SAG3 Gene Research Articles

Causes of mortality and morbidity in the endangered southern brown bandicoot (Isoodon obesulus obesulus).

To investigate and document the causes of death and the parasites of free-ranging populations of southern brown bandicoots in southern, central Victoria, Australia. Gross necropsy, histopathology and identification of ectoparasites and endoparasites were performed on 62 dead southern brown bandicoots opportunistically collected from July 2011 to January 2018. Molecular methods were used to detect and genetically characterise the presence of Toxoplasma gondii in select tissues. The vast majority of animals died as a result of either motor vehicle trauma or predation (45/62). A further seven bandicoots died from varying causes with either diagnostic gross or microscopic evidence of pathology, and there were five ejected pouch young. The cause of death was undetermined in another five cases. Toxoplasma gondii was detected in one southern brown bandicoot, which demonstrated polymorphisms in the B1 and SAG3 genes. This is the first definitively documented case of T. gondii infection in this species in Victoria. All of the identified parasites have previously been reported in southern brown bandicoots, apart from a metastrongyloid lungworm, which is reported here for the first time. Motor vehicle trauma and predation are highlighted as causes of fatality that likely threaten these populations of southern brown bandicoots. Further investigation into the role that disease, particularly toxoplasmosis, may be playing in southern brown bandicoot population decline is recommended.

Multiple Typing Approach to Characterize Toxoplasma gondii Strains from Captive and Livestock Species in Northern Italy Suggests the Circulation of Type-II Variants.

Toxoplasma gondii is a widespread foodborne parasite that affects both humans and animals worldwide. The genetic characterization of this parasite has become crucial due to its epidemiological and clinical implications. The present study focused on the direct genetic characterization of T. gondii-positive DNA samples from Northern Italy, using three standardized genotyping methods. Of the 87 PCR-positive DNA samples obtained from livestock, wild, and captive animals, 9 were confirmed, quantified, and subsequently subjected to typing procedures: microsatellite analysis, PCR-RFLP, and partial sequencing of GRA6 and SAG3 genes. Most of the typed samples, derived from clinical cases of toxoplasmosis, specifically related to abortion outbreaks in small ruminants, as well as one case of systemic toxoplasmosis in lemur (Lemur catta). Predominantly, Type II strains were observed, with non-clonal variants identified in a sheep and a lemur. Sequencing of SAG3 revealed specific single nucleotide polymorphism, previously documented in European type-II variants, with phylogenesis suggesting a potential genetic relatedness between T. gondii population in Italy, Spain, and France. By utilizing three distinct genotyping methods, this study provides the first comprehensive microsatellite profiles of T. gondii in Italy, and additional insights into the molecular variability of T. gondii strains circulating in this country from different epidemiological settings.

Genotyping and Phylogenic tree of Toxoplasma gondii in one-humped camels (Camelus dromedarius) in Al- Diwaniyah province in Iraq

The current study is carry out in Al- Diwaniyah province; in Iraq between (September 2023 - March 2024) for determine the seroprevalence of Toxoplasma gondii in camels and molecular identification with phylogenetic analysis. (140) blood and tissue samples were collected from camels from the slaughter of the Al-Diwaniyah province. The results of total serological prevalence showed that 72 (51.42%) of the camels were infected. Genomic DNA was extracted from 140 camel’s tissues samples and SAG3 gene was amplified by PCR. PCR results showed that camels tissues samples 55/140 (39.28 %) have SAG3 gene. Ten PCR positive products of local Toxoplasma gondii isolates were DNA sequencing and compared with other isolates in GenBank. After that, the phylogenetic relationship was analyzed for this parasite using phylogenetic UPGMA tree (MEGA X version). Sequencing and phylogenetic results of Toxoplasma gondii isolate (No.1, 3, 5, 6, and 10) were showed closed genetic related into Toxoplasma gondii genotype I. The camels T. gondii isolates (No.7) were showed closed genetic related into Toxoplasma gondii genotype II. Whereas, the camels T. gondii (No.2, 4, 8, and 9) were showed closed genetic related into Toxoplasma gondii genotype III. At total genetic change (0.01). The homology sequence identity between local The Toxoplasma gondii isolates and NCBI-BLAST related genotype of T. gondii were reveals that genetic homology sequence identity was ranged (99.32% - 100%).

Phylogenic study of toxoplasmosis in aborted women in Al- Diwnaniya province

The current study included the detection of Toxoplasma gondii from aborted women attented to the Maternity and Childhood Teaching Hospital to detected the rate of abortion due to toxoplasmosis using PCR technique, the samples which was used placenta and fetus, this study also identify the differentiation between it by phylogenic with SAG3 gene.
 A total of 60 ( placenta and fetus) samples were collected randomly from aborted women who received to the Maternity and Childhood Teaching Hospital.
 The present study include molecular method to detected small subunit ribosomal RNA ( ssRNA) using Nested Polymerase Chain Reaction ( nPCR) ,the results of the nPCR for ssRNA gene, the result showed that the rate of toxoplasmosis in the aborted women was Positive 60%. (There is a statistically significant between species in P <0.05).
 The present study also detected for Surface Antigen (SAG3) gene to conform the nPCP result and to send the samples for sequences.
 The SAGA gene showed 100% positive results according to the ssRNA gene .
 T.gondii isolates surface Ag SAG3 gene with NCBI BLAST Toxoplasma gondii isolates, and the DNA nucleotides sequencing analysis of surface Ag SAG3 complete gene was show clear gentic variation between isolates from different host. 

Comparison Between Nested-PCR and ELISA for the Detection of Toxoplasma gondii in Blood and Milk and its Genotyping in Lactating Goats and Aborted Women in Iraq


 
 
 
 The present study aimed to assess enzyme-linked immunosorbent assay (ELISA) and nested-polymerase chain reaction (n-PCR) methods based on B1 gene for the detection of Toxoplasma (T.) gondii in the blood and milk of local Iraqi goats. The SAG3 gene was also used to identify the genotyping of T. gondii in goats and aborted women in Iraq. A total of 240 (80 blood, 80 sera, 80 milk) lactating goats and 30 blood samples from aborted women were included in this study. A total of 17 (21.2%) infected goats were found in blood samples and 23 (28.7%) in milk samples when using n-PCR, while the numbers were 23 (28.7%) and 17 (21.2%) when using ELISA. Aborted women had an overall infection rate of 50% when using ELISA and 33% when using n-PCR. The degree of agreement between n-PCR in milk and blood was almost perfect (Kappa=0.801), with a sensitivity of 100 and a specificity of 90.5, while there was a slight degree of agreement (Kappa=0.14) between n-PCR and ELISA in blood, with 58.8 sensitivity and 74.6 specificity. The results of the comparison between n-PCR in blood and ELISA in milk showed positive samples of 17 (21.2%) for each, with 82.4 sensitivity and 22.2 specificity, and no agreement (Kappa=–0.046). Sequencing of the SAG3 gene of T. gondii from goat and human isolates showed that the similarity ranged from 98.65–99.90% for genotypes I and III. In conclusion, n-PCR may be more accurate than ELISA for detecting T. gondii in blood and milk. In addition, the phylogenetic tree's evidence of a high degree of similarity between human and goat isolates provides further evidence that goats are an important reservoir of T. gondii and that public awareness is necessary.
 
 
 

Molecular detection of Toxoplasma gondii among commensal rodents from the Sahiwal division, Punjab, Pakistan.

Toxoplasmosis is a zoonotic disease in humans and animals caused by the protozoan parasite Toxoplasma gondii. Rodents not only act as intermediate hosts for T. gondii but are also a significant source of T. gondii infection for other hosts. There is limited information about the prevalence of this protozoan within rodents in Pakistan. The current study was conducted to determine the prevalence of T. gondii in commensal rodents as well as associated risk factors. A total of 236 rats including Rattus rattus and Rattus norvegicus were captured from three districts of the Sahiwal division in Punjab province, Pakistan. Brain samples were collected from each specimen. Data was gathered about rodent species, gender, age, location, districts, seasonality, and habitat type. PCR assays were used to screen for T. gondii from the brain samples. A Chi-square analysis was used to investigate the association between positive samples and risk factors. A total of 236 rodent spleen samples were examined for detection of the B1 and SAG3 genes of T. gondii DNA by conventional PCR, and 14 (5.9%) were positive. T. gondii prevalence was higher in R. norvegicus (8.2%) compared to R. rattus (4.6%). All risk factors were statistically non-significant. This study provides evidence of T. gondii in rodents in the Sahiwal division and indicates that other animals and humans are at risk of infection.

Can cloning and sequencing help to genotype positive Toxoplasma gondii clinical samples? Results and validation using SAG3 as a model.

Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.

Detection of toxoplasmosis in (Free living and Industrial) chicken of Qadisiyah province, Iraq

The study aimed to investigate the differences in the genetic tree of types infected (Free living and Industrial chickens) with the Toxoplasma parasite, (brain and liver) collecting samples in Al-Qadisiyah province during the period from October 2021 until the end of March 2022. A total of 90 samples of chicken were collected, 60 samples of brain and 30 samples of liver. The purpose of diagnosis, the impression method was used. It was found that 60 samples of free living chickens were all positive for examination. As for industrial chickens, 20 samples from 30 were positive at rate 88.8%, in rate of (83.3%) 50/60 from the brain and (66.6%) 20/30 from the liver. Nested PCR targeting aspecific region within SAG3 gene technique applied for detect the genotype of T.gondii, the results showed that 30/48 of the chickens (brain and liver) (62.5%) were positive. A genetic match was found between Toxoplasma gondii in chickens SAG3 gene and the gene isolates according to NCBI – BLAST.

Stray dogs in the tropical state of Chiapas, Mexico, harbour atypical and novel genotypes of Toxoplasma gondii

Genotyping of Toxoplasma gondii remains a relevant topic of study, since genotypes can be related to the presentation and severity of toxoplasmosis. To date, 292 restriction fragment length polymorphism genotypes have been described around the world. Serosurveys in southeastern Mexico have documented exposure in over 70% of people and certain animals. Recently, we have described new genotypes and mixed infections in feral cats from Quintana Roo. Thus, the aim of this study was to genotype T. gondii and to describe its genetic variability, from naturally infected stray dogs of Chiapas, which has different geographical and climatic conditions from those found at the Yucatan Peninsula and the other parts of the country. Eleven stray dogs were captured and bled to obtain DNA, and then they were euthanized to perform necropsies and to collect target tissues. Diagnosis of T. gondii was done by quantitative real-time PCR (qPCR) and endpoint PCR. Genotyping was carried out, amplifying 12 polymorphic markers and 15 microsatellites. Atypical SAG3 gene products were cloned and sequenced. All blood samples of dogs were positive to T. gondii DNA by PCR. Two isolates were obtained from pooled heart and diaphragm tissue of two dogs. Two complete PCR-RFLP genotypes were identified (type BrIII and #28). Four animals had mixed infections. A new RFLP atypical allele for the SAG3 marker was observed; cloning and sequencing analysis of this locus revealed mixed infection by a strain identical to GT1, and one type I × II intragenic recombinant. The microsatellite analysis revealed that both isolates are atypical. Thus, atypical new genotypes of T. gondii and mixed infections were found in dogs of Chiapas. The results found here and in genotyping studies in México suggest that the southeastern region favours wide genetic diversity of T. gondii and the possible presence of virulent genotypes such as those found in central and South America.

Molecular detection and genotype identification of Toxoplasma gondii in domestic and industrial eggs

AbstractBirds are important intermediated hosts of Toxoplasma gondii. Consumption of chicken meat and its infected products is one of the main sources of human infection with T. gondii. However, little information is available about T. gondii infection in egg. In this study, the contamination of industrial and local eggs with Toxoplasma was investigated by PCR method targeting the RE gene in two tropical cities of Iran. Genotypes of T. gondii were determined by PCR‐RFLP method targeting the SAG3 gene. T. gondii DNA was detected in 11% (22/200) of the eggs. Genotyping of 14 T. gondii isolates revealed that all of the isolates were belonged to genotype III of T. gondii. Sequencing of the isolates by the SAG3 gene showed 95%–100% similarity with the T. gondii isolates in GenBank.Practical applicationsThe findings of this study suggested that consumption of raw or undercooked eggs might play a role in human infection with Toxoplasma. Hence, consumption of adequately cooked eggs should be considered for prevention of human toxoplasmosis.

Toxoplasma genotyping in congenital toxoplasmosis in Upper Egypt: evidence of type I strain.

Toxoplasma gondii has subpopulation structures in different geographical regions caused by less frequent sexual recombination, population sweeps, and biogeography. The majority of strains isolated in North America and Europe fall into one of three clonal lineages, referred to as types I, II, and III. So far, little is known about genetics of Toxoplasma strains in Africa. The present study aimed to determine the genotype of Toxoplasma strains obtained directly from trophoblastic/placental tissues of 29 complicated pregnant women using multilocus nested-PCR-RFLP technique depending on four independent genetic loci (5' SAG2 and 3' SAG2), SAG3, GRA6, and BTUB genes. All samples gave positive amplicons at 5'-3' SAG2 and SAG3 genes. Meanwhile, no amplification products were observed in 12 (41.37%) and 10 (34.48%) samples with GRA6 and BTUB genes, respectively. The restriction pattern revealed the presence of genotype I in all samples, except one sample, which revealed atypical genotype with unusual restriction pattern at 3' SAG2 gene. The negative amplifications in some samples could be due to presence of mutations or polymorphisms in the primer binding sites of these isolates, raising the possibility of mixed or recombinant genotypes. To the best of our knowledge, this is the first time to perform genotype analysis study based on Multiplex nPCR-RFLP technique for genetic characterization of T. gondii in Egypt. Besides, it is the first time to prove that the most prevalent strain of T. gondii, responsible for congenital toxoplasmosis in Upper Egypt, is the highly virulent type I. Atypical genotype was detected as well.

Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion.

Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA.

Genetic Characterization of Toxoplasma gondii Isolates From Pigs in Southwestern China

The genetic diversity of Toxoplasma gondii varies in different geographical regions. Isolates of T. gondii in South America, for example, are genetically and biologically divergent from those in North America and Europe, where the population structure is highly clonal and composed mainly of 3 distinct lineages, i.e., Types I, II, and III. However, little is known of the T. gondii genotypes in the People's Republic of China. Toxoplasma gondii infection in pigs causes significant economic loss and presents a risk for human infection. We conducted a survey to determine the genetic diversity of this parasite in slaughtered pigs from Yunnan Province, southwestern China. In total, 412 DNA samples were extracted from hilar lymph nodes and livers of pigs from slaughterhouses in Yunnan Province in southwest China, 56 of which were found to be positive for the T. gondii SAG3 gene. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 5 isolates were genotyped with complete data for all loci. Only 1 genotype (ToxoDB 9) was identified, previously reported as a widespread lineage from pigs, cats, and human patients in China. The results indicate that this genotype may be the major T. gondii lineage in China and possibly all of eastern Asia. This is the first report of genetic typing of T. gondii isolates from pigs in China's southwestern Yunnan Province, the results of which have implications for the prevention and control of T. gondii infections in humans and other animals.

Acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico

Toxoplasma gondii causes fatal multisystemic disease in New World primates, with respiratory failure and multifocal necrotic lesions. Although cases and outbreaks of toxoplasmosis have been described, there are few genotyping studies and none has included parasite load quantification. In this article, we describe two cases of lethal acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) of Mexico city. The main pathological findings included pulmonary edema, interstitial pneumonia, hepatitis and necrotizing lymphadenitis, and structures similar to T. gondii tachyzoites observed by histopathology in these organs. Diagnosis was confirmed by immunohistochemistry, transmission electron microscopy and both end point and real time PCR. The load was between <14 and 23 parasites/mg tissue. Digestion of the SAG3 gene amplicon showed similar bands to type I reference strains. These are the first cases of toxoplasmosis in primates studied in Mexico, with clinical features similar to others reported in Israel and French Guiana, although apparently caused by a different T. gondii variant.