Evaluation of In Vitro Cytotoxic and Apoptotic Effects of Miltefosine on the Toxoplasma gondii RH Strain.

Background: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on Toxoplasma gondii RH strain by various techniques. Methods: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to T. gondii tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on T. gondii tachyzoites challenged with IC50 and IC90 doses of miltefosine and unchallenged cells. DNA fragments in T. gondii tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method. Results: Overall, 256, 64, 32, and 16 µg concentrations of miltefosine, respectively could kill more than 50% of viable T. gondii tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (P< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (P<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11µg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms. Conclusion: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.

Prevalence and genetic characterization of Cryptosporidium in pre-weaned cattle in Urmia (Northwestern Iran).

INTRODUCTION: Cryptosporidiosis is a zoonotic disease causing digestive problems in pre-weaned calves. Considering the zoonosis of the parasite and its importance in veterinary medicine, we evaluated the prevalence and genotyping of Cryptosporidium spp. in diarrheic pre-weaned calves in the northwest of Iran. METHODOLOGY: A total of 100 stool samples of the infant calves with diarrhea were collected from industrial and conventional livestock farms in Urmia City. All the samples were tested with acid-fast staining, ELISA, and PCR. Positive samples of the PCR method were sequenced to determine the Cryptosporidium species. The obtained results were compared for the mentioned methods based on statistical factors, sensitivity, specificity, positive and negative predictive values, as well as duration of the experiment and the costs of testing. RESULTS: The results of this study showed that the prevalence of Cryptosporidium spp. in diarrheic infant calves in Urmia city was 5%, and C. parvum species of Cryptosporidium was detected in all the sequenced samples. According to the findings of the current study, the most appropriate method for the detection of the parasite is the ELISA that has a higher sensitivity and predictive value than acid-fast staining method and should be used in veterinary laboratories. CONCLUSIONS: In the current investigation, C. parvum was identified as the only infectious agent in the region and could be the main cause of human infection. More studies are needed to find the source of infection for establishing the control measures.

Comparison of diagnostic methods (wet mount, trichrome staining, formol-ether, PCR, and xenic in vitro culture) for the detection of Blastocystis in stool samples in Urmia educational hospitals, the Northwest of Iran.

Blastocystis spp. is known as a common intestinal protozoan parasite in human and animals. The parasite has a worldwide distribution and is frequently detected in faecal samples in clinical parasitology laboratories. The goal of the study was to compare the sensitivity and specificity of formol-ether technique (FECT), trichrome staining, xenic in vitro culture (XIVC), microscopy of faecal smears, and polymerase chain reaction (PCR) methods for detecting Blastocystis spp. in human stool samples. The prevalence of the parasite in the stool samples referred to educational hospitals was also determined. A total of 575 cases were assessed to detect the parasite. After collecting from patients referring to Urmia educational hospitals, the samples were examined by microscopy of faecal smears, trichrome staining, FECT, XIVC using Jones' medium, and PCR, to evaluate the presence of Blastocystis spp. Microscopy of faecal smears, trichrome staining, FECT, and PCR technique detected 94, 100, 96, and 44 positive cases, with the sensitivity of 71.3%, 74.4%, 74.4%, and 80.4% and the specificity of 99.6%, 99.1%, 100%, and 93.1%, respectively. XIVC method identified the highest number of positive cases (129 cases) among the other methods. Our findings indicates that XIVC technique is more sensitive method for the detection of Blastocystis spp. in human stool, as compared to direct smear, trichrome staining, FECT, and PCR methods.

Association of Toxoplasma gondii infection with cardiovascular diseases: a cross-sectional study among patients with heart failure diseases in Urmia, North-West of Iran.

Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii. Infection with T. gondii is one of the most common parasitic diseases in humans and other warm-blooded animals with global distribution and generally, one-third of human populations are estimated to be contaminated with this parasite. The prevalence of infection varies according to age, geographical location and dietary habits. The socioeconomic losses caused by the disease can be costly for the community. Acquired toxoplasmosis is potentially associated with schizophrenia, Alzheimer's disease, driving accidents, self-injury and suicide. Also, unusual strains of parasite that are genetically different from the rest (atypical strains) are responsible for several cases of lethal acquired parasites in people with safe immunity, which highlights the potential danger of this parasite in public health. As there is no comprehensive study on the association between toxoplasmosis and cardiovascular diseases in Iran, therefore, current study aimed at assessing the relationship between cardiovascular disease and toxoplasmosis among cardiac patients at the Seyyed al-Shohada specialist Cardiology Centre, Urmia, Iran. This study investigated the seropositivity rate for anti-Toxoplasma IgG antibodies by ELISA in patients with cardiovascular diseases. So, 375 patients with cardiovascular diseases and 336 healthy volunteers were selected for this investigation. The seropositivity rate of anti-Toxoplasma IgG antibodies was significantly higher in cardiovascular patients (63.73%) than in healthy volunteers (37.64%) (P<0.001). Also, a positive association was observed between anti-T. gondii IgG antibody seropositivity and cat contact (P≤0.001, OR: 5.178; 95% CI: 1.97-13.57), consumption of raw or undercooked meat (P≤0.001, OR: 0.3; 95% CI: 0.15-0.61), and consumption of not boiled milk (P≤0.001, OR: 0.26, 95% CI: 0.12-0.54). Our results indicate that T. gondii infection is associated with heart disease and suggest that heart disease might be related with a chronic infection. Risk factors associated with T. gondii exposure found in the present study may help design future prevention strategies against T. gondii infection.

Evaluation of Immunogenic Effect of Toxoplasma gondii Recombinant SAG-1 Antigen with Propranolol as Adjuvant in BALB/c Mice.

Purpose: Propranolol as a novel adjuvant, was used to evaluate the immunogenic effect of three doses of recombinant SAG-1 (rSAG-1) antigen of Toxoplasma gondii in BALB/c mice for finding the optimal dose, and was compared with efficacy of tachyzoite lysate antigen (TLA). Methods: Eight different groups of 15 BALB/c mice received different volumes of the immunogenic material (three doses of r SAG-1 and one dose of TLA antigens), with or without propranolol adjuvant, subcutaneously. The control group mice received only PBS. Three weeks after the last immunization, the serum levels of IgG2a, IgG1 and IgG total antibodies against TLA, splenic interleukin-5 (IL-5) and Interferon-gamma (IFN-γ) (produced against TLA) and the splenic lymphocyte proliferation after adding TLA were measured to evaluate humoral and cellular immune responses. Challenge test was performed by subcutaneously injection of 1000 alive and active tachyzoites in to five mice per each group and survival days for each group of mice were recorded. Results: The mice group that received propranolol adjuvant and 20 µg of r SAG-1 antigen per dose of injection showed significantly more IFN-γ production, more proliferation of splenic lymphocytes and higher anti-TLA-specific IgG2a production (three main indexes for cell mediated immunity) in comparison with other groups. Moreover, in the challenge test, this group of mice had a significantly increased survival time, indicating the positive effect of propranolol in the more stimulating of cellular immunity that is necessary for toxoplasmosis prevention or suppress. Conclusion: Our results showed that T. gondii rSAG-1 antigen in combination with propranolol as adjuvant (which can induce Th1 related responses) are good candidates for further study to a vaccine design.

Designing and standardizing Toxocara serologic diagnostic kit and determining anti-Toxocara antibodies frequency in patients referred to health care centers in Urmia (northwest of Iran).

Toxocariasis is a helminthic zoonosis caused by larval stages of the roundworm of dog, Toxocara canis, and less frequently by T. cati, the roundworm of cats. Eosinophilia in peripheral blood may be indicative of a disease; however, it does not necessarily represent toxocariasis. Therefore, it is necessary to investigate the relationship between eosinophilia and toxocariasis in a region. The aims of this study were the diagnosis of hyperosinophilia patients using traditional ELISA kit and also by a handmade ELISA kit produced by T. canis excretory-secretory (TCES) antigens as well as the determination of the abundance of anti-Toxocara antibodies among people referred to Urmia, northwest of Iran care centers. Traditional ELISA kit was used to determine anti-TCES-specific IgG antibodies on 180 hypereosinophilic samples. These antibodies were evaluated in 1002 samples, including 180 hypereosinophilic samples and 822 random samples without eosinophilia by a handmade ELISA kit produced by TCES antigens. A Western-blot confirmatory test was performed on ELISA-positive samples. Our results showed a 17.22% prevalence rate of Toxocara antibodies among hypereosinophilic samples with traditional ELISA kit, and this rate was 3.89% in the 1002 study population with random sampling (with or without eosinophilia). Also, there was a good match between the results of handmade ELISA with those of traditional kit. The positive results in the ELISA method were confirmed by the Western-blot analysis. Our findings show that although the high eosinophil count is not necessarily a sign of toxocariasis, in Urmia distric, about one-fifth of eosinophilia cases have anti-toxocariasis antibodies. In addition, the abundance of anti-Toxocara antibodies in this area was 3.89%.