Seroprevalence of Toxoplasma gondii Infection in Dogs in Tehran,Iran

Background

Toxoplasma gondii infects a wide range of animals; felines are definitive hosts and other animals including the dogs are intermediate hosts. The aim of this study was to determine the seroprevalence of T. gondii infection in dogs in Tehran, capital of Iran and to investigate possible associated risk factors.

Methods

Three hundreds ninety six serum samples were collected during 2007–8 from the dogs. Collected samples were tested using an indirect fluorescent antibody test (IFAT) in dilutions of 1:16 and more. All procedures were carried out in Shahrekord University, Iran. All the data were analyzed using SPSS software, qui square test with confidence interval of 0.95.

Results

From evaluated samples, 89 (22.47%) were positive in titers of at least 1:16. further evaluations in other dilutions showed positive results in dilutions of maximum 1:16, 1:32, 1:64, 1:128 and 1:256 in 38, 29, 15, 2 and 5 dogs respectively. Investigation of the role of risk factors showed no sex predisposition while infection rate was significantly higher in dogs older than one year old. Living places were of significant importance; infection rate was significantly higher in stray or guard dogs in compare with household dogs (P<0.05).

Conclusion

Relatively high seroprevalence of T. gondii infection in dogs in Tehran shows high environmental contamination. It is recommended that the dogs with suspected clinical signs be tested for T. gondii infection.     

High Seroprevalence of Toxoplasma gondii Antibody in HIV/AIDS Individuals from North of Iran

Background:

Toxoplasmosis in immunocompetent people is generally asymptomatic but in immunocompromised patients including HIV/AIDS, cancer patients, and organ transplant recipients, etc. it can lead to serious pathological problems. The objective of current study was to determine the seroprevalence of T. gondii IgG and IgM antibodies in HIV/AIDS patients using ELISA technique in Mazandaran Province, northern Iran.

Methods:

Overall, 82 serum samples (61 males and 21 females) were collected from HIV/AIDS patients in Mazandaran Provinces, in 2013. Sera were surveyed employing ELISA assay. Data were analyzed using Chi-Square or Fisher exact test. In addition, before sampling a questionnaire was filled out for each subject.

Results:

Overall seroprevalence of examined sera was 96.3% for IgG antibody but none of the sera shown IgM antibody against T. gondii. The seroprevalence of toxoplasmosis in males and females was 96.7% and 95.2%, respectively. An antibody titer of >1 IU/ml was considered as positive. Furthermore, none of the included variables statistically was significant.

Conclusions:

Seroprevalence of chronic (latent) toxoplasmosis in HIV/AIDS patients in Mazandaran Province is high compared to toxoplasmosis in general population. Consequently, the risk of acquiring Toxoplasma encephalitis in examined seropositive HIV/AIDS patients of Toxoplasma is high.     

Molecular Survey of Toxoplasma gondii in Sheep,Cattle and Meat Products in Chaharmahal va Bakhtiari Province,Southwest of Iran

Background

Toxoplasmosis is a worldwide spread disease. The present study examined the prevalence of Toxoplasma gondii infection among animals of edible meat (cattle and sheep) in Chaharmahal va Bakhtiari Province (Southwest of Iran) in 2012. Furthermore, we attempted for the first time to identify this parasite from the meat products in the province.

Methods

The tongue, brain, femur muscle and liver of 50 sheep and 70 cattle as well as 50 samples of meat products were selected and collected to perform molecular survey using Nested-PCR method.

Results

Of the studied sheep, 38% were infected. The infection rate in the age groups under 1 year, 1-2 years, and more than 2 years was 25%, 35.29% and 52.94%, respectively. The infection rate in femur muscle, brain, liver and tongue was 28%, 32%, 30% and 16%, respectively. Of the studied cattle, 8.57% were infected. The infection rate in the age groups 1-2 years, 2-4 years, and more than 4 years was 3.7%, 9.09% and 14.28%, respectively. Sheep was infected 6 times more than cattle (OR = 6.53 CI = 2.374-18.005).The infection rate among samples of meat products was 12% (6 samples out of 50 samples).

Conclusion

Due to the high rate of this parasitic infection among the slaughtered animals as well as meat products in this region, the use of infected material can be one of the main risk factors of transmission of the parasite to humans.     

Seroprevalence of Toxoplasma gondii in Pregnant Women and Bioassay of IgM Positive Cases in Zanjan,Northwest of Iran

Background

The aim of this study was to determine the Toxoplasma antibodies in pregnant women in Zanjan, by ELISA method.

Methods

Blood samples were taken from 500 pregnant women referred to the health centers of Zanjan City, North West Iran, IgM and IgG titers were primarily evaluated. The collected data were analyzed with SPSS 11.5 using Chi-Square test.

Results

Anti Toxoplasma IgM and IgG were positive in 1.4% and 37.2% respectively. Seropositive subjects were more frequently seen in those with >30 years old compared to younger women (<20 years old). No significant relationship was found between the seroprevalence of T. gondii infection and level of education, residence area, history of abortion and gestational age.

Conclusion

The rate of IgM positive was low; however, a large number of the studied population were IgG positive, indicative of having a latent infection due to the past exposure to Toxoplasma parasite in this region.     

Recombinant SAG1 Antigen to Detect Toxoplasma gondii Specific Immunoglobulin G in Human Sera by ELISA Test

Background

Although some serological tests for the detection of Toxoplasma gondii-specific immunoglobulin are commercially available, better diagnostic tools are needed. The aim of present study was to evaluate the usefulness of the recombinant Toxoplasma gondii SAG1 antigen for the recognition of toxoplasmosis by ELISA.

Methods

This study was conducted in Cellular and Molecular Biology Research Centers, Shahid Beheshti University, M.C., Tehran, Iran in 2008-2009. Surface antigen 1 (SAG1), a tachyzoite stage-specific protein, was subcloned into an expression vector and was subsequently transformed into BL21 (DE3) pLysS competent bacterial cells. After inducing expression of the recombinant antigen, the protein product was purified using Ni-affinity chromatography. The immunoreactivity of recombinant SAG1 (rSAG1) was analyzed by SDS-PAGE and western blotting. The reactivity of the rec-SAG1 protein was evaluated using an ELISA.

Result

Sensitivity and specificity of the generated recombinant-ELISA (rec-ELISA) compared to a commercially available ELISA (com-ELISA) were 88.4% and 88%, respectively.

Conclusion

Recombinant SAG1 produced in E. coli is a promising antigen that can be used in diagnostic assays for the detection of specific antibodies against T. gondii.     

Seroprevalence of Toxoplasma gondii Infections in Pregnant Women in Gorgan City,Golestan Province,Northern Iran-2012

Background

Toxoplasma gondii is one of the most prevalent parasites of human and warm- blooded animals. Toxoplasmosis is important especially in two groups: pregnant women and immunocompromised patients. If women acquire the primary infection during the pregnancy, it would be life threatening or remains severe disorders for the fetus. This study was performed to evaluate the seroprevalence of T. gondii infection in pregnant women referred to Health Center in Gorgan City, Golestan Province, northern Iran.

Methods

Serum samples were collected from pregnant women referred to Health Center in Gorgan City, south eastern Caspian Sea. Anti- Toxoplasma IgG and IgM antibodies were determined by commercially ELISA kits and the relation of infection with socio-demographic and risk factors such as age, education, occupation, cat ownership, soil contact and some other factors was studied.

Results

From 555 tested sera of pregnant women referred to Health Center in Gorgan, 39.8% had IgG antibodies against T. gondii and 3.4% were positive for IgM antibodies. A significant correlation was seen between T. gondii infection with age and soil contact.

Conclusion

About 60% of pregnant women in Gorgan City are seronegative against T. gondii, so they should considered as at risk persons.     

Genotyping of Echinococcus granulosus Isolates from Human Clinical Samples Based on Sequencing of Mitochondrial Genes in Iran,Tehran

Background

The present study was aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nad1 in Iran.

Methods

Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair (bp) for cox1 and nad1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already deposited in GenBank.

Results

Sixteen, (53.3%), 13 (43.3%), and 1 (3.3%) samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% (n=26) and 10% (n=3) were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 (n=9), G3 (n=1) and G6 (n=1).

Conclusion

In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping.     

Frequency of Toxoplasma gondii in HIV Positive Patients from West of Iran by ELISA and PCR

Background

Toxoplasma gondii, the obligate intracellular parasite is life threatening in AIDS patients. Diagnosis of toxoplasmosis is based on serological methods especially increasing of IgM and IgG titers, but finding of parasite or its components (antigenemia) may be beneficial method in order to detection of acute toxoplasmosis in immunocompromised patients.

Methods

Ninety-four serum samples from HIV positive patients were collected from Sanandaj, Kordistan west of Iran. These patients were lived in Sanandaj of whom 26 were prisoners infected with HIV virus in prison. Toxoplasma gondii antibodies were determined by IgG ELISA. T. gondii antigen was identified by capture-ELISA. PCR was performed on samples with T. gondii antigenemia. CD4+ T cells counts had been determined by flowcytometry and were obtained from records of each patient.

Results

Among the examined HIV seropositive individuals, 19.1% (18/94) and 5.3% (5/94) were positive for Toxoplasma-IgG and antigenemia, respectively. Besides, one of the samples was positively detected by PCR method. Mean age of participants was 37.9 ± 9.5 year. Prevalence of IgG antibody and antgenemia was higher in age group of 40-50 years old. The Mean of CD4+ T cells counts of participants (total of HIV+ patients, IgG positive patients and patients with antigenemia) was 699.2 ± 345.2, 655.1 ± 237.9 and 620.2 ± 215.1 respectively.

Conclusion

Capture-ELISA and PCR could confirm the T. gondii acute infection in HIV positive patients. For precise diagnosis of acute toxoplasmosis in HIV positive patient, performance of more studies based on more sensitive types of PCR is suggested.     

Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii,in Vitro

Background:

The aim of this study was to evaluate the effects of conjugated linoleic acid (CLA) on apoptosis of tachyzoites of T. gondii, RH strain (type I) and the cyst-forming Tehran strain (type II) in vitro.

Methods:

Toxoplasma strains were injected into the peritoneal cavity of BALB/c mice. The Tehran strain forms cysts in the brain of mice. Bradyzoites within the cysts are reactivated to proliferative tachyzoites, by dexamethasone. Tachyzoites were aspirated from the peritoneum of infected mice, and the percentage of viable parasites was estimated with trypan blue staining. Tachyzoites were inoculated into HeLa cells cultivated in DMEM medium. Different concentrations of CLA were evaluated on T. gondii in HeLa cells by the tetrazolium (MTT) colorimetric assay. Differentiation between apoptosis and cell death was determined by flow cytometry using Annexin V and propidium iodide (PI) double staining. The statistical analysis performed by GraphPad Prism version 6.00.

Results:

CLA induces apoptosis in virulent (RH) and avirulent (Tehran) strains of T. gondii. The results of MTT indicated that CLA could decrease the proliferation of tachyzoites of both strains in HeLa cells.

Conclusion:

Conjugated linoleic acid has anti-toxoplasmacidal activity on tachyzoites of T. gondii. Therefore, we recommended further studies on this component in order to achieve a new drug against the parasite.     

Evaluation of Recombinant SAG1 Protein for Detection of Toxoplasma gondii Specific Immunoglobulin M by ELISA Test

Background

Toxoplasmosis is a serious disease in immunocompromised patients and pregnant women. Differentiation of acute and chronic infection is a major challenge in serodiagnosis of the disease. Since the aim of this study was to assess the diagnostic utility of recombinant SAG1 (rec-SAG1) for the detection of Toxoplasma-specific IgM antibodies in human sera, by an enzyme-linked immunosorbent assay (ELISA).

Methods

The purified recombinant protein SAG1 was applied in house ELISA test and the ability of it in binding to specific immunoglobulin M in 30 serum samples of acute infected patients was evaluated. The results obtained by assays with the recombinant SAG1 and standard commercial assays were compared.

Results

The sensitivity and specificity of in house ELISA compared to a standard commercial ELISA (com-ELISA) were 80% and 90%, respectively.

Conclusion

It was concluded that the rec-SAG1 could be an alternative marker for detection of anti Toxoplasma-specific IgM and diagnosis of acute infection.     

Production and Evaluation of Toxoplasma gondii Recombinant Surface Antigen 1 (SAG1) for Serodiagnosis of Acute and Chronic Toxoplasma Infection in Human Sera

Background

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis.

Methods

This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain) was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D – thiogalactopyranoside (IPTG). A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA) as gold standard prior to testing them with the recombinant antigen.

Results

Tested sera were divided into the following groups:(a) The 74 T. gondii IgG positive (b) 70 T.gondii IgM positive (c) 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5), malaria (N=14), leishmaniasis (N=7),fasciolasis (N=4), sterengyloidiasis (N=1). Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA) were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively.

Conclusion

The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.     

Toxoplasma gondii infection in the United States, 1999-2000

Infection with Toxoplasma gondii can lead to congenital and acquired disease, resulting in loss of vision and neurologic illness. We tested sera collected in the National Health and Examination Survey (NHANES) from 1999-2000 for T. gondii-specific immunoglobulin G antibodies and compared these results with results from sera obtained in the NHANES III survey (1988-1994). NHANES collects data on a nationally representative sample of the U.S. civilian population. Of 4,234 persons 12-49 years of age in NHANES 1999-2000, 15.8= (age-adjusted, 95% confidence limits [CL] 13.5, 18.1) were antibody positive; among women (n = 2,221) 14.9= (age-adjusted, 95% CL 12.5, 17.4) were antibody positive. T. gondii antibody prevalence was higher among non-Hispanic black persons than among non-Hispanic white persons (age-adjusted prevalence 19.2% vs. 12.1%, p = 0.003) and increased with age. No statistically significant differences were found between T. gondii antibody prevalence in NHANES 1999-2000, and NHANES III. T. gondii antibody prevalence has remained stable over the past 10 years in the United States.     

Detection and Identification of Toxoplasma gondii Type One Infection in Sheep Aborted Fetuses in Qazvin Province of Iran

Background

The aim of this study was to apply the nested-PCR and bioassay methods in detection and genotyping of Toxoplasma gondii infection in provided sheep aborted fetus samples from Qazvin Province of Iran.

Methods

Eighteen sheep aborted fetal samples were studied by nested-PCR-RFLP, histopathological observation and microbiological assay. Bioassay in mice was carried out by inoculating the brain samples intraperitoneally.

Results

The results demonstrated the frequency of 66% infected sheep aborted fetal samples with T. gondii type one. Although we could not isolate any parasite from inoculated mice even after three passages, but it was confirmed histopathologically formation of cyst like bodies in prepared mice brain sections.

Conclusion

The results of the performed nested-PCR and formation of brain cyst in inoculated mice exhibited that T. gondii type one infection might be considered as one of the major causative agents for abortion in ewes.     

Elevated Toxoplasma gondii Infection Rates for Retinas from Eye Banks,Southern Brazil

We found significantly higher incidence of Toxoplasma gondii DNA in eye bank specimens from Joinville in southern Brazil (13/15, 87%) than in São Paulo (3/42, 7%; p = 2.1 × 10E–8). PCR DNA sequence analysis was more sensitive at locus NTS2 than at locus B1; a high frequency of mixed co-infections was detected.     

Genetic Characterization of Toxoplasma gondii from Zoo Wildlife and Pet Birds in Fujian,China

Background:

Toxoplasmosis, a worldwide zoonotic disease, is caused by Toxoplasma gondii. The distribution of genetic diversity of T. gondii in wild animals is of great importance to understand the transmission of the parasite in the environment. However, little is known about T. gondii prevalence in wild animals and birds in China.

Methods:

We conducted the genetic characterization of T. gondii isolated from Zoo Wild Animals and Pet Birds in Fujian Province, Southeastern China. Heart tissues were collected from 45 zoo animals and 140 pet birds. After identified using B1 gene, the genetic diversity of T. gondii isolates were typed at 11 genetic markers, including SAG1, 5’ and 3’-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3.

Results:

Seven of 45 zoo animals and 3 of 140 pet birds were positive by PCR amplification using T. gondii B1 gene specific primers. Of these positive isolates, 3 isolates from Black-capped (Cebus apella), Peacock (Peafowl) and Budgerigar (Melopsittacus undulatus) were successfully genotyped at 11 genetic loci, and grouped to three distinct genotypes: ToxoDB Genotype #9, #2 and #10, respectively.

Conclusion:

This is the first genotyping of T. gondii isolated from zoo wild animals and pet birds in Fujian, China. There is a potential risk for the transmission of this parasite through zoo wild animals and pet birds in this region.     

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background

Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.

Methods

Sixty-three BALB/c mice were injected intra-peritoneal with 5×10³ tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were injected with phosphate buffer saline as control group. Dot–ELISA was performed for detection of T.gondii antigen in mice sera and capture – ELISA was done as golden standard assay too.

Results

Toxoplasma gondii antigen was detected from day 2 in mice sera; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigenemia by dot – ELISA, no positive result was detected in control mice by dot- ELISA.

Conclusion

Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with capture-ELISA.     

Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant

Previous immunological studies from our laboratory have demonstrated the potential role of Toxoplasma gondii antigens SAG1 and GRA2 as vaccine candidates. To further evaluate the vaccine's effects, a series of recombinant DNA vaccines pVAX1-SAG1, pVAX1-GRA2 and pVAX1-SAG1-GRA2, termed pSAG1, pGRA2 and pSAG1-GRA2, respectively, were constructed. A plasmid pVAX1-S/PreS2, termed pSPreS2 encoding hepatitis B virus (HBV) surface antigen (HBsAg) S and PreS2 as a novel genetic adjuvant, was also constructed. The expression abilities of those DNA plasmids were examined in HFF cells by Western blotting. Then BALB/c mice were intramuscularly immunized with DNA plasmids and followed by challenging with the highly virulent T. gondii RH strain. The results demonstrated that the recombinant DNA vaccine pSAG1-GRA2 was capable of eliciting high levels of antibodies, a Th1 type of immune response with significant production of IFN-γ and low levels of IL-4 or IL-10 in BALB/c mice, and partial protection against the acute phase of toxoplasmosis as compared to pSAG1, pGRA2 and controls. In addition, the adjuvant pSPreS2 formulated with DNA vaccine induced a Th1 type of immune response and therefore might be a novel genetic adjuvant to DNA vaccine for further investigation.     

Effects of Toxoplasma gondii Infection in Level of Serum Testosterone in Males with Chronic Toxoplasmosis

Background

Toxoplasma gondii is an intracellular protozoan parasite that infects human and animals. Toxoplasma parasites are isolated from different parts of animals even from semen but there are little information about the effect of toxoplasmosis on fertility in animals and humans. In present study, the effect of chronic toxoplasmosis on serum levels of testosterone in men was studied.

Methods

In this case-control study, 1026 men referred to Arak Post Marriage Center were selected. Three ml of blood samples were collected and sera separated by centrifugation at room temperature. These sera were analyzed for detection of anti-T. gondii IgG antibody. Next 365 positive sera were selected as cases and also the same number of negative sera (365) as controls. Finally the level of testosterone was analyzed for the cases and controls samples.

Result

Serological tests on the sera of 1,026 men in Arak City showed that 365 of them had anti-Toxoplasma antibody. Comparison of testosterone concentration in case and control groups showed that testosterone concentration in case group was less than control group and this difference was statistically significant (P<0.05).

Conclusion

The chronic toxoplasmosis could affect reproductive parameters in men.     

Histological Changes of the Ovary in Pregnant Mice Vaginally Exposed to Toxoplasma gondii

Background:

Congenital toxoplasmosis is one cause of abortion. Infection can disrupt ovarian cycles and because toxoplasmosis is an infectious disease may have a similar effect on the ovaries. The purpose of this study was to investigate the pathological changes in the ovaries due to toxoplasmosis.

Methods:

Tachyzoites of Toxoplasma gondii were harvested from peritoneal fluid of mice, experimentally infected. Two females and one male mouse were housed per cage for mating in the overnight. The pregnant mice were divided into experimental and control groups. Experimental group were infected by parasite but the control group received the normal saline. The experimental and control mice were euthanized. Ovaries and uterine horns of animals were removed and prepared for light microscopy.

Results:

Ovaries of infected pregnant mice presented gross morphological differences compared to the control groups. In ovaries of experimental groups, changes of corpus luteum were observed. The comparison of experimental and control groups revealed that the number of primary follicles, secondary follicle, atretic primary follicles and atretic secondary follicles had significant differences (P≤0.001).

Conclusion:

Toxoplasma gondii alters ovarian follicular growth and development in mice. In addition, it alters number of different phases of follicles and corpus luteum in ovaries of mice.     

Prevalence of Toxocara Spp. eggs in Public Parks in Tehran City,Iran

Background

The objective of the present research was to determine the frequency of Toxocara spp. eggs in soil samples of public parks, in the city of Tehran, Iran.

Methods

A total of 600 soil samples were taken from 120 parks between Aprils to November, 2008. Soil samples were collected from 5 distinct sites in the parks. The samples were washed with saline solution and the collected sediment from each park were equally divided and examined by floatation and Petri dish methods for Toxocara eggs.

Results

Ten percent were contaminated with Toxocara spp. eggs. The number of observed Toxocara eggs in each microscopic field was varied from 1-3. No significant differences were observed between floatation and Petri dish methods.

Conclusion

Our public parks showed a high risk of toxocariasis and the need for preventive studies.