Ovicidal, larvicidal and adulticidal activity of black pepper (Piper nigrum L.) essential oil and tea tree oil (Melaleuca alternifolia) against Haemonchus contortus.

Haemonchosis is a worldwide helminthic disease affecting ruminants. The anthelminthic resistance has become raised. Medicinal plants are safely used as synthetic anthelmintics. Currently, the efficacy of black pepper essential oil (BPO) and tea tree oil (TTO) were in vitro evaluated against Haemonchus contortus adults, eggs and larvae at concentrations of 1.25, 2.5 and 5 mg/ml in addition to the commercially used albendazole at a concentration of 10 µg/ml. Oils were used in both normal and nanoparticles-loaded forms. Oxidative stress enzymes of worms were estimated. Scanning electron microscopy (SEM) for treated worms was done. Both normal and nanoemulsion forms of both BPO and TTO stopped the adult motility [BPO 2.5 h (hrs), NBPO 1.5 h, TTO 3 h, NTTO 1.5 h] and induced a marked decrease in the oviposition. Post treatment, the egg development and hatching were significantly (P ≤ 0.05) reduced. The damage of the egg shell, embryonal cessation and destruction of larvae occurred. Noticeable elevated antioxidant enzymes (catalase CAT, glutathione transferase GST and glutathione GSH) were found, while oxidative enzymes (lipid peroxidation LPO and nitric oxide synthase NOS) decreased. Scanning electron microscopy (SEM) for both oil-treated worms revealed anterior ends damage and several cuts associated with cuticular pores. The use of albendazole induced more or less anthelmintic and enzymatic activities with less morphological alterations of adults revealed by SEM. This study proved the marked anthelmintic potency of the BPO and TTO and their nanoemulsion forms against H. contortus rather than the widely used anthelmintic drugs.

Efficacy and safety of geranium-oregano-thymol formulations to control of dog tick Rhipicephalus sanguineus sensu lato under laboratory and field conditions.

The present study evaluated, in laboratory and field, the efficacy and safety of formulations of Pelargonium graveolens (geranium - G), Origanum majorana (oregano - O) commercial essential oils (EO) and thymol (T) to control of Rhipicephalus sanguineus sensu lato. In the laboratory, three formulas (A: 2% tween 80%, B: powder and C: nanoemulsion) by a mixture of these components (GOT) were prepared and evaluated, and the best one was used to assess its safety and field application against R. sanguineus s. l. on naturally infested dogs. Besides the major compounds of the EO used were identified. The results of the lab study showed that formula A (2.5 g of each G + O + T + 2% tween 80 to complete 100 mL) was significantly more effective than the other two formulas tested and exhibited highly effective adulticidal, larvicidal, and ovicidal activity against R. sanguineus s.l. Significant LC50 and LC90 values of GOT were evaluated (13.4 and 21.5 mg/mL, respectively) for the adulticidal activity, (2.81 and 4.46 mg/mL, respectively) for ovicidal activity and (2.44 and 4.45 mg/mL, respectively) for larvicidal activity. The safety of formula A has been proven by the absence of its cytotoxicity on a cell line of human epidermoid carcinoma. Citronella and carvacrol were the major compounds identified in the commercial essential oils of P. graveolens and O. majorana, respectively. Formula A was used in a field control trial for almost 8 months, during the tick infestation season (April to November, 2022). Fourteen naturally infested dogs were divided into two groups, each with seven dogs. One group received formula A spraying five times during an experiment that continued for 8 months, while the other group received treatment with commercially available malathion acaricide. The animals were sprayed on five occasions throughout the experiment (April, June, July, August, and September). The results showed a substantial percentage of effectiveness after the first application of formula A with a 99.3% reduction in tick count at day 28 post-application (PA). In the case of severe infestation 60 days after the first application of formula A (more than 180 ticks per dog), the second application was done, achieving an efficacy of 54.9% at day 3 PA, so an emergency spray was done at day 5 PA to combat the rest of the tick infestation, achieving efficacy of 99% after 3 days. Consequently, a regular spray (third, fourth, and fifth application) was done every 35 days. This regular spray revealed 100% effectiveness at 14 days PA. Biochemical parameters of treated dogs were evaluated to confirm the safety of formula A. Creatinine, ALT, and albumin of the dogs treated with formula A were within the normal range of dogs, while urea and AST were higher than the normal range. In conclusion, formula A can safely treat R. sanguineus s.l. infestations in dogs with regular application every 5 weeks.

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

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

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.

The prevalence and molecular detection of bovine cysticercosis and its impact on slaughtered cattle in Egypt.

Cysticercus bovis is the metacestode of the commonly defined foodborne cestode, Taenia saginata. It infects heart, masseter muscles and other muscular sites of cattle causing bovine cysticercosis. So, a retrospective study using archival documents from authorized agencies is done during the period of 2018-2020 with estimating economic losses. Cysts were collected from municipal Beni-Suef and Basatin abattoirs. Molecular approach including cPCR and sequencing analysis is used to prove the species. The prevalence was 0.34% in Beni-Suef abattoir and 5.70% in Basatin abattoir including heads, hearts or all carcasses condemned. Among 27 provinces surveyed, the highest prevalence was recorded in Aswan followed by Cairo, Suez and Red Sea. Moreover, 19 provinces (Cairo, Alexandria, Suez, Dakahlia, Sharkia, Qalyobia, Kafrelsheikh, Gharbia, Menofiya, Ismailia, Giza, Beni-Suef, Assiut, Sohag, Qena, Aswan, Red Sea, South Sinai and El-Wadi El-Gadid) recorded bovine cysticercosis in 2018, 2019 and 2020. On the other hand, Luxor and North Sinai had no infections. The percentages of condemnation among slaughtered cattle were 1.38, 1.49 and 0.87% in 2018, 2019 and 2020, respectively. Condemnations significantly varied among north, middle and south districts of Egypt, and also varied annually. Molecularly, a diagnostic band at an amplicon size 253 bp targeting the COI gene specific for Cycticercus bovis was revealed. The obtained sequences showed 100% identity with the different Taenia saginata COI GB sequence isolates in many countries worldwide. The phylogenetic analysis method showed that the obtained sequences originating from the same clade of Taenia saginata GB isolates globally. Careful meat inspection as well as strict hygienic measures is recommended for both veterinarians and public.

Detection of unusual Cryptosporidium parvum subtype in patients with gastrointestinal cancer in Egypt.

While the importance of cryptosporidiosis in immunocompromised persons is well known, the prevalence of Cryptosporidium spp. in cancer patients is not clear. The current study was designed to assess the occurrence and genetic characteristics of Cryptosporidium spp. in patients with gastrointestinal (GI) cancer in Egypt. Stool samples were collected from 100 patients with GI malignancies and 20 healthy individuals without any GI manifestations (control group). They were screened by microscopy and the immunochromatographic RIDA®QUICK Cryptosporidium kit. Subtyping of Cryptosporidium spp. was conducted by sequence analysis of the glycoprotein 60 (gp60) locus. Sociodemographic, environmental data and information on GI symptoms, cancer types, and clinical treatment were obtained via a questionnaire. By microscopy and RIDA®QUICK, only 7% (7/100) of GI cancer patients were positive for Cryptosporidium, compared with 40% (40/100) by gp60 nPCR. No positives were obtained from the control group. Male sex (P = 0.02) and younger age (P = 0.004) were major Cryptosporidium risk factors for infection. The occurrence of Cryptosporidium was also significantly more frequent (P = 0.003) in watery stool samples. Sequence analysis of the gp60 amplicons (~ 400 bp) identified a novel C. parvum subtype with nine TCA repeats and eleven ACATCA repeats. A formal subtype designation could not be made due to the short sequence length. More studies should be conducted to verify the common occurrence of this unusual C. parvum subtype and establish its genetic identity.

Safety and Efficacy of Pure and a Nanosuspension of D-limonene for Controlling Pigeon Lice.

This study investigated the safety and efficacy of two forms of D-limonene (DL) against Columbicola columbae (pigeon feather lice); pure and a nanoemulsion formulation (DLN). The cell cytotoxicity of the prepared forms of DL/DLN was investigated using skin cell lines. In vitro and ex vivo bioassays were applied on lice. The ex vivo bioassay was done on cut feathers containing lice eggs. The in vivo experiment was conducted on pigeons naturally infested by lice. The infested pigeons were treated with DL, DLN, or deltamethrin (D) as a positive control. Both forms of D-limonene were found to be safe when applied to the normal human skin fibroblast cell line, but DLN was toxic to skin cell carcinoma. The in vitro and ex vivo results of both DL and DLN forms were similar. All eggs treated with DL, DLN, and D failed to hatch (100%). The in vivo results showed complete elimination of lice 24 h post-treatment (PT), and biochemical analysis showed that the treated birds retained normal kidney and liver functions. Treated groups also showed improved productivity in the 4 months PT. In conclusion, DL and DLN are safe and effective in controlling feather lice infestation in pigeons and successful treatment encourages bird productivity.

D-limonene nanoemulsion: lousicidal activity, stability, and effect on the cuticle of Columbicola columbae.

The current study was conducted to investigate the efficacy and stability of D-limonene (DL) and its nanoemulsion (DLN) against pigeon feather lice (Columbicola columbae) and their mode of action. DL pure form and DLN were prepared and characterized freshly and after storage for 50 days. In vitro bioassay on live lice was conducted with different concentrations of DL, DLN, and deltamethrin (DM). The results revealed significant mortality rates in the DL-, DLN-, DM-treated groups when compared with the control (p < 0.05). The scanning electron micrographs of lice treated with DL and DLN revealed collapsed bodies with destruction in the cuticle of the mouthparts and damaged antennae. The 50 days stored DLN showed stability in their effectiveness when compared with the freshly prepared formulation. DL and DLN caused significant inhibition (p ≤ 0.05) in acetylcholinesterase activity (AchE). Malondialdehyde level (MDA) was significantly increased while glutathione was significantly decreased in DL- and DLN-treated lice. In conclusion, DL and DLN have significant lousicidal activities. DLN showed better stability than DL after storage for 50 days. In addition, the mode of action of DL may associate with its effect on the cuticle of the lice body, inhibition of AchE, and increasing oxidative stress in the treated lice.

Toxoplasma gondii and Neospora caninum Antibodies in Dogs and Cats from Egypt and Risk Factor Analysis.

BACKGROUND: Toxoplasma gondii and Neospora caninum are major protozoan parasites of worldwide distribution and significance in veterinary medicine and, for T. gondii, in public health. Cats and dogs, as final hosts for T. gondii and N. caninum, respectively, have a key function in environmental contamination with oocysts and, thus, in parasite transmission. Very little is known about the prevalence of T. gondii infections in dogs and cats in Egypt, and even less about the prevalence of N. caninum in the same hosts. METHODS: In the current study, 223 serum samples of both dogs (n = 172) and cats (n = 51) were investigated for specific antibodies to T. gondii and N. caninum using commercially available ELISAs. A risk factor analysis was conducted to identify factors associated with seropositivity. RESULTS & DISCUSSION: Exposure to T. gondii was reported in 23.3% of the dogs and in 9.8% of the cats, respectively. In addition, N. caninum-specific antibodies were recorded in 5.8% of dogs and in 3.4% of cats. A mixed infection was found in two dogs (1.2%) and in one cat (2%). Antibodies to T. gondii in dogs were significantly more frequent in dogs aged 3 years or more and in male German Shepherds. As this breed is often used as watchdogs and was the most sampled breed in Alexandria governorate, the purpose "watchdog" (compared to "stray" or "companion"), the male sex, and the governorate "Alexandria" also had a significantly higher seroprevalence for T. gondii. No factors associated with antibodies to N. caninum could be identified in dogs, and no significant factors were determined in cats for either T. gondii or N. caninum infection. Our study substantially adds to the knowledge of T. gondii infection in dogs and cats and presents data on N. caninum infection in cats for the first and in dogs in Egypt for the second time.

Economic losses, morpho-molecular identification, and identity of Fasciola species recovered from Egypt.

A retrospective study to estimate economic losses caused by livers condemnation, due to fascioliasis, of slaughtered cattle and buffaloes in Egypt during the period of 2016-2020, was done. Moreover, a morpho-molecular identification of collected liver flukes from slaughtered animals in municipal abattoirs was conducted. Livers of naturally infected carcasses were obtained from slaughtered animals in Beni-Suef, Cairo and Tanta provinces, Egypt during 2019-2020 for phenotypic characterization of recovered Fasciola species and molecular identification of collected worms using PCR targeting the ITS-1 region. Findings of the retrospective study revealed that percentages of livers condemnation of cattle and buffaloes ranged from 0.79 to 0.66% during the period from 2016 to 2020. The highest percentages were detected in the south Egypt (2.5-6.0%) with the highest economic losses (261850-616300 USD annually). Morphometrically, collected flukes categorized into Fasciola hepatica and Fasciola gigantica. No intermediate forms (Fasciola sp.-like) were detected. Sequencing analysis of ITS-1 PCR products showed that only Fasciola hepatica (26/34) and F. gigantica (8/34) isolates were found, with no intermediate forms, Fasciola sp.-like, could be identified. Currently, Fasciola hepatica was 100% identical with the Egyptian species (LC076196 and JF294998), French species (JF294999), and Iranian species (MF969009 and MK377150). Moreover, the obtained F. gigantica species showed 100% identity with Egyptian ones (LC076125, LC076108 and KX198619), Iranian (KF982047 and MF372919), and other GenBank specimens from Vietnam, Cameroon and India. In conclusion, South Egypt showed the highest economic losses due to fascioliasis, especially Aswan province. Fasciola hepatica was more common than F. gigantica, while the hybrid form was not detected.

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.

Tunable Polymeric Mixed Micellar Nanoassemblies of Lutrol F127/Gelucire 44/14 for Oral Delivery of Praziquantel: A Promising Nanovector against Hymenolepis nana in Experimentally-Infected Rats.

Hymenolepiasis represents a parasitic infection of common prevalence in pediatrics with intimidating impacts, particularly amongst immunocompromised patients. The present work aimed to snowball the curative outcomes of the current mainstay of hymenolepiasis chemotherapy, praziquantel (PRZ), through assembly of polymeric mixed micelles (PMMs). Such innovative nano-cargo could consolidate PRZ hydrosolubility, extend its circulation time and eventually upraise its bioavailability, thus accomplishing a nanoparadigm for hymenolepiasis tackling at lower dose levels. For consummating this goal, PRZ-PMMs were tailored via thin-film hydration technique integrating a binary system of Lutrol F127 and Gelucire 44/14. Box-Behnken design was planned for optimizing the nanoformulation variables employing Design-Expert® software. Also, in Hymenolepis nana-infected rats, the pharmacodynamics of the optimal micellar formulation versus the analogous crude PRZ suspension were scrutinized on the 1st and 3rd days after administration of a single oral dose (12.5 or 25 mg/kg). Moreover, in vitro ovicidal activity of the monitored formulations was estimated utilizing Fuchsin vital stain. Furthermore, the in vivo pharmacokinetics were assessed in rats. The optimum PRZ-PMMs disclosed conciliation between thermodynamic and kinetic stability, high entrapment efficiency (86.29%), spherical nanosized morphology (15.18 nm), and controlled-release characteristics over 24 h (78.22%). 1H NMR studies verified PRZ assimilation within the micellar core. Additionally, the in vivo results highlighted a significant boosted efficacy of PRZ-PMMs manifested by fecal eggs output and worm burden reduction, which was clearly evident at the lesser PRZ dose, besides a reversed effect for the intestinal histological disruptions. At 50 µg/mL, PRZ-PMMs increased the percent of non-viable eggs to 100% versus 47% for crude PRZ, whilst shell destruction and loss of embryo were only clear with the applied nano-cargo. Moreover, superior bioavailability by 3.43-fold with elongated residence time was measured for PRZ-PMMs compared to PRZ suspension. Practically, our results unravel the potential of PRZ-PMMs as an oral promising tolerable lower dose nanoplatform for more competent PRZ mass chemotherapy.

Assessment of the efficacy of thymol against Toxocara vitulorum in experimentally infected rats.

The effect of thymol and ivermectin on the development and embryonation of Toxocara vitulorum (T. vitulorum) eggs, as well as their migration in albino rats was investigated both in vitro and in vivo. A total of forty male albino rats were divided into four groups for an in vivo experiment. The first group was uninfected; the second group was infected but left untreated; the third group was infected and received thymol at a dose of 40 mg/kg; and the fourth group was infected and received ivermectin (0.2 mg/kg). In vitro, thymol inhibited the development of Toxocara larvae within the eggs. However, ivermectin, produced inconsistent results. The in vivo results indicated that the recovery rates of Toxocara larvae from the liver and lungs on day 7 post-infection were significantly lower in the thymol or ivermectin-treated groups than in the infected untreated control. Albumin levels were significantly increased in the thymol-treated group as compared to the positive control and ivermectin groups. Nitric oxide, IL-4, and IFN- levels in the serum of the thymol or ivermectin-treated groups were significantly lower than that of the positive control group. Histopathological examination demonstrated that thymol and ivermectin were effective in reducing larval load, reducing the number and size of granulomas in the absence of larvae, and improving tissue architecture. The current study concluded that thymol possessed anti-Toxocara activity in a rat model. Additionally, thymol possessed ovicidal properties and may be used as a disinfectant.

Acaricidal activity of Foeniculum vulgare against Rhipicephalus annulatus is mainly dependent on its constituent from trans-anethone.

Globally, the economic losses due to hard ticks infestation and the control of the associated diseases have been calculated at USD $13.9-18.7 billion per year. The economic impact is related to its direct damage to the skins, blood loss, anemia, severe immunological reactions and indirect losses that related to the effects of hemoparasites, cost of treatment for clinical cases and expenses incurred in the control of ticks. The current study evaluated the acaricidal activities of fennel Foeniculum vulgare essential oil and its main components; trans-anethole and fenchone; against R. annulatus. GC-MS analysis revealed that this oil contained 16 components representing 99.9% of the total identified compounds with E-anethole being the predominant component(64.29%), followed by fenchone (9.94%). The fennel oil and trans-anethole showed significant acaricidal activities. The LC50 of the fennel oil was attained at concentrations of 12.96% for adult ticks and 1.75% for tick larvae meanwhile the LC50 of trans-anethole was reached at concentrations of 2.36% for adult tick and 0.56% for tick larvae. On the contrary, fenchone showed no any significant adulticidal activities and its LC50 attained at a concentration of 9.11% for tick larvae. Regarding repellence activities, trans-anethole achieved 100% repellency at the concentration of 10% while fennel showed 86% repellency at the same concentration. Fenchone showed no repellency effect. Treatment of larvae with fennel, trans-anethole, and fenchone LC50 concentrations significantly inhibited the acetylcholinesterase activity. Meanwhile, glutathione s-transferase activity was significantly decreased in fennel treated larvae but no significant effect was found in the larvae of trans-anethole and fenchone groups. These results indicate that the acaricide effect of fennel oil may attributed to its high content of trans-anethole. This was supported by potent adulticidal, larvicidal, and repellency effects of trans-anethole against Rhipeciphalus annulatus tick and therefore it could be included in the list of acaricide of plant origin.

Remarks on Eimeria labbeana infection in domestic pigeons "Columbia livia domestica".

The purpose of this study was to determine the course of infection in pigeons with Eimeria labbeana. Thirty-five squabs (4-6 weeks old) were brought from the local poultry market and examined for coccidial infection for 7 days to ensure they were coccidia free. A negative control group of five squabs was used, and thirty squabs were infected orally with 2.5 × 104 sporulated E. labbeana oocysts. From day 1-8 post-infection (PI), three squabs were scarified daily to track the endogenous stages in the intestinal tissue. Furthermore, six squabs were preserved to track the patent period and calculate daily oocyst shedding. The parasite stages were differentiated using paraffin-embedded intestinal tissues that were sectioned and stained. On day 5 PI, the infected squabs had greenish watery diarrhea, weakness, rough feathers, and decreased food intake. The pre-patent and patent durations were six and fourteen days PI, respectively. The shedding of oocysts began on day 6 PI and peaked on day 8 PI. In the duodenum and jejunum of the small intestine, histopathological investigation indicated the presence of three schizont stages, macro- and micro-gametes, and oocysts. To the best of our knowledge, this is the first study in Egypt to explore the course of E. labbeana infection in domestic pigeons.

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.

Haemonchus contortus Susceptibility and Resistance to Anthelmintics in Naturally Infected Egyptian Sheep.

PURPOSE: Susceptibility and resistance of Haemonchus contortus to anthelmintic drugs, including ivermectin, levamisole, and albendazole in naturally infected sheep were investigated. METHODS: Three sets of assays were conducted to detect drug efficacy. Firstly, in vivo estimation of drug resistance to H. contortus was explored in 80 sheep naturally infected with H. contortus. Sheep were divided into four equal groups (20 sheep for each group): the first group was treated with albendazole (5.00 mg/kg BW), the second with levamisole (7.50 mg/kg BW), the third with ivermectin (0.20 mg/kg BW), and the fourth group served as the untreated control. Fecal egg reduction test (FERT) was done at days 7 and 14 after treatment. Secondly, for in vitro egg hatching assay (EHA), H. contortus eggs from naturally infected sheep were collected and treated with 0.0002, 0.002, 0.02, 0.2, and 2.0 µg/mL albendazole. Thirdly, molecular detection of the albendazole resistance gene in adult male H. contortus worms and larvae from infected sheep was carried out using allele-specific PCR. RESULTS: The FECRT results showed that the drug efficacy was 86.84% for albendazole and 100% for both levamisole and ivermectin. The result of EHA showed that eggs did not hatch at 2.0 µg/mL albendazole concentration. Molecular findings showed two forms, including H. contortus homozygous susceptible (SS) and heterozygous (RS) of "ß-tubulin" gene at 200 sites, which were recorded in both single male worms and larvae. CONCLUSION: H. contortus, which was susceptible to levamisole and ivermectin, had developed resistance to albendazole.

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.

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.

Molecular Detection, Phylogenetic Analysis, and Genetic Diversity of Theileria annulata, Babesia bigemina, and Anaplasma marginale in Cattle in Three Districts of Egypt.

BACKGROUND: Under the poor hygienic conditions, tick-borne pathogens cause severe economic losses to the cattle industry. PURPOSE: The current study investigated the presence of Theileria annulata, Babesia bigemina, and Anaplasma marginale, the most relevant tick-borne pathogens in cattle, in 3 provinces of Egypt utilizing species-specific PCR assays. METHODS: PCR was conducted, on bovine blood specimens, using primers targeting the T. annulata merozoite-piroplasm surface antigen (Tams1, 768 bp), A. marginale major surface protein-1b gene (msp1b, 265 bp), and B. bigemina small subunit ribosomal RNA gene (SSrRNA, 543 bp). RESULTS: PCR findings revealed overall prevalences of T. annulata, B. bigemina, and A. marginale as 22.0% (33/150), 19.33% (29/150), and 10.6% (16/150), respectively. The co-infection with two or three pathogens was detected in 20.0% (30/150) of examined specimens. Sequence analyses indicated that T. annulata and A. marginale varied from those of corresponding GenBank sequences revealing percent identities ranging from 90.68 to 97.75% and from 94.98 to 98.63%, respectively. On the other hand, the obtained B. bigemina sequences showed a high similarity with those previously reported in GenBank with a percent identity ranging from 98.85 to 100%. CONCLUSION: T. annulata was the most prevalent tick-borne pathogen in examined bovine specimens. The genetic diversity of markers used for identification of T. annulata and A. marginale should be highly considered.