Exploiting the Macrophage Production of IL-12 in Improvement of Vaccine Development against Toxoplasma gondii and Neospora caninum Infections.

Toxoplasmosis and neosporosis are major protozoan diseases of global distribution. Toxoplasma gondii is the cause of toxoplasmosis, which affects almost all warm-blooded animals, including humans, while Neospora caninum induces neosporosis in many animal species, especially cattle. The current defective situation with control measures is hindering all efforts to overcome the health hazards and economic losses of toxoplasmosis and neosporosis. Adequate understanding of host-parasite interactions and host strategies to combat such infections can be exploited in establishing potent control measures, including vaccine development. Macrophages are the first defense line of innate immunity, which is responsible for the successful elimination of T.gondii or N. caninum. This action is exerted via the immunoregulatory interleukin-12 (IL-12), which orchestrates the production of interferon gamma (IFN-γ) from various immune cells. Cellular immune response and IFN-γ production is the hallmark for successful vaccine candidates against both T. gondii and N. caninum. However, the discovery of potential vaccine candidates is a highly laborious, time-consuming and expensive procedure. In this review, we will try to exploit previous knowledge and our research experience to establish an efficient immunological approach for exploring potential vaccine candidates against T. gondii and N. caninum. Our previous studies on vaccine development against both T. gondii and N. caninum revealed a strong association between the successful and potential vaccine antigens and their ability to promote the macrophage secretion of IL-12 using a murine model. This phenomenon was emphasized using different recombinant antigens, parasites, and experimental approaches. Upon these data and research trials, IL-12 production from murine macrophages can be used as an initial predictor for judgment of vaccine efficacy before further evaluation in time-consuming and laborious in vivo experiments. However, more studies and research are required to conceptualize this immunological approach.

High relatedness of bioinformatic data and realistic experimental works on the potentials of Fasciola hepatica and F. gigantica cathepsin L1 as a diagnostic and vaccine antigen.

Introduction: Fascioliasis is a parasitic foodborne disease caused by the liver flukes, Fasciola hepatica and F. gigantica. Such parasites cause serious illness in numerous domestic animals and also in humans. Following infection, the parasite secretes a variety of molecules that immediately interact with the host immunity to establish successful infection. These molecules include cathepsin L peptidase 1 (CatL1); the highly investigated diagnostic and vaccine antigens using various animal models. However, a few studies have analyzed the potentials of FhCatL1 as a diagnostic or vaccine antigen using bioinformatic tools and much less for FgCatL1. The present study provides inclusive and exclusive information on the physico-chemical, antigenic and immunogenic properties of F. hepatica cathepsin L1 (FhCatL1) protein using multiple bioinformatic analysis tools and several online web servers. Also, the validation of our employed available online servers was conducted against a huge collection of previously published studies focusing on the properties of FhCatL1as a diagnostic and vaccine antigen. Methods: For this purpose, the secondary, tertiary, and quaternary structure of FhCatL1 protein were also predicted and analyzed using the SWISS-MODEL server. Validation of the modeled structures was performed by Ramachandran plots. The antigenic epitopes of the protein were predicted by IEDB server. Results and discussion: Our findings revealed the low similarity of FhCatL1 with mammalian CatL1, lacking signal peptides or transmembrane domain, and the presence of 33 phosphorylation sites. Also, the containment of FhCatL1 for many topological, physico-chemical, immunological properties that favored its function of solubility and interaction with the immune components were reported. In addition, the earlier worldwide reports documented the high efficacy of FhCatL1 as a diagnostic and vaccine antigen in different animals. Altogether, FhCatL1 is considered an excellent candidate for using in commercialized diagnostic assays or vaccine products against fascioliasis in different animal species. Our assessment also included FgCatL1 and reported very similar findings and outputs to those of FhCatL1.

Prevalence of Neospora caninum and Toxoplasma gondii Antibodies and DNA in Raw Milk of Various Ruminants in Egypt.

The prevalence of Neospora caninum and Toxoplasma gondii antibodies in raw milk samples was estimated in different ruminants and Egyptian governorates. Of 13 bulk milk samples tested by ELISA, five (38.5%) were positive for antibodies to N. caninum, and two samples were additionally positive for antibodies to T. gondii, resulting in a seroprevalence of 15.4% for both T. gondii and co-infection. In individual milk samples (n = 171) from the same bulks, antibodies to N. caninum were detected in 25.7%, to T. gondii in 14%, and 3.5% had antibodies to both parasites. A strong correlation between the OD values of the bulk samples and of the relevant individual milk samples was found for T. gondii (Pearson r = 0.9759) and moderately strong for N. caninum (Pearson r = 0.5801). Risk factor assessment for individual milk samples revealed that antibodies to T. gondii were significantly influenced by animal species, while no risk factors were detected for N. caninum antibodies. Additionally, DNA of N. caninum was detected in a bulk milk sample of cattle for the first time in Egypt, and DNA of T. gondii was found in bulk milk samples of cattle, sheep and goats. This is the first study in Egypt in which bulk milk samples of different ruminants were tested for the presence of N. caninum and T. gondii antibodies and DNA. Both individual and bulk milk samples are useful tools for monitoring antibody response to N. caninum and T. gondii infections in different ruminants in Egypt.

Molecular detection and identification of Babesia bovis and Trypanosoma spp. in one-humped camel (Camelus dromedarius) breeds in Egypt.

BACKGROUND AND AIM: Camels are a unique source of milk and meat, which helps recover from several diseases that affect humans worldwide. In Egypt, one of the great obstacles for this industry is tick-borne diseases. This study aimed to characterize blood parasite infections, such as Babesia (B.) bovis and Trypanosoma (T.) spp. in one-humped camel (Camelus dromedarius) (n=142) breeds in Halayeb and Shalateen, Egypt, through phylogenetic analysis. MATERIALS AND METHODS: The prevalence of B. bovis and Trypanosoma spp. was identified in camels using polymerase chain reaction (PCR) assays targeting the Rhoptry-Associated Protein-1 and internal transcribed spacer 1 genes, respectively. A nested PCR technique was conducted to detect B. bovis. At the same time, KIN multispecies PCR assay was employed to diagnose and classify trypanosome DNA in camels. RESULTS: B. bovis was detected in 4/142 camels with an infection rate of 2.81%. Sequencing and phylogenetic analyses revealed that the strain of B. bovis isolated from this population was closely related to strains isolated from Argentine, the United States, and Brazil. Moreover, Trypanosoma evansi was detected in 8/142 camels with an infection rate of 5.63%. Sequencing and phylogenetic analyses revealed that this isolated strain T. evansi was closely related to Trypanosoma theileri detected from cattle in Brazil. CONCLUSION: The obtained data indicated the existence of B. bovis and T. evansi in camels from two provinces of Egypt. The obtained findings have economic significance and reflect the importance of implementing effective prevention and control methods across Egypt to reduce the incidence of B. bovis and T. evansi in camels.

Phylogenetic study of Theileria ovis and Theileria lestoquardi in sheep from Egypt: Molecular evidence and genetic characterization.

BACKGROUND AND AIM: Ovine theileriosis caused by Theileria ovis and Theileria lestoquardi is an important infectious disease affecting small ruminants in regions of the tropic and subtropic zones. There is limited studies about ovine theileriosis in Egypt; so the present study aims to assess the occurrence of ovine theileriosis in Egypt at the molecular level. MATERIALS AND METHODS: Blood samples were collected from 115 randomly selected sheep, which were apparently healthy; the ages of the sampled sheep ranged from 1 to 5 years old, from a local breed (barkae and balade), and showed no symptoms indicating infection with Theileria spp. The study was conducted in three governorates representing Lower Egypt (Menoufia and Beheira) and Upper Egypt (El-Wady El-Geded). All blood samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to target Theileria spp. 18S rRNA genes. Positive samples were sequenced, and these sequences were analyzed using nucleotidebasic local alignment search tool (BLAST). RESULTS: Six animals (5.22%) were PCR-positive carriers for ovine theileriosis. Nucleotide BLAST and phylogenetic analyses of the six obtained sequences showed that T. ovis was present in five animals (4.37%) in Menoufia (n=2) and El-Wady El-Geded (n=3), whereas T. lestoquardi was detected in 1 animal (0.87%) in El-Wady El-Geded. CONCLUSION: This study is the first to provide molecular evidence, genetic characterization, and phylogenetic analysis of ovine Theileria spp. in Egypt. Specifically, T. lestoquardi and T. ovis carrier statuses of sheep were confirmed. These results highlight the importance of developing an effective control strategy against ovine theileriosis carriers that might develop and/or spread theileriosis.

Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt.

Fasciolosis is an important food and water-borne parasitic infection caused by the two trematode species, Fasciola hepatica, and F. gigantica. The present study aimed to identify the phenotypic features and genetic characterization of adult fasciolid that infecting buffaloes were studied in Aswan, Egypt. The genetic identity of Fasciola species was investigated by the analysis of forward and reverse sequences of the ITS-2 of the rDNA gene. The Fasciola isolates were obtained from sheep, buffaloes, and cows in the regions of Aswan. The sequence of ITS2 gene isolates obtained from the present investigation were compared with GenBank reference sequences of F. hepatica, F. gigantica, and intermediate Fasciola. The obtained results were based on morphometric and genetic data which revealed the existence of F. gigantica, F. hepatica, and an intermediate form of Fasciola. Several variable sites were encountered among the investigated isolates in the Aswan, that were compared with the Fasciola species acquiesced in Gene Bank. Furthermore, the relationships between Egyptian Fasciola and Fasciola spp. from various other nations were discussed in the study.

Exploring Prokaryotic and Eukaryotic Microbiomes Helps in Detecting Tick-Borne Infectious Agents in the Blood of Camels.

Dromedary camels (Camelus dromedarius) are widely distributed in Africa, the Middle East and northern India. In this study, we aimed to detect tick-borne pathogens through investigating prokaryotic and eukaryotic microorganisms in camel blood based on a metagenomic approach and then to characterize potentially pathogenic organisms using traditional molecular techniques. We showed that the bacteria circulating in the blood of camels is dominated by Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. At the genus level, Sediminibacterium, Hydrotalea, Bradyrhizobium and Anaplasma were the most abundant taxa. Eukaryotic profile was dominated by Fungi, Charophyta and Apicomplexa. At the genus level, Theileria was detected in 10 out of 18 samples, while Sarcocystis, Hoplorhynchus and Stylocephalus were detected in one sample each. Our metagenomic approach was successful in the detection of several pathogens or potential pathogens including Anaplasma sp., Theileria ovis, Th. separata, Th. annulate, Th. mutans-like and uncharacterized Theileria sp. For further characterization, we provided the partial sequences of citrate synthase (gltA) and heat-shock protein (groEL) genes of Candidatus Anaplasma camelii. We also detected Trypanosoma evansi type A using polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) region. This combined metagenomic and traditional approach will contribute to a better understanding of the epidemiology of pathogens including tick-borne bacteria and protozoa in animals.

Epizootiology of zoonotic parasites in Middle East: a comprehensive review.

Parasitic zoonosis (PZs) have a cosmopolitan significant impact on public health but they are often omitted in discussions, especially in developing countries. Zoonotic parasites include protozoa, cestodes, nematodes, trematodes and arthropods, and notably in African and Arabian countries have a high prevalence among livestock and man. Through this comprehensive review, we summarize the extant published research of the most significant zoonotic parasites present in some countries of Arabic world and we identify the epidemiology and risk factors for significant infections and suggest some effective control measures. This review might help the researches, governments about the zoonotic impact of these neglected infections for future considerations and application for real control programs.

Myrrh Oil in Vitro Inhibitory Growth on Bovine and Equine Piroplasm Parasites and Babesia microti of Mice.

The present experimental study was conducted for the assessment of the efficacy of in vitro inhibition of myrrh oil on the propagation of Babesia bovis, B. divergens, B. bigemina, Theileria equi, and B. caballi and in vivo efficacy on B. microti in mice through fluorescence assay based on SYBR green I. The culture of B. divergens B. bovis and was used to evaluate the in vitro possible interaction between myrrh oil and other commercial compound, such as pyronaridine tetraphosphate (PYR), diminazene aceturate (DA), or luteolin. Nested-polymerase chain reaction protocol using primers of the small-subunit rRNA of B. microti was employed to detect any remnants of DNA for studied parasitic species either in blood or tissues. Results elucidated that; Myrrh oil significantly inhibit the growth at 1% of parasitic blood level for all bovine and equine piroplasm under the study. Parasitic regrowth was inhibited subsequently by viability test at 2 µg/mL for B. bigemina and B. bovis, and there was a significant improvement in the in vitro growth inhibition by myrrh oil when combined with DA, PYR, and luteolin. At the same time; mice treated with a combination of myrrh oil/DA showed a higher inhibition in emitted fluorescence signals than the group that challenged with 25 mg/kg of diminazene aceturate at 10 and 12 days post-infection. In conclusion, this study has recommended the myrrh oil to treat animal piroplasmosis, especially in combination with low doses of DA.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

Evaluation of in vitro inhibitory effect of enoxacin on Babesia and Theileria parasites.

Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by micro molar concentrations of enoxacin (IC50 values = 33.5, 15.2, 7.5 and 23.2 µM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Enoxacin IC50 values for Babesia and Theileria parasites were satisfactory as the drug is potent antibacterial drug with minimum side effects. Therefore, enoxacin might be used for treatment of Babesiosis and Theileriosis especially in case of mixed infections with bacterial diseases or incase of animal sensitivity against diminazin toxicity.

Molecular and phylogenetic study on Toxocara vitulorum from cattle in the mid-Delta of Egypt.

Toxocara vitulorum is an important intestinal nematode that commonly infects ruminants world-wide, notably in tropical and sub-tropical regions. In Egypt, T. vitulorum has a high prevalence rate in cattle and buffaloes calves. The current work aims to identify and verify T. vitulorum collected from cattle in El-Mahlla El-Kubra city in the mid-Delta of Egypt, using molecular and phylogenetic tools. The first internal transcribed spacer (ITS-1) and 18S genes of ribosomal DNA were amplified, sequenced, and compared with nucleotide sequences deposited in data bases, and also used to construct the phylogenetic trees. Our results confirm that T. vitulorum isolated from cattle in Egypt is genetically identical to those recorded in other countries. Moreover, the phylogenetic trees show a close relationship among different species of Toxocara, including the zoonotic species. Our results show that ITS genes can be targeted as genetic markers to diagnose and discriminate among different Toxocara spp. The data presented here may be helpful in the pursuit of further molecular and genetic studies of Toxocara species.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

Microscopical and serological studies on Sarcocystis infection with first report of S. cruzi in buffaloes (Bubalus bubalis) in Assiut, Egypt.

This study was performed for the purpose of investigating the prevalence and the species composition of Sarcocystis spp. in buffaloes in Assiut province, Egypt. Macroscopically we reported the infection of buffaloes with Sarcocystis fusiformis, while microscopically three Sarcocystis species (Sarcocystis cruzi, Sarcocystis levinei and Sarcocystis hominis) cysts were recognized, and were differentiated by their morphological features using both histopathological sections and electron microscope scanning. Regarding the prevalence of Sarcocystis species among buffaloes in Assiut province, we reported that, using gross examination of 90 buffaloes' esophagus, only 23 samples out of 90 (25.5 %) were found to be infected; on the other hand, by using microscopical examination, the prevalence was 27.7 % (25 samples out of 90 samples were found to be infected). Using ELISA, 85 samples out of 90 (94.4 %) were found positive, an overall prevalence of 94.4 %. In this work we concluded that customary meat inspection methods in abattoirs in Egypt are insufficient for detecting Sarcocystis infection. Due to the presence of hidden or microscopic cysts, we strongly recommend the use of combined microscopical examination and ELISA for Sarcocystis diagnosis, to avoid human infection of such zoonotic parasite and to control the consequent disease. In addition, this study introduced the first report of S. cruzi in buffaloes in Egypt, and proved the hypothesis that S. cruzi is able to use animals such as water buffalo as intermediate hosts.

Molecular and phylogenetic status of Fasciola sp., of cattle in Qena, Upper Egypt.

The species of liver fluke of the genus Fasciola (phylum platyhelminthes, order Digenea, Family Fasciolidae) are obligatory parasites that inhabit the large biliary ducts of herbivore animals as well as man. Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. In the current study a precise identification of Fasciola isolates from cattle in Qena province, Upper Egypt was done based on examination of the second Internal Transcribed Spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI). Amplification, sequencing and phylogenetic examination revealed that the collected Fasciola isolates represent only one species which is Fasciola hepatica.