showing both high antigenicity and cross-reactive binding abilities with other related antigens of L. sericata larvae for obtaining a promising candidate vaccine antigen. The ELISA results primary concluded that among the excretory secretory (ES) and midgut (MG) antigens of the different larval instars of L. sericata, MGL2 could be characterized as antigen which was able to reflect the highest level of antigenicity and cross-reactivity with the other tested L. sericata antigens. The results were extended to spot the light on the relation between different protein bands in MGL2 and rabbit hyper- immune sera (HIS) raised against the other tested antigens using SDS-PAGE and Western blot technique. Analysis by SDS-PAGE of ES and MG antigens of the different larval instars of L. sericata revealed common protein bands at molecular weights of about 10, 12, 16, 20, 28, 33 and 46 kDa. Western blotting of MGL2 antigen transferred to nitrocellulose sheet revealed reaction by MGL2 HIS to five polypeptide bands; 20, 28, 33, 46 and 63 kDa. Three bands of 28, 33 and 63 kDa were the most prominent bands detected whereas; there was a weak reaction with bands of 20 and 46 kDa. But what was apparent in Western blot was a strong reaction of all tested HIS with a polypeptide band of 63 kDa. This band might be considered to be the main cause of cross reactive binding ability of MGL2 antigen that had been recorded previously in ELISA technique.