Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine.

INTRODUCTION: Cholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. MATERIAL AND METHODS: B and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. RESULTS: The titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. CONCLUSION: This study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Evaluation of the biodiversity and distribution of sand flies and tracking of Leishmania major in them in central Iran.

Many areas of Iran are endemic regarding Cutaneous leishmaniasis (CL) as a parasitic disease transmitted by a female sand fly vector's bite. The present study investigated the distribution of sand flies in Harand and Egieh in Isfahan province, Central Iran. Overall, 408 out of 1260 collected sand flies were identified morphologically, among which 353 and 55 were isolated from Harand and Egieh, respectively. Also, 66.4% and 33.6% of the sand flies were female and male, respectively. The most prevalent sand fly species were Phlebotomus papatasi (52%), followed by Ph. caucasicus (40.4%), Sergentomyia sintoni (4.9%), and Ph. kazeruni (0.7%). Among 180 molecularly-analyzed sand flies, 14 (7.77%) were found infected with L. major, with 9 out of 103 (8.73%) Ph. papatasi and 5 out of 75 (6.66%) Ph. caucasicus.

Seroprevalence of Toxoplasma infection in individuals occupationally exposed to livestock and raw meat: A case-control study.

BACKGROUND: The expectancy of Toxoplasma gondii transmitted from livestock and raw meat to humans is a public health problem and is an example of the One Health theory. OBJECTIVES: This survey aimed to determine the seroprevalence and risk factors related to this common infection in individuals occupationally exposed (IOE) to livestock, raw meat and viscera in industrial slaughterhouses and livestock fields in Isfahan province, central Iran. METHODS: This study is a case-control survey carried out on the 401 serum samples of IOE (including slaughterhouse workers, butchers, veterinarians, veterinary technicians, livestock farmers and farm workers) compared to 401 archived samples of the general population (that all matched with cases by region, age and gender). All 802 samples were investigated for anti-T. gondii IgM and anti-T. gondii IgG using enzyme-linked immunosorbent assay. RESULTS: A statistically significant higher anti-T. gondii IgG occurrence (p < 0.001) was observed in IOE compared to the control group (46.1% vs. 31.4%). According to our knowledge, this is the first case-control study on the seroprevalence of anti-T. gondii in IOE to livestock in central Iran. CONCLUSIONS: These findings show a potentially significant association between T. gondii seropositivity and occupational exposure to livestock. Therefore, it is essential to develop guidelines for preventing disease transmission among IOE to livestock, raw meat and viscera in industrial slaughterhouses and livestock fields.

A large-scale study on the seroprevalence of Toxoplasma gondii infection in humans in Iran.

It is estimated that nearly one-third of the world's human population is infected with Toxoplasma gondii (Nicolle et Manceaux, 1908). Human infection is commonly asymptomatic, multifaceted, and can manifest in severe pathological forms in congenital toxoplasmosis and immunocompromised individuals. This study attempted to recognise the seroprevalence of T. gondii infection in Iranian residents referred to medical laboratories for toxoplasmosis tests throughout the country. This retrospective cross-sectional study was conducted from 2015 to 2019 on individuals referred to diagnostic laboratories in 26 provinces, and these laboratories sent their samples to the referral centres. Accordingly, data associated with serodiagnosis of toxoplasmosis, age, sex, anti-T. gondii IgG, and IgM status in Iranian residents were collected from two referral diagnostic laboratories. All individuals were evaluated using the antibody immunocapture-chemiluminescence assay (CLIA) method with the Immulite®2000s XPi system. In this study, the first large-scale assay of T. gondii infection in Iran, an overall seroprevalence of 30.4% was among 35,047 patients examined. The highest IgM seropositivity rate was in the 10-20 years old patients. In addition, this study showed a very different prevalence of T. gondii across the country, highest in the humid areas, such as the Caspian Sea basin in the North, and the North West with seroprevalence of 48.6%.

Evaluation of the antileishmanial effect of polyclonal antibodies and cationic antimicrobial peptides.

Leishmaniasis is one of the tropical and subtropical diseases which, according to WHO, has the priority of control. The list of anti-leishmanial drugs is limited and requires side effects, high costs, and long-term treatments. Various species, parasite resistance, and simultaneous diseases are among the factors that affect the effectiveness of treatment. Due to these problems and based on satisfactory records of previous studies using antimicrobial peptides (AMPs) against infectious diseases, this study aimed to evaluate the antileishmanial effect of Leishmania-infected macrophage polyclonal antibody (LIMPA) with or without different concentrations (2, 4, 6, 8, 10, 20, 40, 60, and 100 µg/ml) of CM11 and (40, 80, and 100 µg/ml) BufIIIb, two AMPs, in vitro and their therapeutic effects against CL of Balb/c mice. Results showed that LIMPA induced an anti-proliferative effect on Leishmania major growth in macrophages in vitro and intramacrophage-amastigotes in vivo. CM11 with IC50 of 8.73 and 10.10 µg/ml at 48 hours, and BufIIIb with IC50 of 66.83 and 80.26 µg/ml, at 24 hours showed the most significant inhibition of L. major promastigotes and amastigotes. In addition, the CM11 and BufIIIb, with a CC50 of 9.7 µg/ml and 40.34 µg/ml, showed the most significant inhibition effect on the J774.A1 cell line at 48 hours, respectively. In addition, in vivo experiments using LIMPA with a 0.01 mg/kg dosage showed a significant difference (p < 0.001) in the last week of the measurement compared to the control. The results of this study may be a promising prospect for further investigations.

Induction of apoptosis by essential oil of Dracocephalum kotschyi on Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is a protist parasite that causes trichomoniasis, a sexually transmitted disease. Metronidazole is the current treatment for trichomoniasis. However, this drug can provoke severe side effects, and some strains present resistance, making the development of alternative treatments for trichomoniasis urgent. OBJECTIVES: We investigate the use of essential oil obtained from Dracocephalum kotschyi on T. vaginalis. D. kotschyi has antispasmodic and analgesic properties and is well known in Iran. METHODS: The essential oil was obtained by hydrodistillation from 1000 g of the powdered plant. Gas chromatography-mass spectrometry analysis was used for the chemical composition of the essential oil, and 11 substances were identified, corresponding to 91.5% of the oil. Copaene (22.15%), Methyl geranate (16.31%), Geranial (13.78%) and Carvone (11.34%) were the main substances. A cell viability test was used to determine the percentage of growth inhibition (GI%) and the half-maximal inhibitory concentration (IC50) on T. vaginalis after incubation with the prepared essential oil. RESULTS: The oil induced an IC50 of 84.07 µg/ml after 24 h contact with trophozoites. Cytotoxicity was determined by MTT assay on the J774.A1 haematopoietic cell line. In addition, the initial stage of apoptosis was assayed using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. Evaluation of the in vitro anti-trichomonal properties of D. kotschyi essential oils showed that it effectively induces apoptosis on T. vaginalis between 100 and 700 µg/ml after 48 h without toxicity on haematopoietic cells, suggesting that D. kotschyi essential oil can induce programmed death in T. vaginalis. CONCLUSIONS: The anti-trichomonal properties of D. kotschyi essential oil indicate that they could be suitable for new pharmacologic studies after new tests with human vaginal epithelial cells.

Molecular characterization and genotyping of Toxoplasma gondii in free-living animals in Iran: Effect of One Health.

To understand the transmission of Toxoplasma gondii, this parasite's genetic diversity distribution in free-living hosts is essential. This research's objective is the molecular genotyping of T. gondii isolates from the brain and muscles of Columbidae, Corvidae, Rattus, and Felidae of Mianeh County, East-Azerbaijan Province, Northwest Iran. Three hundred fifty samples were taken. For the genotyping of T. gondii, the GRA6 gene was amplified and digested by the Tru1I (MseI) enzyme. Results of RFLP were confirmed by sequencing and phylogenetic analysis. In total, 52%, 34%, 24%, and 50% of Columbidae, Corvidae, Rattus, and Felidae were positive for T. gondii DNA, respectively. All isolated Columbidae were identified as genotype III (100%). Also, 94.1% and 5.9% of Corvidae isolates, 84.4% and 15.6% of the Rattus isolates, and 51.7% and 48.3% of the Felidae isolates belonged to genotypes III and II, respectively. This study is the first to evaluate genetic similarity and phylogenetic analysis between many definitive and intermediated hosts in northwestern Iran. The finding indicates that the T. gondii cycle is maintained among these hosts. As a result, their presence in the environment can be a risk factor for transmitting the infection to humans. Due to demographic and geographic differences in various regions, further studies are required to determine the genetic population structure.

Isolation and identification of 6-methoxy parillin and coniferin from the bulbs of Allium affine L.

Background and purpose: Phytochemically, Allium species are a rich source of important secondary metabolites especially steroidal saponin and sapogenins, flavonoids, and sulfur compounds. As a member of this genus, Allium affine, which is locally known as "tareh kouhi", is an endemic plant of middle Asian countries. Experimental approach: Bulbs of A. affine were collected and air-dried in the shade. The chloroform-methanol (9:1) extract of the sample was subjected to purification by MPLC and HPLC. Structure elucidation of isolated compounds was done using comprehensive spectroscopic methods including 1D-NMR, 2D-NMR, and MS. Findings/Results: A steroidal saponin structurally related to parillin and a phenylpropanoid glycoside (coniferin) were isolated and identified from the plant chloroform-methanol extract. Conclusion and implication: To the best of our knowledge isolation of these potentially medicinal compounds from A. affine was reported for the first time in this study.

Study of therapeutic effect of different concentrations of imatinib on Balb/c model of cutaneous leishmaniasis.

Leishmaniasis, as a tropical and subtropical disease, is endemic in more than 90 countries around the world. Today, cutaneous leishmaniasis (CL) that affects more than 1.5 million people per year lacks a definitive treatment approach. Imatinib is an anticancer drug that inhibits the abnormal function of Bcr-Abl due to its tyrosine kinase inhibitor, and that was the reason why the drug was tested for CL treatment because protein kinases are essential enzymes in the Leishmania genus. In this study, the L. major CL model of Balb/c mice was produced by injection of the cultured metacyclic form of parasite at the base of the tail. The possible recovery of CL ulcers and determination of the optimum dose of imatinib against Leishmania amastigotes were evaluated. A significant decrease was observed in mice treated with amphotericin B (positive control group) as well as imatinib 50 mg/kg compared to the unreceived drug, negative control group (P<0.05). This study could be promising in gaining insight into the potential of imatinib as an effective treatment approach against CL.

Evaluation of Different Concentrations of Imatinib on the Viability of Leishmania major: An In Vitro Study.

BACKGROUND: Leishmaniasis is an infectious disease caused by an intracellular parasite of Leishmania and is transmitted through the female sandflies bite and may lead to severe skin lesions. Although drugs such as antimony compounds are available, their side effects such as toxicity, low efficacy, and emergence of resistance have raised the importance of effective replacement. Imatinib, as an inhibitor of tyrosine kinase (TK) of Leishmania, stops abnormal function of TK such as Bcr-Abl through assembling into transmembrane pores in a sterol-dependent manner. Hence, the evaluation of killing effects of different concentrations of imatinib against Leishmania major amastigotes and promastigotes in vitro were the objectives of the present study. MATERIALS AND METHODS: The killing effects of different concentrations of imatinib (25, 50, and 100 µg) and 25 µg amphotericin B (as positive control) were evaluated against RPMI 1640-cultured promastigotes and the amastigote/macrophage model by MTS cell proliferation assay kit (ab197010) and Giemsa staining method during 24, 48, and 72 h. RESULTS: The results showed anti-Leishmania effect of imatinib in concentration and time-dependent manner. The lowest number of live promastigotes and amastigotes were obtained due to treat with 100 µg/ml imatinib at 72 h. Furthermore, 100 µg concentration of imatinib had the same effect as 25 µg amphotericin B on both L. major promastigotes and amastigotes (P < 0.001). CONCLUSION: The anti-Leishmania effect of imatinib was confirmed by MTS and direct microscopy. Further study is recommended for evaluating possible therapeutic effects of imatinib on leishmaniasis in vivo.