Evaluation of Adjuvant Effectiveness of Alum-Propranolol Mixture on the Immunogenicity of Excreted/Secreted Antigens of Toxoplasma gondii RH Strain.

Purpose: The introduction of novel adjuvants is an important step in attempts to develop a safe and more efficient vaccine. The present study was performed to determine whether the use of a mixed beta-adrenergic receptor antagonist propranolol (PRP) and aluminum (alum), as an adjuvant, have efficacy for Toxoplasma gondii vaccine to induce protective immunity in a mouse model. Methods: Female BALB/c mice divided into five groups were immunized with excretorys-ecretory antigens (ESA) vaccine, alum-ESA vaccine, PRP-ESA vaccine, and alum-PRP ESA vaccine, as well as with phosphate buffered saline (PBS), as a negative control group. The immune responses were evaluated by lymphocyte proliferation assay for measuring delayedtype hypersensitivity (DTH) response and by cytokine assay for evaluating IFN-γ and IL-5 levels. The survival rate of mice in all groups was assessed during a three-week monitoring period after an intraperitoneal challenge with T. gondii tachyzoites. Results: The results showed that mice immunized with PRP, as an adjuvant, could secret a higher level of IFN-γ, which was significant in comparison to other groups. However, mice vaccinated with alum-precipitated ESA antigen had ability to produce an elevated level of IL-5 compared to other mouse groups (P ≤ 0.05). Moreover, alum-PRP co-administration together with ESA vaccine resulted in the longer survival of mice. Conclusion: The findings of this study revealed that the combination of alum-PRP adjuvants and ESA vaccine of T. gondii elicits both humoral and cellular immune responses, which are comparable to either alum or PRP alone.

Sero-epidemiology of Hydatidosis Among General Population of Jolfa County, Northwestern Iran Using IHA, ELISA and Western Blot (2017-2018).

BACKGROUND: Human hydatidosis is mostly a latent and neglected disease with known endemicity in Iran. AIMS: Due to the importance of this infection in the country and its latent nature, we aimed to evaluate the serological status of hydatid cyst in northwestern Iran. OBJECTIVES: Herein, we evaluated the serological status of hydatid cyst in urban and rural inhabitants of Jolfa county, northwestern Iran during 2017-2018. METHODS: In total, 1296 blood samples were obtained from human individuals and the presence of anti-E. granulosus antibodies was investigated using IHA, ELISA and WB. RESULTS: Based on results, 25 IHA positive person were detected in the examined population, however ELISA test showed 14 of 25 IHA positive patients as negative. Also, 269 IHA negative fellows were shown as negative by ELISA. WB analysis of sera from 25 IHA positive subjects revealed consistent results with the ELISA test, and the most reactive SHCF Ag was a 37 KDa protein. The age-standardized seroprevalence of hydatidosis among Jolfa's general population was 1.12% with 95%CI: 1.02-1.20. Moreover, there existed a significant association between keeping/- contact with dogs (P = 0.022) as well as vegetable consumption (P < 0.001) with ELISA positive test results. CONCLUSION: Along with such serological evidence in this region, we highly suggest physical examination and applying imaging techniques for suspected cases in the area for a better understanding of CE.

Seroprevalence of Toxocara Infection in Association with Different Risk Factors among Children of 4-12 Years Old Referred to Some Medical Centers in Aras Free Zone, Northwest Iran.

BACKGROUND: Toxocariosis is a parasitic disease caused by the larval stage of Toxocara species from dog and cat. It has a worldwide distribution with higher prevalence in children. This study aimed to determine seroprevalence of Toxocara infection and its association with some risk factors among children of Aras Free Zone (Jolfa City) in Northwest of Iran. METHODS: Sera were collected from 514 children aged 4-12 yr old attending to some medical centers in the study area from May 2018 to Feb 2019. Anti-Toxocara IgG antibodies assay was performed using commercial ELISA kit (Nova Tec, Germany). The seropositivity rate was determined and its association with different demographic criteria and risk factors were statistically analyzed. RESULTS: The overall seroprevalence was 2.3% (12/514). Risk factors of children's age group and contact with either pet animals (dog and cat) and/or soil were significantly associated with seropositivity. However, there was not any relationship between Toxocara infection and gender of children, place of residency (urban or rural) and their mothers' education level. CONCLUSION: Both girls and boys are at risk of Toxocara infection in the study area. Younger age of childhood and contact with sources of infection were important associated factors. More probably, additional criteria are involved in the initiation of infection.

In Vitro and in Vivo Scolicidal Activities of Holothuria leucospilota Extract and CeO2 Nanoparticles against Hydatid Cyst.

BACKGROUND: We aimed to investigate the scolicidal effects of Holothuria leucospilota extract and CeO2 nanoparticles against protoscoleces of hydatid cysts in-vitro and in-vivo. METHODS: Hydatid cysts were collected from, Urmia slaughterhouses between years 2016-2017 and the hydatid fluid aspirated from the fertile cysts. Various concentration of H. leucospilota extract, CeO2 NPs and combination of CeO2-NPs/H. leucospilota were used for 10-60 min to evaluate the viability of protoscoleces by 0.1% eosin method. CASPASE -3 activity measured for assessment of cell apoptosis in treated protoscoleces. BALB/c mice were infected intraperitoneally with 2000 viable protoscoleces and treated daily for 4 wk by intragastrical inoculation with H. leucospilota, CeO2 NPs, combination of CeO2 NPs/H. leucospilota and Albendazole. Cyst development was macroscopically analyzed. RESULTS: H. leucospilota extract and combination of CeO2 NPs/H. leucospilota have potent scolicidal activity at concentration of 20 mg/ml and 15 mg/ml after 60 min treatment. Maximum caspase-3 activity was observed when protoscoleces expose with H. leucospilota and combination H. leucospilota & CeO2 NPs. After treatment of cyst infected mice with extract and CeO2 NPs, combination of CeO2 NPs/H leucospilota and albendazole, a significant decrease in number of cysts, size and volume of cyst (P<0.05) was observed. CONCLUSION: This result shows an antihydatic and scolicidal effects of H. leucospilota extract and CeO2 NPs.

In silico and in vitro comparative activity of green tea components against Leishmania infantum.

OBJECTIVES: Green tea contains a predominant set of polyphenolic compounds with biological activities. The aim of this study was to investigate the antileishmanial activities of the main components of green tea, including catechin, (-)-epicatechin, epicatechin gallate (ECG) and (-)-epigallocatechin 3-O-gallate (EGCG), against Leishmania infantum promastigotes. METHODS: Green tea ligands and the control drug pentamidine were docked using AutoDock 4.3 software into the active sites of trypanothione synthetase and arginase, which were modelled using homology modelling programs. The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to measure L. infantum promastigotes at different concentrations of green tea compounds in a concentration- and time-dependent manner. Results were expressed as 50% and 90% inhibitory concentrations (IC50 and IC90, respectively). RESULTS: In silico and in vitro assays showed that all of the green tea compounds have antileishmanial activity. EGCG and ECG were the most active compounds against L. infantum promastigotes, with IC50 values of 27.7µM and 75µM and IC90 values of 88.4µM and 188.7µM, respectively. Pentamidine displayed greater growth inhibition than all of the other tested compounds in a concentration- and time-dependent manner. CONCLUSION: In this study, in silico and docking results were in accordance with the in vitro activity of the compounds. Moreover, EGCG and ECG showed reasonable levels of selectivity for Leishmania.

The Anti-leishmanial Efficacy of Artemisia dracunculus Ethanolic Extract in Vitro and Its Effects on IFN-γ and IL-4 Response.

BACKGROUND: Leishmaniasis is a parasitic disease that appears with a range of symptoms including cutaneous, mucocutaneous, and visceral leishmaniasis. The present study sought to determine the antileishmanial effect of the extract of Artemisia dracunculus (Tarragon) compared to control treatment with pentavalent antimony (meglumine). METHODS: This experimental study was performed in 2014-2015. A. dracunculus were collected from West Azerbaijan Province, Iran and dried; then the ethanolic extract of the plant was prepared. The effect of different concentrations of Artemisia's extract was compared with Glucantime ® in the stationary phase by MTT colorimetric assay and Trypan blue staining. Human peripheral blood mononuclear cells (HPBMCs) treated with L. major and production of IFN-γ and IL-4 cytokines measured at concentrations of 25, 20, 10 and 5µg/ml A. dracunculus. RESULTS: Treatment with the extract did not affect the survival of the parasites during the first 48 h; however, on the third day (72 h), all concentrations significantly reduced the number of parasites with an efficacy of more than 50% at 10 µg/ml (P<0.01), 20µg/ml (P<0.001), and 25 µg/ml (P<0.0001). Moreover, IFN-γ and IL-4 secretion from the HPBMCs was significantly affected in a dose-dependent manner, compared to the control (no extract). The IFN-γ/IL-4 ratio further confirmed this notion. CONCLUSION: A. dracunculus extract cannot only exert potent antileishmanial activity but may also enhance cellular immunity to this parasite. Further studies are required to determine the main compound(s) responsible for these effects of the plant.

Toxoplasmosis in rodents: A systematic review and meta-analysis in Iran.

During recent years, implication of rodents in the epidemiology of Toxoplasma gondii is overlooked in Iran; thus, we performed a systematic review and meta-analysis to evaluate the prevalence of toxoplasmosis in rodents of Iran. For this purpose, following the general methodology recommended for systematic reviews and meta-analysis, 5 English and 3 Persian databases were explored from 1 January 2000 till 10 September 2016 using related keywords. Finally, 9 out of 291 citations were met to be included in this study. Due to significant heterogeneity, the random-effects model was conducted (I2=93.55%). During the years, 661 rodents were trapped, and 121 of them were identified positive for T. gondii 15% (95% CI=5-27). Moreover, overall prevalence using direct microscopic examination (1/230), PCR-based techniques (41/246) and serological tests (83/437) was obtained 0.1% (95% CI=0.0-1.5), 18% (95% CI=4-39) and 15% (95% CI=3-33), respectively. Our study revealed the prevalence of toxoplasmosis in rodents is remarkable. Considering this fact, they play a key role in the life cycle of T. gondii and should not be neglected. Further surveys is needed to better recognize the role of various rodent species in distribution of toxoplasmosis.

Genetic Characterization of Fasciola Isolates from West Azerbaijan Province Iran Based on ITS1 and ITS2 Sequence of Ribosomal DNA.

BACKGROUND: Fascioliasis, caused by Fasciola hepatica and F. gigantica, has medical and economic importance in the world. Molecular approaches comparing traditional methods using for identification and characterization of Fasciola spp. are precise and reliable. The aims of current study were molecular characterization of Fasciola spp. in West Azerbaijan Province, Iran and then comparative analysis of them using GenBank sequences. METHODS: A total number of 580 isolates were collected from different hosts in five cities of West Azerbaijan Province, in 2014 from 90 slaughtered cattle (n=50) and sheep (n=40). After morphological identification and DNA extraction, designing specific primer were used to amplification of ITS1, 5.8s and ITS2 regions, 50 samples were conducted to sequence, randomly. RESULT: Using morphometric characters 99.14% and 0.86% of isolates identified as F. hepatica and F. gigantica, respectively. PCR amplification of 1081 bp fragment and sequencing result showed 100% similarity with F. hepatica in ITS1 (428 bp), 5.8s (158 bp), and ITS2 (366 bp) regions. Sequence comparison among current study sequences and GenBank data showed 98% identity with 11 nucleotide mismatches. However, in phylogenetic tree F. hepatica sequences of West Azerbaijan Province, Iran, were in a close relationship with Iranian, Asian, and African isolates. CONCLUSIONS: Only F. hepatica species is distributed among sheep and cattle in West Azerbaijan Province Iran. However, 5 and 6 bp variation in ITS1 and ITS2 regions, respectively, is not enough to separate of Fasciola spp. Therefore, more studies are essential for designing new molecular markers to correct species identification.

Mixture of Alum--Naloxone and Alum--Naltrexone as a novel adjuvant elicits immune responses for Toxoplasma gondii lysate antigen in BALB /c mice.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite. Treatment of the infection induced by this parasite is not straightforward due to the toxic side effects of the available drugs. Vaccine development could be a solution to this problem. In the present study, T.gondii Lysate Antigen (TLA), as a model vaccine, in combination with the Alum-NLT (Aluminum phosphate-Naltrexone) and Alum-NLX (Aluminum phosphate-Naloxone) were evaluated for immunization BALB/c. 147 female BALB/c mice which were divided into seven groups of 21, were allocated to immunization experiments. The first group was selected as the negative control group, followed by the second, third, fourth, fifth, sixth and seventh groups which were immunized with Vac, Vac-Alum, Vac-NLX, Vac-NLT, Vac-Alum-NLX, Vac-Alum-NLT, respectively. Ten days after the final immunization, mice in all groups were divided into three groups for evaluating cellular immune responses, measuring the delayed-type hypersensitivity responses (DTHs) and evaluating survival. The DTH and cellular immune responses showed that in mice immunized with the TLA vaccine combined with the Alum-NLT mixture, the efficacy improved by increasing the production of Interleukin-5(IL-5) and Interferon gamma. This consequently shifted the immune responses toward a Th1 profile by increasing the IFN-γ/IL-5 ratios. In challenge experiments, immunized mice with the Alum-NLT-Vac mixture survived for a longer period of time which indicated an improvement in protective immunity against T. gondii. Administration of the Alum-NLT mixture adjuvant in combination with TLA vaccine enhanced the cellular immunity by shifting the immune response to a Th1 pattern. This shift to the Th1 pattern plays an important role in the induction of cellular.

Evaluation of the adjuvant activity of propranolol, a Beta-adrenergic receptor antagonist, on efficacy of a malaria vaccine model in BALB/c mice.

We have previously shown the adjuvant activity of propranolol (PRP) (a beta-adrenergic receptor antagonist) using a vaccine model for Salmonella typhimurium. In this study PRP was used as an adjuvant in combination with Plasmodium berghei (P. berghei) whole blood stage (PWBS) antigens. BALB/c mice were immunized three times with a 2-week interval, either PWBS vaccine alone or in combination with the adjuvant alum or propranolol. The control group received phosphate buffered saline. Evaluation of the cellular and humoral immunity was performed by measurement of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, lymphocyte proliferation, total IgG and IgG2a in the control and immunized groups. Furthermore, Clinical evaluations were carried out by analyze survival rate and parasitemia of the mice. Our results showed that the mice immunized with propranolol induced higher levels of antibody, IFN-γ and TNF-α as well as stronger lymphocyte proliferative responses compared with other groups. This resulted in improved protective immunity against Plasmodium berghei. Administration of the PRP as an adjuvant in combination with the PWBS Antigen vaccine can shift the immune responses to a T helper1 pattern and enhance the protective immunity.

Evaluation of Alum-Naltrexone Adjuvant Activity, on Efficacy of Anti-Leishmania Immunization with Autoclaved Leishmania major (MRHO/IR/75/ER) Antigens in BALB/C Mice.

BACKGROUND: Naltrexone, an opioid receptor antagonist shifts the immune response toward a Th1 profile. In the current study, we evaluated the efficacy of the mixture of NTX and alum, as a new adjuvant, to enhance immune response and induce protection against Leishmania major in a mouse model. METHODS: BALB/c mice were immunized three times either autoclaved L. major promastigotes' antigens alone or in combination with the adjuvant alum, naltrexone or the alum-naltrexone mixture. Both humoral and cellular immune responses were assessed two weeks after the last immunization and compared with control mice. RESULTS: The administration of alum- NTX in combination with the parasite antigen, significantly increased production of IFN-γ IFN-γ /IL-5 ratio, lymphocyte proliferation and improved DTH response against L. major. There was no significant difference in survival following challenge among groups. CONCLUSION: Immunization with the alum- naltrexone mixture as an adjuvant, in combination with the autoclaved L. major promastigotes antigens, can enhance cellular immunity and shift the immune responses to a Th1 pattern.