GRA6 Gene Research Articles

Serological and molecular survey of Toxoplasma Gondii in aborted livestock fetuses from Northeast Iran

BackgroundToxoplasmosis not only leads to abortion in humans but also in herbivores, which causes significant financial and quality-adjusted life-year losses. The present study aimed to determine the prevalence of toxoplasmosis in aborted fetuses via serological and molecular assays. Moreover, the genotypes of the obtained isolates were detected.MethodsSerological and molecular methods were used to study aborted fetuses from Bojnourd City, North Khorasan Province, Iran, which included 52 ovines and 16 bovines. Nested PCR of the B1 gene was used to detect parasite DNA in brain tissues. The PCR-RFLP method for the GRA6 gene was used to determine the genotype of T. gondii.ResultsOut of 68 aborted fetuses, 16.1% showed the presence of anti-T. gondii IgG. Among these, 11.7% were identified in bovine fetuses and 4.4% in ovine fetuses. Additionally, two (2.94%) samples of ovine tested positive for anti-T. gondii IgM. Our PCR analysis detected parasite DNA in two cases (2.94%) among 11 IgG-positive samples. All obtained isolates belong to type I of T. gondii.ConclusionInfection with Type I of T. gondii during the neonatal period may partly be responsible for abortion and economic losses in livestock farming in our studied region. To understand the molecular epidemiology and genotypes of T. gondii associated with abortion, further evaluation of aborted samples from different geographical locations is necessary.

Seromolecular study on the prevalence and risk factors of Toxoplasma gondii infection in pregnant women referred to a gynecology hospital in Urmia, northwest part of Iran in 2022

Toxoplasmosis is a frequent infection among the human population. The infection can cause devastating complications for the fetus during pregnancy. The present study aimed to determine the serological and molecular prevalence of the infection and molecular characterization of Toxoplasma gondii isolates among pregnant women referred to Kowsar Hospital, Urmia, Iran. In a cross-sectional study, 340 blood samples were collected from pregnant women referred to Kowsar Hospital, Urmia, Iran from May to July 2022. Anti-T. gondii IgG and IgM seropositivity were determined by enzyme-linked immunosorbent assay. PCR was carried out by targeting the GRA6 gene of the parasite on all patients’ buffy coats. Anti-T. gondii IgG and IgM antibodies were positive in two (0.6%) women, and 101 (29.7%) women had anti-T. gondii IgG and 70.3% were seronegative. PCR was positive in two IgM-positive women, and both isolates belonged to T. gondii carrying the GRA6 allele of lineage I. The risk of infection was significantly higher in women who had constant contact with cats and soil, and who were residents of rural areas. The two IgM-positive women were asymptomatic regarding acute toxoplasmosis. According to the results of the present study, the prevalence of toxoplasmosis in pregnant women in Urmia is similar to its prevalence in other areas in northwestern Iran, and despite the low prevalence of acute infection, it should not be ignored.

Molecular Detection of Toxoplasma gondii Oocytes in Soil Samples from Guilan Province, Northern Iran.

Soil is an appropriate substrate for the storage and transmission of oocytes of Toxoplasma gondii. Ingestion of soil contaminated with T. gondii oocysts is a major transmission route of human and animal toxoplasmosis. The present study was carried out to investigate soil contamination with T. gondii oocysts in urban and rural areas of Guilan Province, northern Iran. Overall, 208 soil samples were collected from 16 cities and villages in Guilan Province, northern Iran from Oct 2020 to Nov 2021. Soil samples were investigated using modified sucrose flotation technique. Real-time polymerase chain reaction was used to detect presence of T. gondii DNAs in the samples. Positive samples were further analyzed using nested polymerase chain reaction for GRA6 gene. Moreover, six selected positive samples were used for amplifying and sequencing of the GRA6 gene. Overall, 31 samples were positive for T. gondii with frequency of 14.9% and ranging from 10.9% in rural areas to 16.3% in urban areas. Statistical analysis showed significant differences between the seasons (P=0.003). The phylogenetic analysis illustrated that our six sequences were similar and closely related to Type I strain of T. gondii. Results showed relatively high levels (14.9%) of T. gondii oocytes in soil samples of Guilan Province, northern Iran, which provided essential data for the effective prevention and control of toxoplasmosis in the region.

Prevalence and risk factors of Toxoplasma gondii infection among women with miscarriage and their aborted fetuses in the northwest of Iran.

Toxoplasmosis is a worldwide disease of various animals and human and results from infection with the Toxoplasma gondii parasite. Abortion and congenital defects are important consequences of the T. gondii infection. The aim of this study was to determine the Toxoplasma-induced abortions among women with miscarriage and the presence of T. gondii in their aborted fetuses in Urmia, the northwest of Iran. This cross-sectional study was conducted with 215 women with abortion and their aborted fetuses, from 2020 to 2021. Seroprevalence of anti-Toxoplasma IgG and IgM were determined using the sera of the aforesaid women. Nested PCR was carried out using RE-529 gene sequences, and sequencing was performed using the T. gondii GRA6 gene on the remnant of pregnancy after abortion. The tissue positive samples were then subjected to another PCR on GRA6 gene and sequenced for genotyping. Among 215 serum samples of women with abortion, 70 (32.6%) were positive for anti-Toxoplasma IgG, and three (1.4%) were positive for IgM. The RE-529 sequence of T. gondii was positive in three (1.4%) of the aborted fetuses. The analysis of GRA6 gene indicated that all three positive samples carried a GRA6 allele (GRA6I) of T. gondii type I genotype. Our findings suggest that T. gondii is one of the causative agents of spontaneous abortion in West Azerbijan Province, the northwest of Iran.

Genetic Diversity of Toxoplasma gondii by Serological and Molecular Analyzes in Different Sheep and Goat Tissues in Northeastern Iran.

Toxoplasmosis is a parasitic disease caused by compilation protozoan agent Toxoplasma gondii, leading to significant financial and quality-adjusted life-year losses. Overcooked or raw meat consumption has been a considerable transmission route. The present study was conducted to determine the seropositivity rate of T. gondii in sheep and goats by serological and molecular tests and genotyping of obtained isolates in northeast Iran. Blood and tissue samples (diaphragm, heart) of 296 animals (including 168 sheep and 128 goats) were collected from the slaughterhouse in Quchan Country from august 2016 to April 2017. Modified agglutination test (MAT) and the PCR method performed to detect parasite DNA on tissues.PCR-RFLP method of GRA6 gene was used to determine the genotype of T. gondii. In addition, sequencing analysis was performed to evaluate the Toxoplasma type strains. Serum positive for MAT results were found in 27.4% of sheep and 23.4% of goats. Positive PCR of B1 gene results in diaphragm and heart tissues of sheep and goats was 47.8% and 26.1%, 40% and 23.3%, respectively. PCR of GRA6 gene results were positive in 10 samples that RFLP technique results using MseI enzyme revealed genotype I. Sequencing and phylogenetic analysis revealed DNA of all samples was closely related to Toxoplasma type I. Concerning the high seropositivity rate of toxoplasmosis, undertaking an appropriate preventive program for reducing the prevalence of T. gondii infection by raw or undercooked meat consumption of livestock is recommended. Our study supports the notion that these animals' consumption of raw and undercooked meat can be a probable source of human toxoplasmosis.

Molecular prevalence and genetic diversity of Toxoplasma gondii in free-range chicken in Northeastern Libya.

Toxoplasma gondii is one of the zoonotic protozoa parasites. It can prevalently infect humans and warm-blooded animals, causing human health problems and substantial economic losses to the livestock industry worldwide. Chicken is one of the potential sources of toxoplasmosis, but there is no report of the prevalence of toxoplasmosis and their genotypes in free-range chickens in Libya. This study aims to conduct a survey of molecular prevalence and identify the T. gondii genotype in free-range chickens and its association with the risk factors of age, gender, and region in Northeastern Libya. This study was conducted by examining a total of 315 free-range chicken organs (brain and heart) derived from three administrative districts in Northeastern Libya. The molecular prevalence was determined by PCR technique using B1 gene amplification. and the T. gondii genotype was determined by nested PCR-RFLP of GRA6 gene amplicon with restriction enzymes (MseI). The overall molecular prevalence of T. gondii in free-range chicken in all three districts was 9.5% (30/315), and the highest (15.4%) was in the Al-Marj district (p = 0.01; x 2 = 9.238). The highest prevalence of T. gondii by age was in chickens aged more than 2 years (p = 0.001; x 2 = 15.530). The difference in T. gondii prevalence in male and female chickens was not significant (p = 0.372; x 2 = 0.798). The predominant genotype I (93.3%) had identified at position 544 and 194 bp at the GRA6 marker, and only two positives were from genotype II (6.7%) at 700 and 100 bp fragments. The molecular prevalence of toxoplasmosis in free-range chicken in three districts in Northeastern Libya was 9.5%, and the highest rate was shown in the Al Marj district. Chicken by age more than 2 years had more risk to transmit toxoplasmosis in human. There was no different infection risk by consuming male or female free-range chicken. It is the first report to determine the predominant genotype, which was genotype I.

Genetic characterization of Toxoplasma gondii isolates from human spontaneous aborted fetuses in Jahrom, southern Iran

Toxoplasma gondii (T. gondii) is an intracellular protozoan that infects the fetus through the placenta and leads to severe complications in the fetus. One of the complications of congenital toxoplasmosis is spontaneous abortion. The prevalence of toxoplasmosis infection was investigated among spontaneously aborted fetuses (SAFs), and the genotypes of parasite isolates were determined in the present study. Placentas from 330 samples of SAFs were collected in Jahrom (Fars province) from February to September 2018. DNA was extracted from each placental tissue. The T. gondii infection was detected using nested polymerase chain reaction (Nested-PCR) assay based on a 529 bp repeat element (RE) gene. Afterward, Toxoplasma was genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) based on the GRA6 gene. The frequency of T. gondii infection was found to be 14.5% (48 out of 330 samples). Genotyping of nine T. gondii isolates revealed that all belonged to genotype II. Statistically, the prevalence of T. gondii infection was significantly correlated with the education levels of the mothers and the age of the fetus (P < 0.05). The lowest prevalence of Toxoplasma infection belonged to mothers with university education and the highest frequency of infection was observed among the fetuses in the age group of 8–9 weeks. The findings of the present study suggest a significant role for toxoplasmosis in SAFs in Jahrom city.

Genotyping of Toxoplasma gondii in sheep and cattle meat using PCR-RFLP technique

Toxoplasma gondii is an obligate, intracellular parasite, with worldwide distribution. The main source of infection for humans is livestock and meat-producer animals. The relationships between Toxoplasma genotype and biological characteristics of the parasite have already been identified. According to the pathogenicity of the parasite in laboratory animals, Toxoplasma is divided into three genotypes included type I, II and III. Understanding the genotype of the parasite, could help us to predict clinical features and severity of disease. The aim of this study was to identify genotypes of T. gondii in cattle and sheep meat and meat products in Ahvaz city southwest of Iran.One hundred and ninety samples of tongue, heart and muscles of sheep and cattle and meat products, including sausages and burgers, were collected from slaughterhouses and stores. To identify Toxoplasma gondii, DNA were extracted from samples and B1 gene were amplified by specific primers. To determine the genotype of T.gondii, PCR-RFLP was done on positive samples using by amplifying GRA6 gene and endonuclease Msel enzyme. Data analysis showed that the strain of the parasite in all positive samples belonged to genotype I.In this study the predominant Toxoplasma genotype was type I which can cause severe clinical symptoms in immunocompromised patients. Further research is needed to determine the genotype of the parasite in humans and other animals.

Toxoplasma gondii antibody prevalence and isolation in free-ranging cats in Okinawa, Japan.

Cats are an important host of Toxoplasma gondii from an epidemiological perspective because they are the only definitive hosts that excrete oocysts in their feces. In this study, 201 free-ranging cats in Okinawa were examined for T. gondii infection. Using the latex agglutination test, we detected antibodies against T. gondii in 26.9% (54/201) of the cats. Oocysts of T. gondii were not detected upon microscopic examination of the feces of 128 cats. T. gondii was isolated from the tissues of 9 out of 24 seropositive or pseudo-seropositive cats with a bioassay using laboratory mice. Genotyping for the GRA6 gene revealed that five and four of the isolates were type I and II, respectively.

Fatal systemic toxoplasmosis in a 3-month-old young tibetan goat (Capra hircus)

BackgroundToxoplasmosis is one of the most common parasitic infections in both humans and animals. It is a frequent cause of abortion and stillbirth in intermediate hosts, especially sheep and goats but rarely causes fatal clinical form in adult animals.Case presentationIn contrast, the study reports an unusual fatal case of toxoplasmosis in a young goat naturally infected with type II strain of Toxoplasma gondii. A three-month-old female goat was presented with dyspnea and died few days later. Grossly, lungs were firm, edematous and mottled with disseminated whitish areas. Generalized lymphadenopathy was found. The histopathological examination showed necrotic interstitial bronchopneumonia and necrotizing lymphadenitis with intralesional free and clustered within macrophages tachyzoites of T. gondii. DNA extracted from lungs and lymph nodes was positive for T. gondii by a fast qPCR. PCR-RFLP analysis and sequencing of GRA6 gene showed that the isolated strains belonged to type II genotype.ConclusionsThis is an unusual report of acute systemic toxoplasmosis caused by the type II strain of T. gondii with a fatal outcome in a young goat.

Isolation and Genotypic Characterization of Toxoplasma gondii based on GRA6 Gene from Environmental Soil Samples in Mazandaran Province, North of Iran

Soil is one of the environmental sources of Toxoplasma gondii oocysts. The other hand, genotype of the parasite is one of the important factors for its pathogenicity. Due to the importance of toxoplasmosis on public health, this study aimed to isolation and genotyping of T. gondii in environmental soil samples of Mazandaran Province, north of Iran. Overall, 192 soil samples were collected from different areas in Mazandaran Province from Apr to Sep 2014. The flotation method was used for recovering oocysts. Then, soil samples were investigated for DNA detection of T. gondii using nested PCR of RE gene, genotyping with Semi-nested PCR of GRA6 gene and restriction fragment length polymorphism (RFLP) analysis. Results were analyzed using Chi-squared test. A significant difference was considered with a P<0.05. From 192 soil samples, T. gondii DNA was detected in 150 samples (78.1%). Then genotype of 23 samples was determined (91.3% type I and 8.7% type II). Prevalence of T. gondii in soil samples of Mazandaran province, north of Iran is high and T. gondii GRA6 type I is predominant. Soil can be the most important source of severe toxoplasmosis in this province.

Toxoplasma infection in patients with myocardial infarction.

Toxoplasma gondiiis awidespreadprotozoanparasitethat infects one third of the globalhumanpopulation. Very little information is known about the impact of T.gondii on patients with heart disease. The aim of the present study was to determine the association between T.gondii exposure and patients suffering from myocardial infarction.The infection rate of anti-Toxoplasma IgG antibodies in 86patients with myocardial infarction (troponin‑T positive) and 86age and gender-matched controls (troponin‑T negative) was examined using enzyme-linked immunoassays. The DNA extraction was performed on separated buffy coats of serologically positive blood samples (32samples with high titer of anti-Toxoplasma IgG).The GRA6 gene ofT. gondii was amplified using PCR. The existence of polymorphic restriction sites for endonuclease MseI was used with the PCR-RFLP method and the bases of GRA6 gene were sequenced to determine the type of strains (I, II and III).A positive anti-Toxoplasma IgG level was found in 61.6% of the myocardial infarction samples and in 24.4% of the healthy controls (P-value < 0.05). The PCR results showed that only 3 of the anti-Toxoplasma IgG positive patients were found to be positive with GRA6 gene for T.gondii. The PCR-RFLP results showed that 2 of the 3positive sample had 75bp and 623 bp DNA fragments belonging to typeII genotype. The sequencing result confirmed the genotype II of T. gondii. Toxoplasma infection should be considered in myocardial infarction cases.

Prevalence of Toxoplasma gondii Infections in Chicken Hearts from Farmers' Markets and Supermarkets in the Tai'an Region of China

The detection of Toxoplasma gondii in quick-frozen chickens is a good indicator of the possible transmission risk that this parasite poses to human consumers. The aim of this study was to investigate the occurrence of T. gondii in quick-frozen chicken hearts using a nested PCR assay. Heart samples (n = 720) from farmers' markets and supermarkets in the Tai'an region, People's Republic of China, were collected, and the DNA extracted was analyzed for the presence of T. gondii. The overall prevalence of T. gondii in all samples was 10.7%, but the rates for samples from farmers' markets (19.2%) and supermarkets (2.2%) were significantly different (P < 0.01). Nested PCR restriction fragment length polymorphism genotyping was performed on the 77 positive samples based on the T. gondii SAG3 and GRA6 gene loci. SAG3 genotyping revealed a mixed infection rate of 89.6% for type I and type I/II strains, and GRA6 genotyping revealed an infection rate of 98.7% for type I strains. Our results revealed a high prevalence of T. gondii in chicken hearts from farmers' markets, and most strains were mainly type I strains. Further studies are needed to determine whether quick-frozen chicken hearts are involved in disease transmission to human consumers in this area.

Molecular Genotyping of Toxoplasma gondii in Sheep Aborted Fetuses Reveals Predominance of Type I Infection in Southwest of Iran.

Background:We aimed to detect Toxoplasma gondii in ovine aborted fetuses and evaluate its genetic variations in the southwest of Iran.Methods:This cross-sectional study was performed on 100 aborted ovine fetuses collected from the different region of Kohgiluyeh and Boyer-Ahmad Province, Iran, in lambing season during 2017 and 2018. DNA was extracted from the brain samples of all of the aborted fetuses and PCR amplified, targeting a 529 bp repetitive element gene of T. gondii. Moreover, to find out the heterogeneity of the positive samples, PCR-DNA amplification of the two main genetic markers, B1 and GRA6, of T. gondii were performed. Nucleotide sequencing and phylogenetic analysis were performed, using the BLAST program and MEGA-X software.Results:The 529 bp gene of T. gondii was detected in 2 out of 100 (2%) of the ovine aborted samples. The sequences analysis of GRA6 and B1 genes revealed that both isolates from the aborted fetuses of sheep belonged to type I of T. gondii. Intra-divergence was more seen in GRA6 gene whereas less divergence was observed in B1 gene.Conclusion:Congenital infection with Type I of T. gondii during the neonatal period is associated with abortion in ovine. Evaluation of more aborted samples from broader geographical areas is needed to elucidate the molecular epidemiology and also the genotypes of T. gondii associated with abortion.

Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate.

Aim:The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid.Materials and Methods:Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells.Results:The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304.Conclusion:This research cloned rGRA-4 in pET SUMO plasmid.

Molecular Identification of Toxoplasma gondii in the Native Slaughtered Cattle of Tehran Province, Iran

Background: Toxoplasmosis, caused by Toxoplasma gondii, is a common parasitic disease, affecting almost one-third of the world’s population. It is transmitted by ingestion of food or water contaminated with oocysts excreted by cats and the consumption of raw or undercooked meat from ruminants. This study aimed at molecular characterization of T. gondii in native cattle from West of Tehran, Iran. Methods: A total of 180 samples were collected from the cattle diaphragms (n=80) and heart muscles (n=100) from multiple slaughterhouses. The nested Polymerase Chain Reaction (PCR) assay was carried out to amplify the GRA6 gene of T. gondii. The PCR-Restriction Fragment Length Polymerase (PCR-RFLP) assay was also performed on positive samples, using Tru1I (MseI) restriction enzyme. Data were statistically analyzed using SPSS (v.15.0). Results: T. gondii was found in 38 out of 180 (21.1%) samples. The infection rate in heart muscle samples (16.66%) was significantly (p&lt;0.05) higher than the diaphragm samples (4.44%). The PCR-RFLP pattern by MseI enzyme showed that 13 (7.22%) samples were genotype II, while 25 (13.88%) were genotype III, having statistically meaningful difference (p&lt;0.05). No genotype I was found in the studied isolates. Conclusion: Based on our findings, the frequency of T. gondii was high in the study area. Therefore, educational programs need to be implemented in order to inform people about the risks of raw or undercooked meat consumption.

Toxoplasma gondii in Aborted Fetuses of Sheep in Ardebil Ar-ea, North-West of Iran

One of the severe complications of toxoplasmosis is the induction of abortion in sheep, goats, and human beings. The rate of abortions related to toxoplasmosis in sheep varies between 10% and 23% in the United States and European countries. In Iran, Toxoplasma infection were diagnosed in aborted ovine fetuses between 13.5% and 69% in the brain samples based on PCR. The purpose of this study was to investigate the prevalence of abortions related to T. gondii among the aborted fetuses of sheep in Ardabil area, North-West of Iran. The brains of 75 aborted sheep fetuses in Ardebil area were investigated with nested PCR method using fragments of the GRA6 gene during lambing seasons in 2014-2015. Meanwhile, thoracic and abdominal fluids of aborted fetuses and 200 serum samples collected from the herds affected by abortion were examined for the existence of anti-T. gondii IgG. For 48 out of the 75 fetal brain samples (64%), the results of nested PCR test were found to be positive. Furthermore, antibodies against Toxoplasma was observed in 136 (68%) collected samples of sheep and 21 samples (28%) of fetal fluids. There is the significant role of T. gondii in the abortions of sheep in Ardabil area. Meanwhile, this condition can also be dangerous for human beings because of their consumption of sheep meat.

The dense granule protein 8 (GRA8) is a component of the sub-pellicular cytoskeleton in Toxoplasma gondii.

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma β-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.

Mixed Toxoplasma gondii infection and new genotypes in feral cats of Quintana Roo, México

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii that infects homeothermic animals, including humans. To date, as many as 287 genotypes have been described worldwide. Genetic characterization of the parasite is crucial because the parasite type can determine the presentation and severity of toxoplasmosis. Previously, we reported that the Yucatán Peninsula has a frequency of infection of over 70% in humans and other animals; moreover, there are seven species of felids, including domestic cats; thus, we hypothesized that this might be a region with a high diversity of the parasite. Nevertheless, no genotyping of this protozoan has been performed in this region. Thus, the aim of this study was to genotype T. gondii from naturally infected feral cats of Quintana Roo, within the Yucatán Peninsula, and to describe its genetic variability. Eleven feral cats were captured and bled to obtain the buffy coat; then, they were euthanized to collect target organs or tissues to extract DNA. Samples were processed by PCR for diagnosis, and ten polymorphic markers were genotyped by PCR-RFLP. Atypical GRA6 gene products were cloned and sequenced. Ten of the eleven cats were PCR positive for toxoplasmosis in blood; of these, seven had mixed infections. Also, two isolates were obtained from the heart and diaphragm of two animals. At least 23 different genotypes were detected, from which 18 are new worldwide. From the atypical GRA6 gene cloning and sequencing analysis, a mixed infection was discovered, due to one strain identical to GT1 and another to VAND. In conclusion, T. gondii genetic diversity in the region is high and different from that in other regions, with new genotypes exclusive to México and some others shared with USA and South America.

New prevalence surveillance of Toxoplasma gondii among rodents and stray cats by ELISA avidity and nested PCR methods, Northeast of Iran.

Rodents and stray cats are the sources of many parasitic infections including T. gondii, for other animals and human. Toxoplasmosis has a wide range of laboratory factors in its intermediate and definite hosts. Regarding the importance of rodents and stray cats as the hosts that spread the Toxoplasma gondii, it is necessary to obtain comprehensive information about these animals in the life cycle of T. gondii. The objective was to investigate the new prevalence of toxoplasmosis among target animals in Iran, using GRA6 gene in combinacion with ELISA avidity. In this study, 286 rodents and 210 stray cats were collected and their heart tissues extracted to obtain DNA, blood samples and IgG Ab of T.gondii parasite. We detected the positive tissue samples in our study by the nested-PCR method. Then, we examined T. gondii IgG ELISA avidity for assessment of toxoplasmosis among rodents and stray cats. This study, was conducted in January to March 2017, based on the prevalence study. The findings revealed that 246/286 (86.01%) of rodents and 180/210 (85.71%) of stray cats were positive by IgG ELISA avidity methods. moreover, 68 rodents samples and 38 stray cats samples were positive concerning the GRA6 Toxoplasma gene; and these positive samples were at intermediate levels for IgG avidity. We concluded that the new prevalence of toxoplasmosis among rodents and stray cats was at high levels, using the serologic method in Northeast of Iran and the results of quantitative ELISA avidity were as the same as those of the nested-PCR for detecting recent toxoplasmosis in these hosts.