High potency of linalool-zinc oxide nanocomposite as a new agent for cystic echinococcosis treatment.

This current in vitro, ex vivo, and in vivo research aims to evaluate and analyze the linalool-zinc oxide nanocomposite (Lin-ZNP) for treating cystic echinococcosis. Lin-ZNP was synthesized using an ethanolic solution of polyvinyl alcohol. The protoscolicidal effects of Lin-ZNP were tested on hydatid cyst protoscoleces (PTS) in both in vitro and ex vivo by eosin exclusion test. The study also examined the impact on caspase-3 gene expression and the external structure of PTS. The in vivo effect was measured by examining hydatid cysts' quantity, dimensions, and weight in mice intraperitoneally infected with 0.5 mL of PTS solution containing 1,000 PTS. The antioxidant and inflammatory cytokine gene expression levels were examined using real-time PCR. Lin-ZNP significantly (P < 0.001) killed the PTS in both in vitro and ex vivo in a dose- and time-dependent manner. The treated PTS exhibited creases and protrusions as a result of bleb formation and upregulation in the gene expression of caspase-3. Upon treatment with Lin-ZNP, there was a significant (P < 0.001) reduction in the number, diameter, and weight of the hydatid cysts. Treatment with Lin-ZNP nanocomposite led to a significant increase in the expression of antioxidant genes and a notable decrease in oxidative stress markers, and the expression levels of IL-4 and IL-10. Lin-ZNP has the potential to act as a scolicidal agent and demonstrates promise in controlling hydatid cysts in a mouse model, attributed to its antioxidant and anti-inflammatory properties. However, additional studies in clinical trials are needed to confirm the use of Lin-ZNP for treating hydatidosis.

Linalool-zinc oxide nanocomposite controls Toxoplasma gondii infection through inhibiting inflammation, oxidative stress, and pathogenicity.

The present in vitro and in vivo study aimed to fabricate and characterize linalool-zinc oxide nanoparticles (Lin-ZNP) and evaluate their effectiveness against Toxoplasma gondii infection in terms of inflammation, oxidative stress, and pathogenicity. Lin-ZNP was synthesized using an ethanolic solution of polyvinyl alcohol. The anti-Toxoplasma and cytotoxicity activities of Lin-ZNP were investigated, along with its effects on nitric oxide (NO) production, caspase-3 activity, and pro-inflammatory genes. After treating T. gondii-infected mice with Lin-ZNP for 14 days, the number and size of tissue cysts, antioxidant potential, pro-inflammatory cytokines, and T. gondii pathogenicity-related genes were evaluated by real-time polymerase chain reaction and Western blot analysis. The Lin-ZNP composite showed a reduced tendency with an average size of 105 nm. Lin-ZNP significantly reduced the viability of tachyzoites. The obtained selectivity index higher than 10, indicating high specificity for parasites with low cytotoxicity to normal cells. The Lin-ZNP significantly (p < 0.05) increased the production of NO, caspase-3 activity, and the expression levels of pro-inflammatory genes. Lin-ZNP significantly (p < 0.001) decreased the size and number of tissue cysts and caused a significant reduction in the level of malondialdehyde and a considerable increase (p < 0.001) in antioxidant enzymes and their expression genes. Lin-ZNP significantly downregulated both mRNA and protein expression of the inflammation-related markers associated with the TLRs/NF-κB pathway. The expression levels of the T. gondii pathogenicity-related genes were significantly downregulated (p < 0.05). The recent survey indicated that Lin-ZNP manages T. gondii infection by its antioxidant activity and inhibiting the TLRs/NF-κB pathway without toxicity in mice.

Anti-Cancer Effects of Queen Bee Acid (10-Hydroxy-2-Decenoic Acid) and Its Cellular Mechanisms against Human Hepatoma Cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer that occurs in hepatocytes. Although many chemical drugs, e.g., cisplatin, methotrexate, taxis, and doxorubicin are used to treat HCC, there have been numerous reports related to the side effects of these drugs (e.g., emerging drug resistance, bone marrow failure, and gastrointestinal disorders). These issues led scientists to search for the novel anti-cancer drugs, mainly in natural products with greater efficiency and less toxicity. The current survey was intended to assess the anti-cancer effects of queen bee acid (10-Hydroxy-2-Decenoic Acid, 10-HDA) and its cellular mechanisms against the human hepatoma cell line HepG2. MATERIALS AND METHODS: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to evaluate the effect of 10-HDA on the viability of HepG2 cells. The initial and late apoptosis in the HepG2 cells treated with 10-HDA were assessed by the Annexin-V (AV) assay. The level of the gene and protein expression of some apoptosis genes (e.g., caspase-3, Bcl-2-associated X protein (BAX), and B-cell lymphoma protein 2 (Bcl-2)), Poly (ADP-ribose) polymerases (PARP), and miRNA-34a (miR-34a), were measured by real-time PCR and Western blot. RESULTS: The obtained findings revealed that HepG2 cell viability was markedly reduced (p < 0.01) following exposure to 10-HDA in a dose-dependent matter. The calculated half maximal cytotoxic concentration (CC50) value of 10-HDA was 59.6 µg/mL for HepG2 cells, while this value for normal THLE-3 cells was 106.4 µg/mL. We found that 10-HDA markedly elevated (p < 0.01) the percentage of necrotic and apoptotic cells from 0.94 to 9.7 and 27.6%, respectively. The real-time PCR results showed that the expression levels of the caspase-3, Bax, and miR-34a genes were significantly (p < 0.001) elevated. Contrary to these results, a significant (p < 0.01) reduction in the expression level of the Bcl2 gene was observed. The levels of protein expression of Caspase-3, PARP, and Bax were markedly elevated following exposure of HepG2 cells to 10-HDA at » CC50, ½ CC50, and CC50. The level of protein expression of Bcl-2 was markedly reduced following exposure of HepG2 cells to 10-HDA at » CC50, ½ CC50, and CC50 (p < 0.01). CONCLUSION: The current results confirmed the potent in vitro cytotoxic effects of 10-HDA on HepG2 cells with no significant cytotoxic effects on normal cells. Although its mechanisms of action have not been fully studied, the induction of apoptosis via different pathways was determined as one of the principle mechanisms of action of 10-HDA against HepG2 cells. Nevertheless, additional surveys must be performed to clearly understand the mechanisms of action and safety of this fatty acid.

Structure-based in silico design and in vitro acaricidal activity assessment of Acacia nilotica and Psidium guajava extracts against Sarcoptes scabiei var. cuniculi.

Infestation by Sarcoptes scabiei var. cuniculi mite causes scabies in humans and mange in animals. Alternative methods for developing environmentally friendly and effective plant-based acaricides are now a priority. The purpose of this research was the in silico design and in vitro evaluation of the efficacy of ethanol extracts of Acacia nilotica and Psidium guajava plant leaves against S. scabiei. Chem-Draw ultra-software (v. 12.0.2.1076.2010) was used to draw 36 distinct compounds from these plants that were employed as ligands in docking tests against S. scabiei Aspartic protease (SsAP). With docking scores of - 6.50993 and - 6.16359, respectively, clionasterol (PubChem CID 457801) and mangiferin (PubChem CID 5281647) from A. nilotica inhibited the targeted protein SsAP, while only beta-sitosterol (PubChem CID 222284) from P. guajava interacted with the SsAP active site with a docking score of - 6.20532. Mortality in contact bioassay at concentrations of 0.25, 0.5, 1.0, and 2.0 g/ml was determined to calculate median lethal time (LT50) and median lethal concentration (LC50) values. Acacia nilotica extract had an LC50 value of 0.218 g/ml compared to P. guajava extract, which had an LC50 value of 0.829 g/ml at 6 h. These results suggest that A. nilotica extract is more effective in killing mites, and these plants may have novel acaricidal properties against S. scabiei. Further research should focus on A. nilotica as a potential substitute for clinically available acaricides against resistant mites.

Molecular characterization of Leishmania species from stray dogs and human patients in Saudi Arabia.

Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. To assess the frequency and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania infection were examined for CL by a nested polymerase chain reaction (nPCR). Seven (25.9%) out of 27 human patients scored positive for Leishmania spp. (i.e., L. major in five patients from Riyadh and L. tropica in two patients from Al-Qaseem). Out of 311 dogs, five (1.6%) were infected by L. tropica. Data herein presented demonstrate the occurrence of L. tropica in dogs and humans in Saudi Arabia, as well as the occurrence of L. major in humans.

Chemical Composition, Apoptotic Activity, and Antiparasitic Effects of Ferula macrecolea Essential Oil against Echinococcus granulosus Protoscoleces.

BACKGROUND: Today, the present protoscolicidals used to minimize the serious risks during hydatid cyst surgery are not completely safe and have various adverse side effects. The present study aimed to evaluate the chemical composition and apoptotic activity of Ferula macrecolea essential oil (FMEO) as well as its in vitro and ex vivo protoscolicidal effects against hydatid cyst protoscoleces. METHODS: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of FMEO. Protoscoleces of hydatid cysts were collected from liver fertile hydatid cysts of infected sheep and were then treated with various concentrations of the essential oil (75, 150, and 300 µL/mL) for 5-60 min in vitro and ex vivo. Then, by using the eosin exclusion test, the viability of the protoscoleces was studied. The caspase-3-like activity of the FMEO-treated protoscoleces was also evaluated through the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: According to GC/MS, the main constituents of the essential oil were terpinolene (77.72%), n-nonanal (4.47%), and linalool (4.35%), respectively. In vitro, the maximum protoscolicidal activity of FMEO was observed at the concentrations of 150 and 300 µL/mL, such that 100% of the protoscoleces were killed after 30 and 20 min of exposure, respectively. Based on the obtained findings, the results demonstrate that FMEO required a longer time to kill protoscoleces ex vivo; after 12 min of exposure to FMEO, only 13.4% of the protoscoleces remained alive. After 48 h of the treatment of protoscoleces, FMEO, in a dose-dependent manner and at doses of 75, 150, and 300 µL/mL, induced the activation of the caspase enzyme by 24.3, 35.3, and 48.3%, respectively. CONCLUSIONS: Our findings demonstrate the potent protoscolicidal effects of FMEO in vitro and ex vivo; however, further studies are required to assess the safety and the efficiency of FMEO as a promising scolicidal agent in a preclinical model and clinical setting.

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.

Non-invasive surveillance of Plasmodium infection by real-time PCR analysis of ethanol preserved faeces from Ugandan school children with intestinal schistosomiasis.

BACKGROUND: As part of ongoing co-surveillance of intestinal schistosomiasis and malaria in Ugandan school children, a non-invasive detection method for amplification of Plasmodium DNA using real-time (rt)PCR analysis of ethanol preserved faeces (EPF) was assessed. For diagnostic tabulations, results were compared to rtPCR analysis of dried blood spots (DBS) and field-based point-of-care (POC) rapid diagnostic tests (RDTs). METHODS: A total of 247 school children from 5 primary schools along the shoreline of Lake Albert were examined with matched EPF and DBS obtained. Mean prevalence and prevalence by school was calculated by detection of Plasmodium DNA by rtPCR using a 18S rDNA Taqman® probe. Diagnostic sensitivity, specificity, positive and negative predictive values were tabulated and compared against RDTs. RESULTS: By rtPCR of EPF and DBS, 158 (63.9%; 95% CI 57.8-69.7) and 198 (80.1%, 95% CI 74.7-84.6) children were positive for Plasmodium spp. By RDT, 138 (55.8%; 95% CI 49.6-61.9) and 45 (18.2%; 95% CI 13.9-23.5) children were positive for Plasmodium falciparum, and with non-P. falciparum co-infections, respectively. Using RDT results as a convenient field-based reference, the sensitivity of rtPCR of EPF and DBS was 73.1% (95% CI 65.2-79.8) and 94.2% (95% CI 88.9-97.0) while specificity was 47.7% (95% CI 38.5-57.0) and 37.6% (95% CI 29.0-46.9), respectively. With one exception, school prevalence estimated by analysis of EPF was higher than that by RDT. Positive and negative predictive values were compared and discussed. CONCLUSIONS: In this high transmission setting, EPF sampling with rtPCR analysis has satisfactory diagnostic performance in estimation of mean prevalence and prevalence by school upon direct comparison with POC-RDTs. Although analysis of EPF was judged inferior to that of DBS, it permits an alternative non-invasive sampling regime that could be implemented alongside general monitoring and surveillance for other faecal parasites. EPF analysis may also have future value in passive surveillance of low transmission settings.

A comprehensive evaluation and first molecular report of Theileria ovis infection in small ruminants in Saudi Arabia.

A total of 1000 clinically healthy small ruminants comprising 500 sheep and 500 goats from five districts within Riyadh Province in Saudi Arabia were investigated by routine Giemsa staining for hematozoan parasites. Out of these, 100 sheep and 95 goat samples were investigated by PCR using three pairs of hemoprotozoan-specific primers. Based on microscopic examination, 33.2% of sheep and 25.2% of goats were found infected with hemoprotozoan parasites, while PCR detected hematozoan infection in 46% of sheep and 33.7% of goats. Extensive molecular characterization of hematozoan infection using six pairs of species-specific primers revealed the dominance of Theileria ovis, rather than any other species, which is recorded for the first time in small ruminants in Saudi Arabia. Prevalence of T. ovis in sheep and goats was found to be the highest in Riyadh (32, 48%) followed by AL-Kharj (31, 35%), Ad-Dawadimi (31, 33%), AL-Majmaah (15, 27%), and Rumah (17, 23%). The highest parasite prevalence was recorded in the 3 years of age and > 4 years of age ruminants, while the lowest prevalence was recorded in < 1 year of age ruminants. No noticeable differences in parasite prevalence between male or female ruminants were recorded. Partial sequencing of 18S rRNA gene revealed the infection of the studied ruminants with four new isolates of T. ovis. Further characterization of the pathogenicity and the clinical effects of these T. ovis isolates in sheep and goats is highly needed. The current results can be helpful in protecting and improving livestock industry in the countries that depend on a high number of small ruminants.

Unveiling of the antileishmanial activities of Linalool loaded zinc oxide nanocomposite through its potent antioxidant and immunomodulatory effects.

This study aimed to produce linalool loaded zinc oxide nanocomposite (LZNPs) and assess its in vitro and in vivo antileishmanial effects against Leishmania major. LZNPs was produced through the synthesis of an ethanolic solution containing polyvinyl alcohol. The average size of LZNPs was determined to be 105 nm. The findings indicated that LZNPs displayed significant (p < 0.01) antileishmanial effects on promastigotes and amastigotes. Following exposure of promastigotes to LZNPs, there was a notable rise in the percentage of early and late apoptotic cells from 9.0 to 57.2 %. The gene expression levels of iNOS, IFN-γ, and TNF-α in macrophages were upregulated in a dose-dependent approach following exposure to LZNPs. LZNPs alone and in conjunction with glucantime (Glu) resulted in a reduction in the diameter and parasite load of CL lesions in infected mice. Treatment of the CL-infected mice with LZNPs at 25 and 50 mg/kg mainly in combination with Glu-reduced the tissue level of malondialdehyde (MDA), increased both gene and protein expression of the antioxidant enzymes as well as raised the expression level of IFN-γ and IL-12 cytokines, whereas caused a significant reduction in the expression level of IL-4. The present study shows that LZNPs has potent antileishmanial effects and controls CL in a mice model through its antioxidant and immunomodulatory properties. Further investigation, especially in clinical trials, could explore the potential use of this nanocomposite in managing and treating CL.

High Efficacy of Green Synthesized Silver Nanoparticles for Treatment of Toxoplasma Gondii Infection Through Their Immunomodulatory, Anti-Inflammatory, and Antioxidant Potency.

The present experimental survey designed to green synthesis, characterization, as well as in vitro and in vivo anti-Toxplasma gondii activity of silver nanoparticles (SLN) green synthesized by Lupinus arcticus extract. SLN were green synthesized based on the reducing by L. arcticus extract through the precipitation technique. In vitro lethal effects of SLN on T. gondii tachyzoites, infectivity rate, parasites inside of the human macrophage cells (THP-1 cells), nitric oxide (NO) triggering, and iNOS and interferon gamma (IFN-γ) expression genes were evaluated. In vivo, after establishment of toxoplasmosis in BALB/c mice via T. gondii ME49 strain, mice received SLN at 10 and 20 mg/kg/day alone and combined to pyrimethamine at 5 mg/kg for 14 days. SLN exhibited a spherical form with a size ranging from 25 to 90 nm. The 50% inhibitory concentration (IC50) value of SLN and pyrimethamine against tachyzoites was 29.1 and 25.7 µg/mL, respectively. While, the 50% cytotoxic concentration (CC50) value of SLN and pyrimethamine against THP-1 cells was 412.3 µg/mL and 269.5 µg/mL, respectively. SLN in combined with pyrimethamine obviously (p < 0.05) decreased the number and size of the T. gondii cysts in the infected mice. The level of NO, iNOS and IFN-γ genes was obviously (p < 0.001) upregulated. SLN obviously (p < 0.05) decreased the liver level of oxidative stress and increased the level of antioxidant factors. The findings displayed the promising beneficial effects of SLN mainly in combination with current synthetic drugs against latent T. gondii infection in mice. But we need more experiments to approve these findings, clarifying all possible mechanisms, and its efficiency in clinical phases.

Zinc oxide nanoparticles loaded with linalool as a potential control agent of malaria infection.

This research aimed to produce and analyze zinc oxide nanoparticles (ZNPs) loaded with linalool (LZNPs), and to evaluate their in vitro and in vivo efficacy through targeting the inflammation and oxidative stress. LZNPs were synthesized using an ethanolic solution of polyvinyl alcohol. The Malstat technique was used to evaluate the effectiveness of LZNPs against both sensitive and resistant strains of Plasmosium falciparum. In vivo effects of ZNPs and LZNPs on parasite growth suppression, survival rate, oxidative stress markers, antioxidant genes, and gene and protein levels of inflammatory cytokines were evaluated by Real-time PCR and Western blot techniques. The results indicated that LZNPs demonstrated noteworthy (P<0.001) antiplasmodial activity against both susceptible and resistant strains of P. falciparum. P. berghei NK65 strain-infected mice treated with the ZNPs and LZNPs at doses of 5-15 mg/kg notably (p<0.001) increased the survival rates and parasite growth suppression. LZNPs at 5-15 mg/kg demonstrated a significant (p<0.001) decrease in oxidative stress markers, increased the expression level of antioxidant genes, and reduced the gene and protein expression level of inflammatory cytokines. The current experimental study demonstrated the potent in vitro antiplasmodial activity of LZNPs against chloroquine-resistant and sensitive strains of P. falciparum compared to ZNPs alone. Additionally, the study identified the potential benefits of this nanocomposite in suppressing the parasite and extending the survival rate in mice infected with P. berghei by targeting inflammation and oxidative stress. It also showed minimal toxicity in liver and kidney function in healthy mice. Nevertheless, further research is essential to elucidate the comprehensive mechanisms and practical effectiveness of LZNPs.

Molecular prevalence of vector borne bacterial pathogens in the blood samples of wild rodent species trapped from Saudi Arabia.

Order Rodentia is the most speciose among mammals and the members of this order are known to host more than 60 zoonotic diseases and rodents are a potential health threat to humans. This study was designed to report the molecular prevalence and phylogenetic evaluation of various blood borne bacterial pathogens (Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma marginale and Bartonella spp.) in the blood samples of four wild rodent species [Meriones rex (N = 27), Acomys dimidiatus (N = 18), Myomys yemeni (N = 6) and Rattus rattus (N = 3)] that were trapped during August till October 2020 from Al Makhwah governorate in Saudi Arabia. Results revealed by 9/54 (16.6%) rodents amplified Msp4 gene and 2/54 (3.7%) rodents amplified rpoB gene of Anaplasma ovis and Bartonella spp. respectively. Anaplasma phagocytophilum and Anaplasma marginale were not detected among enrolled rodent species. Meriones rex was the most highly infected rodent species. DNA sequencing and BLAST analysis confirmed the presence of Anaplasma ovis and the Bartonella koehlerae in rodent blood samples. Phylogenetic analysis of both pathogens showed that Saudi isolates were clustered together and were closely related to isolates that were reported from worldwide countries. Risk factor analysis revealed that prevalence of both bacterial pathogens was not restricted to a particular rodent species or a rodent sex (P > 0.05). In conclusion, we are reporting for the very first time that Saudi rodents are infected with Anaplasma ovis and rodents can be infected with Bartonella koehlerae. Similar studies at large scale are recommended in all those areas of Saudi Arabia that are unexplored for the incidence and prevalence of bacterial pathogens among the rodents that are living near human dwellings in order to prevent bacterial infections in local people as well as in livestock.

Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants. The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively.  Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries.  Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Immunomodulatory, Antioxidant, and Anti-Inflammatory Activities of Green Synthesized Copper Nanoparticles for Treatment of Chronic Toxoplasma gondii Infection.

BACKGROUND: Nowadays, interest in the use of nanotechnology for medical purposes is increasing. The current experimental investigation is planned for the green synthesis, characterization, and efficacy of copper nanoparticles (CLN) against chronic Toxoplasma gondii infection. METHODS: Green synthesis of CNP was performed using the Lupinus arcticus extract via the precipitation method. The effects of CNP on tachyzoites, infectivity rate, parasites inside THP-1 cells, nitric oxide (NO) triggering, iNOS, and IFN-γ expression genes were evaluated. Following toxoplasmosis in BALB/c mice via the T. gondii ME49 strain, mice received CNP at 5 and 10 mg/kg/day alone and combined with pyrimethamine (PYM) at 5 mg/kg for two weeks. CNP's in vivo effects were evaluated by analyzing the load and size of cysts, oxidant/antioxidant enzymes, and bradyzoite surface antigen 1 (BAG1) expression gene levels. RESULTS: CNP displayed a circular shape ranging from 10 to 85 nm. The IC50 value of CNP and PYM against tachyzoites was 37.2 and 25.7 µg/mL, respectively, whereas the CC50 value of CNP and pyrimethamine against THP-1 cells was 491.4 µg/mL and 269.5 µg/mL, respectively. The rate of infectivity and parasite load among THP-1 cells exposed to CNP was obviously reduced (p < 0.05). CNP at the doses of 5 and 10 mg/kg predominantly along with PYM evidently (p < 0.05) reduced the number and size of the T. gondii cysts in the infected mice. The levels of NO, iNOS, and IFN-γ genes were remarkably (p < 0.001) boosted compared with the cells without treatment. CNP at the doses of 10 and 20 mg/kg drastically (p < 0.05) reduced the oxidative stress markers in the infected mice, whereas CNP significantly elevated the level of antioxidant factors. CNP also revealed no toxicity in the liver and kidney at the tested doses in healthy mice. CONCLUSIONS: Our experimental study reported the beneficial effects of CNP principally along with existing chemical drugs against latent toxoplasmosis in mice, whereas the possible action mechanisms of CNP are controlling oxidative stress, refining antioxidant enzymes, and increasing the production of immunomodulatory cytokines with no toxicity to the function of vital organs. But, additional trials are required to confirm these results, as well as to clarify the accurate mechanisms and their toxicity.

Potent In Vitro and In Vivo Effects of Stachys lavandulifolia Methanolic Extract against Toxoplasma gondii Infection.

The present study aimed to evaluate the in vitro, in vivo, and safety of Stachys lavandulifolia Vahl. methanolic extract (SLME) against acute toxoplasmosis caused by Toxoplasma gondii RH strain in mice. METHODS: MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the in vitro effect of the SLME on T. gondii tachyzoites. Totally, 72 male BALB/c mice (40 mice for in vivo evaluation of SLME and 32 mice for its toxicity effects on liver and kidney serum enzymes) were used for the present investigation. At first, 40 mice were orally pre-treated with the SLME at doses of 25, 50, and 75 mg/kg/day for two weeks. Mice were checked daily, and the rate of survival and the mean number of tachyzoites were recorded. Liver lipid peroxidation (LPO) and nitric oxide (NO) levels, the effects on kidney and liver function, as well as the expression level of the proinflammatory cytokines such as interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), were studied by the quantitative real-time PCR. Flow cytometry analysis was performed on the effects of SLME on the detection of apoptotic and necrotic cells in T. gondii tachyzoites. RESULTS: The SLME at the concentrations 75 and 150 µg/mL completely killed the tachyzoites after 2 hr of incubation. SLME at 25, 50, and 75 mg/kg/day increased the survival rate of infected mice by the sixth, seventh, and eighth days, respectively. SLME also significantly (p < 0.05) decreased the LPO and NO levels and upregulated the IL-1ß and IFN-γ mRNA gene expression levels, whereas no considerable change was observed in the serum level of kidney and liver enzymes. Flow cytometry analysis revealed the prompted early and late apoptosis after exposure to T. gondii tachyzoites with various concentrations of SLME. CONCLUSION: We found the relevant in vitro anti-Toxoplasma effects of SLME against T. gondii. Moreover, the results confirmed the promising in vivo prophylactic effects of SLME. SLME provokes the innate immune system, induces apoptosis, modulates the proinflammatory cytokines, and inhibits hepatic injury in infected mice. With all these descriptions, further surveys are required to support these findings and elucidate this plant's possible mechanisms of action.

Relationship between KIR genotypes and HLA-ligands with SARS-CoV-2 infection in the Saudi population.

Aim: To ascertain whether killer cell immunoglobulin-like receptors (KIR) genes polymorphisms and HLA-I ligands are associated with COVID-19 in Saudi Arabia. Methods: Eighty-seven COVID-19 patients who tested positive for SARS-CoV-2 and one hundred and fourteen healthy controls were enrolled in this study for genotyping of the 16 KIR genes, HLA-C1 and -C2 allotypes and HLA-G 14-bp indels polymorphisms using the sequence specific primer polymerase chain reaction (SSP-PCR) method. KIR genotype frequency differences and combination KIR-HLA-C ligand were tested for significance. Results: Framework genes KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP2 were present in all individuals. The frequencies of KIR2DL2 and KIR2D4 were higher in COVID-19 positive patients than in healthy individuals. The frequencies of the combination KIR2DL2-HLA-C2 was also significantly higher in patients affected by COVID-19 compared with healthy controls. Conclusion: It was found that the inhibitory KIR2DL2 gene in isolation or combined with its HLA-C2 ligand could be associated with susceptibility to COVID-19 in the Saudi population.

Antiparasitic effects of Elettaria cardamomum L. essential oil and its main compounds, 1-8 Cineole alone and in combination with albendazole against Echinococcus granulosus protoscoleces.

Background: The present investigation aims to determine the chemical structure and protoscolicidal effects of Elettaria cardamomum L. essential oil (ECEO) and its main compounds 1-8 cineole alone and along with albendazole (ALZ) against Echinococcus granulosus protoscoleces in vitro and ex vivo. We also decided to evaluate some cellular mechanisms such as the apoptotic activity and the permeability of plasma membrane of protoscoleces treated with ECEO and 1-8 cineole. Methods: Hydatid cyst protoscoleces were divided into seven groups including protoscoleces treated with ECEO 50 µl/mL (T1), protoscoleces treated with ECEO 100 µl/mL (T2), protoscoleces treated with ECEO 200 µl/mL (T3), protoscoleces treated with 1-8 cineole 100 µg/mL (T4), protoscoleces treated with 1-8 cineole 200 µg/mL (T5), protoscoleces treated with 1-8 cineole 100 µg/mL + albendazole 50 µg/mL (T6), and protoscoleces treated with 1-8 cineole 200 µg/mL + albendazole ALZ-50 µg/mL (T7). The viability of protoscoleces were recorded by eosin staining examination. Moreover, the induction of apoptosis and the plasma membrane permeability of the protoscoleces treated with ECEO and 1-8 cineole were evaluated. Results: The highest protoscolicidal effect of ECEO was observed at the dose of 200 µl/ml (T3). 1,8-Cineole alone and combined with ALZ, particularly at the dose of 200 µg/ml (T5 and T7), destroyed the 100% protoscolices after 10 min incubation. The ECEO (T1-T3) and 1-8 cineole alone (T4 and T5) and in combination with ALZ (T6 and T7) took longer to display their protoscolicidal effect ex vivo. The obtained results of relative fuorescent items exhibited that the protoscoleces incubated with ECEO and 1,8-Cineole, alter the permeability of plasma membrane by Sytox Green with increasing the concentration. The findings revealed exhibited that ECEO and 1,8-Cineole increasingly and dose-dependently induced activation of caspase-3 enzyme ranging from 6.8 to 23.3%. Conclusion: Our obtained results revealed that ECEO and its main compound, 1,8-Cineole exhibited the potent protoscolicidal in vitro and ex vivo; and if more research is done on their efficacy and toxicity in animal models and even clinical setting, it can be suggested as a protoscolicidal agent to use during hydatid cyst surgery.

Insecticidal, Antimalarial, and Antileishmanial Effects of Royal Jelly and Its Three Main Fatty Acids, trans-10-Hydroxy-2-decenoic Acid, 10-Hydroxydecanoic Acid, and Sebacic Acid.

Natural products and their derivatives as an inexpensive, accessible, and useful alternative medicine are broadly applied for the treatment of a wide range of diseases and infectious ones. The present study was designed to evaluate the insecticidal, antimalarial, antileishmanial, and cytotoxic effects of royal jelly and its three main fatty acids (trans-10-hydroxy-2-decenoic acid (10-H2DA), 10-hydroxydecanoic acid (10-HDAA), sebacic acid (1,10-decanedioic acid)). Insecticidal activity of RJ and 10-H2DA, 10-HDAA, and sebacic acid was performed against healthy 4th instar larvae at 25 ± 2°C. Antiplasmodial and antileishmanial effects of RJ and 10-H2DA, 10-HDAA, and sebacic acid were also performed against chloroquine-resistant Plasmodium falciparum K1-strain and Leishmania major amastigotes according to the Malstat method and macrophage model, respectively. In addition, the level of nitric oxide (NO) production in J774-A1 macrophages cells, plasma membrane permeability, and caspase-3-like activity and cytotoxicity effects of RJ and 10-H2DA, 10-HDAA, and sebacic acid against human embryonic kidney 293 (HEK239T cells) were evaluated. Considering the insecticidal activity, the results showed that the lethal concentration 50% value for RJ, 10-H2DA, 10-HDAA, and sebacic acid was 24.6, 31.4, 37.8, and 44.7 µg/mL µg/mL, respectively. RJ, 10-H2DA, 10-HDAA, and sebacic acid showed potent (P < 0.0001) antileishmanial effects with IC50 values ranging from 2.4 to 8.4 µg/mL. Various concentrations of RJ, 10-H2DA, 10-HDAA, and sebacic acid significantly (P < 0.05) increased the production of NO, plasma membrane permeability, and caspase-3-like activity level as a dose-dependent response. Considering the cytotoxicity, SIs > 10 of these compounds exhibited their specificity to parasites and safety against human HEK239T normal cells. The results of the present investigation revealed the promising insecticidal, antimalarial, and antileishmanial effects of RJ and its three main fatty acids (10-H2DA, 10-HDAA, and sebacic acid). However, more studies are required to confirm the mechanisms of action mode of these compounds as well as their efficacy in animal models and clinical settings.

Mitochondrial genome of Rhipicephalus cf. camicasi Morel, Mouchet et Rodhain, 1976 from a camel (Camelus dromedarius Linnaeus) in Riyadh, Saudi Arabia.

Rhipicephalus camicasi Morel, Mouchet et Rodhain, 1976 is thought to be distributed across Africa, Arabian Peninsula and the Mediterranean region. It belongs to the Rhipicephalus sanguineus (Latreille, 1806) species complex. Mitochondrial genome sequences are becoming frequently used for the identification and differentiation of tick species. In the present study, the entire mitochondrial genome of R. cf. camicasi (~15 kb) collected from a camel in Saudi Arabia was sequenced and compared with mitogenomes of two species of Rhipicephalus Koch, 1844. The mitochondrial genome is 87.8% and 91.7% identical to the reference genome of R. sanguineus (sensu stricto, former "temperate lineage") and Rhipicephalus linnaei (Audouin, 1826) (former "tropical lineage"). The current study delivers a molecular reference for material that resembles R. camicasi. We propose to consider the current material, including the complete mitogenome, as the reference for R. camicasi, until a revision using topotypical material is available.