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

Background: Toxoplasmosis, a parasitic disease caused by compilation protozoan agent Toxoplasma gondiithat led to significant financial and quality-adjusted life-year losses. Consumption of undercooked or raw meat has been regarded as a major route of transmission. The present study was conducted to determine the seroposevitity rate of T.gondii in sheep and goats by serological and molecular tests and also genotyping of obtained isolatesin northeast of Iran.Methods: Blood and tissue samples (diaphragm, heart) of 296 animals (including 168 sheep and 128 goats) were collected from slaughterhouse in Quechan city from august 2016 to April 2017. Serum samples examined by the Modified agglutination test (MAT) and the Nested-PCR method performed to amplify the fragment of the B1 gene to detect parasite DNA on diaphragm and heart tissues of seropositive animals. PCR-RFLP method of GRA6 gene was used to determine the genotype of T. gondii. Also, sequencing analysis was performed to evaluate the Toxoplasma type strains. Results: Serum positive for MAT results were found in 27.4% (46/168) of Sheep and23.4% (30/128) of goats. Positive Nested-PCR of B1 gene results in diaphragm and heart tissues of sheep and goats was 47.8% (22/46) and 26.1% (12/46), 40% (12/30) and 23.3% (7/30), respectively. Nested-PCR of GRA6 gene results were positive in 10 samples (7 sheep and 3 goats) that RFLP technique results with using MseІ enzyme revealed genotype І. Sequencing and Phylogenetic analysis revealed DNA of all samples were closely related to Toxoplasma type І.Conclusions: Concerning to highseropositivityrate of toxoplasmosis in studied region, undertaking an appropriate preventive program for reducing the prevalence of T. gondii infection by raw or undercooked meat consumption of livestock recommended. Our study supports the notion that consumption of raw and undercooked meat of these animals can be a probable source of human toxoplasmosis.

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

Background: 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. Methods: 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. Results: 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). Conclusion: 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.

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

ABSTRACT 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 &lt; 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. HIGHLIGHTS

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<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<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.

Genetic characterization of Toxoplasma gondii isolates from chickens in India by GRA6 gene sequence analysis

PCR-RFLP and nucleotide sequencing based genotyping of Toxoplasma gondii Indian isolates (Izatnagar and Chennai isolates and Chennai clone) vis-a vis RH-IVRI strain was conducted by targeting GRA6 as genetic marker. The 791 bp GRA6 product was PCR amplified from the genomic DNA of different T. gondii Indian isolates, including the RH-IVRI strain. Tru1I restriction endonuclease based PCR-RFLP of GRA6 sequence produced polymorphic digestion pattern that discriminated the virulent RH-IVRI strain (as type I) from the moderately virulent local isolates as type III. The PCR amplicon of T. gondii GRA6 from RH-IVRI strain as well as from the local isolates were cloned in cloning vector and custom sequenced. The nucleotide and deduced amino acid sequences of T. gondii isolates were aligned with that of the type I, II and III strains (RH, BEVERLEY, ME49, C56, TONT and NED) available in public domain and analyzed in silico using MEGA version 4.0 software. Nucleotide sequencing and phylogenetic analysis of GRA6 marker from the Indian isolates revealed a close genetic relationship with type III strains of T. gondii. Further, detection of a single nucleotide polymorphism (SNP) at positions 162 and 171 of the GRA6 marker, established the lineage of Indian isolates as type III. This is the first report on characterization of T. gondii lineage as type III in selective chicken population of India based on PCR-RFLP and sequence analysis of GRA6 gene.