Gondii Isolates Research Articles

Genotyping of human isolates from human toxoplasmosis outbreak: Restriction Fragment Length Polymorphism and Sanger Sequencing.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii, capable of infecting a wide range of hosts. The parasite exhibits a broad genetic diversity, necessitating genotypic characterization for genotype identification and associations with epidemiological information. Therefore, the Restriction Fragment Length Polymorphism (RFLP) technique is used for characterization. This study aimed to perform genotypic characterization of isolates from pregnant women infected during a human toxoplasmosis outbreak, comparing the RFLP and Sanger Sequencing methodologies. For this purpose, six human isolates were subjected to conventional PCR, Multiplex PCR, Nested PCR, Enzymatic Digestion, and Sanger Sequencing. Additionally, the standard strains GTI (Type I), PTG (Type II), and CTG (Type III) were also subjected to the same techniques described above. Subsequently, the amplified DNA products were compared with the standard strains. As a result, it was observed that Sanger Sequencing provides the same information as RFLP PCR, as well as the possibility of cost reduction for genotypic characterization, and providing greater agility in issuing results. Additionally, Sanger Sequencing of T. gondii isolates allows for detailed evaluation of nucleotide sequences, including the assessment of SNPs and enzymatic restriction sites, which the RFLP technique does not.

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%).

Molecular survey of Toxoplasma gondii B1 gene in pigs from various localities in Korea.

Toxoplasma gondii, a common protozoan parasite, poses significant public health risks due to its potential to cause toxoplasmosis in humans and can be contracted from pigs, which are considered its critical intermediate host. The aim of this study is to evaluate the prevalence of T. gondii in slaughtered pigs for human consumption, emphasizing the zoonotic implications and the need for improved biosecurity and monitoring practices in pig farming. A total of 1,526 pig samples (1,051 whole blood samples and 384 lung tissue samples from the local slaughterhouse and 91 aborted fetus samples from local farms) were collected throughout the whole country of Korea in 2020. Among them, 6 (0.4%) were found to be infected with T. gondii by nested PCR. When compared by sample type, the prevalence of T. gondii was significantly higher in the aborted fetus samples (2.2%, 2/91) than in the blood (0.3%, 3/1,051) and lung tissue samples (0.3%, 1/384). The B1 gene sequence of T. gondii was similar (97.9-99.8%) to that of the other T. gondii isolates. This study represents the first molecular genotyping survey of T. gondii in the lung tissue of fattening pigs and aborted fetuses in Korea. Our findings indicated the importance of adopting preventive measures including the implementation of rigorous farm hygiene protocols and the promotion of public awareness about the risks of consuming undercooked pork. By addressing the gaps in current control strategies and encouraging the One Health approach, this study contributes to the development of more effective strategies to mitigate the transmission of T. gondii from pigs to humans, ultimately safeguarding public health.

First identified Toxoplasma gondii Type I in market-sold ducks in Fujian province, China: a significant for public health

Toxoplasma gondii (T. gondii) is an intracellular protozoan that can cause toxoplasmosis in all warm-blooded hosts. This study focused on the prevalence and genetic characterize of T. gondii in ducks from Fujian province, China. Genomic DNA was extracted from duck tissue samples (heart, liver, lung, and muscle). To assess the genetic diversity of the T. gondii isolates, it was determined by using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. A total of 586 ducks from 5 cities in Fujian province were tested, and 35 (6.0%) of which were found to be positive for the T. gondii B1 gene. Further genotyping of these positive samples at 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using PCR-RFLP revealed that one tissue samples (heart samples from Fuzhou ducks) were identified as Type I (ToxoDB#10). This study is the first report on the prevalence and genetic characterization of T. gondii in ducks in Fujian province, and Type I (ToxoDB#10) is found in ducks in China for the first time. The findings document the genetic characterization of T. gondii in free-range ducks from Fujian Province, thereby enriching the understanding of T. gondii genetic diversity in China. Moreover, these results provide essential data support for further prospective studies and underscores the "One Health" concept, emphasizing the integral link among human, animal, and environmental health.

Rapid genotyping of Toxoplasma gondii isolates via Nanopore-based multi-locus sequencing

Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite’s diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel’s) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

Infection dynamics following experimental challenge of pigs orally dosed with different stages of two archetypal genotypes of Toxoplasma gondii

Toxoplasma gondii is a food-borne zoonotic parasite widespread in a variety of hosts, including humans. With a majority of infections in Europe estimated to be meat-borne, pork, as one of the most consumed meats worldwide, represents a potential risk for consumers. Therefore, we aimed to investigate the progress of T. gondii infection and tissue tropism in experimentally infected pigs, using different T. gondii isolates and infectious stages, i.e. tissue cysts or oocysts. Twenty-four pigs were allocated to treatment in four groups of six, with each group inoculated orally with an estimated low dose of either 400 oocysts or 10 tissue cysts of two European T. gondii isolates, a type II and a type III isolate. The majority of pigs seroconverted two weeks post-inoculation. Pigs infected with the type III isolate had significantly higher levels of anti-T. gondii antibodies compared to those infected with the type II isolate. Histopathological exams revealed reactive hyperplasia of the lymphatic tissue of all pigs. Additionally, a selected set of nine tissues was collected during necropsy at 50 dpi from each of the remaining 22 pigs for T. gondii DNA detection by quantitative real-time PCR. A positive result was obtained in 29.8 % (59/139) of tested tissues. The brain was identified as the most frequently positive tissue in 63.6 % (14/22) of the animals. In contrast, liver samples tested negative in all animals. The highest mean parasite load, calculated by interpolating the average Cq values on the standard curve made of ten-fold serial dilutions of the genomic DNA, corresponding to 100 to 104 tachyzoites/µL, was observed in shoulder musculature with an estimated concentration of 84.4 [0.0–442.5] parasites per gram of tissue. The study highlights the variability in clinical signs and tissue distribution of T. gondii in pigs based on the combination of parasite stages and strains, with type III isolates, particularly oocysts, causing a stronger antibody response and higher tissue parasite burden. These findings suggest the need for further investigation of type III isolates to better understand their potential risks to humans.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

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.

Genotyping of Toxoplasma gondii in domestic animals from Campeche, México, reveals virulent genotypes and a recombinant ROP5 allele.

Toxoplasma gondii has at least 318 genotypes distributed worldwide, and tropical regions usually have greater genetic diversity. Campeche is a state located in the southeastern region of México and has favourable climate conditions for the replication and dissemination of this protozoan, similar to those in South American countries where broad genetic diversity has been described. Thus, in this study, 4 T. gondii isolates were obtained from tissues of stray dogs and free-range chickens in Campeche, México, and were genotyped by Mn-PCR-RFLP with 10 typing markers (SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and 5 virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5) to provide new information about the distribution and virulence prediction of T. gondii genotypes. Two isolates of T. gondii genotype #116 and 2 of genotype #38 were obtained from stray dogs and chickens, respectively. The parasite load found in these species was between <50 and more than 35 000 tachyzoites per mg of tissue. Virulence marker genotyping revealed a recombinant 1&3 ROP5 RFLP pattern in 2 ToxoDB #116 isolates with no prediction of virulence in a murine model, while in the 2 ToxoDB #38 isolates, the ROP18/ROP5 combination predicted high virulence. Considering all the typed markers, there is a predominance of type I and III alleles, as constantly reported for the isolates characterized in various regions of México. It is crucial to determine their phenotype to corroborate the genetic virulence profile of the T. gondii isolates obtained in this study.

Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method

PurposeA new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains.MethodsT. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples.ResultsAmong type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing.ConclusionThe new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Isolation of Toxoplasma gondii in cell culture: an alternative to bioassay

Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii.

In Vitro Virulence Contrast of Seven Genetically Distinct Toxoplasma gondii Isolates After Rejuvenation In Vivo.

In the past for more than 100years at least 300 genotypes of Toxoplasma gondii were recorded and several traditional isolates such as RH, GT1, ME49, PRU and VEG were used repeatedly to clarify the pathogenic mechanisms and the epidemiological significance to human, but for if their virulence was mutative post-iterative passages it remains confused. Therefore, in the study, seven genetically distinct T. gondii including C7 and PYS previously discovered in China were reidentified by sequencing the head of hsp40 locus to distinguish their virulence in vitro post-rejuvenation in vivo. Our data showed the nucleotides were different in 18 positions and 7 of them can be used to type T. gondii isolates. Total 634 plaques of T. gondii without two or more overlaps indicated that RH and GT1 tachyzoites possess stronger power than other five isolates in vitro (p < 0.001), followed by ME49, PRU, C7, PYS, and the weakest VEG. Based on the shapes of plaques, we found the ratio of their width/length was associated with the virulence of T. gondii, and speculated it could be used to judge T. gondii tachyzoites in vitro, whereas the data of simple linear regression analyses did not agree. Together, virulence of seven genetically distinct T. gondii isolates that can be distinguished by seven mutative nucleotides in hsp40 was redefined in vitro post-rejuvenation in vivo, and it cannot be directly judged just according to the shapes of plaques formed in vitro.

Genotyping of toxoplasma gondii isolates from México reveals non-archetypal and potentially virulent strains for mice.

Genotyping and virulence studies of Toxoplasma gondii are essential to investigate the pathogenesis of strains circulating worldwide. In this study, eight T. gondii isolates obtained from a congenitally infected newborn, a calf, two cats, three dogs, and a wallaby from five states of México were genotyped by Mn-PCR-RFLP with 11 typing markers (SAG1, SAG2 5'3', altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico), five virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5), 15 microsatellite markers (TUB-2, W35, TgM-A, B18, B17, M33, IV.1, XI.1, M48, M102, N60, N82, AA, N61, N83), and sequencing. A phylogenetic network was built to determine the relationship between Mexican isolates and those reported worldwide. Six different genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ToxoDB #8, #10, #28 (n = 3), #48, #116, and #282. Genotyping by microsatellite analysis differentiated the three PCR-RFLP genotype #28 isolates into two strains, revealing a total of seven microsatellite genotypes. Three different allele combinations of ROP18/ROP5 virulence markers were also found, 3/3, 1/1, and 4/1. The last two combinations are predicted to be highly virulent in the murine model. According to the phylogenetic network, the T. gondii strains studied here are related to archetypal strains I and III, but none are related to the strains previously reported in México. The genotypes identified in this study in different species of animals demonstrate the great genetic diversity of T. gondii in México. The ToxoDB-PCR-RFLP #28 genotype was found in three isolates from different hosts and states. Additionally, four of the isolates are predicted to be highly virulent in mice. The next step will be to perform in vitro and in vivo assays to determine the phenotype of these T. gondii isolates in murine models.

Toxoplasma gondii Genotypes Isolated from Humans: A Systematic Review

Toxoplasma gondii has the capacity to infect several animals, including humans. This parasite has great genetic variability due to crossing over in felines’ gut. These new atypical and exotic genotypes are complex, requiring genotyping research to help elucidate virulence mechanisms. For the purpose of the study, five English language databases reporting data on T. gondii genotyping in humans were searched. The searching process resulted in the inclusion of 28 articles published from 2000 to September 2020. The data revealed that 1,167 samples were genotyped into 470 T. gondii isolates. In the world, type II was predominant (53.65%, n = 257). Atypical genotypes were the second most common (22.33%, n = 107). The results suggest greater genetic diversity in Asia and America than in the European and African continents. The genotype #9 was prevalent in Asia. Furthermore, atypical strains were predominant in 50 isolates from patients with ocular toxoplasmosis (52%, n = 26). In individuals with congenital toxoplasmosis, type II was predominant presenting a prevalence of 46.49% (n = 73). Type II strains were predominant in immunocompromised with a prevalence of 75.6% (n = 31). In cancer patients, there was a predominance of atypical strains with 67.14% (n = 47) and genotype #17 was specific for this group. In general, this systematic review indicated a great degree of genetic diversity and circulation of virulent T. gondii strains in humans. However, further studies are needed to better understand the population structure of T. gondii and its clinical characteristics.

In vitro and in vivo susceptibility to sulfadiazine and pyrimethamine of Toxoplasma gondii strains isolated from Brazilian free wild birds

Little is known about the existence of drug-resistant Toxoplasma gondii strains and their possible impact on clinic outcomes. To expand our knowledge about the existence of natural variations on drug susceptibility of T. gondii strains in Brazil, we evaluated the in vitro and in vivo susceptibility to sulfadiazine (SDZ) and pyrimethamine (PYR) of three atypical strains (Wild2, Wild3, and Wild4) isolated from free-living wild birds. In vitro susceptibility assay showed that the three strains were equally susceptible to SDZ and PYR but variations in the susceptibility were observed to SDZ plus PYR treatment. Variations in the proliferation rates in vitro and spontaneous conversion to bradyzoites were also accessed for all strains. Wild2 showed a lower cystogenesis capacity compared to Wild3 and Wild4. The in vivo analysis showed that while Wild3 was highly susceptible to all SDZ and PYR doses, and their combination, Wild2 and Wild4 showed low susceptibility to the lower doses of SDZ or PYR. Interestingly, Wild2 presented low susceptibility to the higher doses of SDZ, PYR and their combination. Our results suggest that the variability in treatment response by T. gondii isolates could possibly be related not only to drug resistance but also to the strain cystogenesis capacity.

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.&#x0D; A total of 60 ( placenta and fetus) samples were collected randomly from aborted women who received to the Maternity and Childhood Teaching Hospital.&#x0D; 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 &lt;0.05).&#x0D; The present study also detected for Surface Antigen (SAG3) gene to conform the nPCP result and to send the samples for sequences.&#x0D; The SAGA gene showed 100% positive results according to the ssRNA gene .&#x0D; 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. &#x0D;

Newly detected, virulent Toxoplasma gondii COUG strain causing fatal steatitis and toxoplasmosis in southern sea otters (Enhydra lutris nereis)

From February 2020 to March 2022, four southern sea otters (Enhydra lutris nereis) stranded in California with severe protozoal steatitis and systemic toxoplasmosis. Three of the infected otters stranded within 26 km of each other, and all four animals died during periods of increased rainfall-driven surface water runoff. High parasite burdens were observed in all tissues except the central nervous system, and toxoplasmosis with severe protozoal steatitis was the primary cause of death for all cases. This lesion pattern differs substantially from all prior reports of toxoplasmosis in sea otters. All cases were T. gondii-positive via serology, immunohistochemistry, and PCR. Multilocus sequence typing at 13 loci revealed that all were infected with the same strain of T. gondii, previously characterized as an atypical and rare genotype in North America (TgCgCa1, or COUG). The COUG genotype was first isolated from mountain lions in British Columbia, Canada during investigation of a waterborne outbreak of toxoplasmosis in humans. This genotype has not been previously reported from sea otters, nor any aquatic species. All prior T. gondii strains obtained from &amp;gt;140 southern sea otters represent Type II or Type X strains, or variants of these genotypes. Archival necropsy data (&amp;gt;1,000 animals over 24 years) were negative for prior cases of severe T. gondii-associated steatitis prior to the cases described herein, and no sublethal COUG T. gondii infections have been previously indentified in sea otters. According to prior studies, the T. gondii COUG genotype is highly virulent in mice and is unusual among T. gondii isolates in eliciting a Type I interferon response in murine and human cells in vitro; this unusual immunomodulatory response could explain the apparent high virulence of this atypical T. gondii strain. Our findings reveal a novel and concerning lesion pattern for sea otters with toxoplasmosis. Due to high zoonotic potential and the risk of infection via shared marine food resources, these findings may also indicate potential health threats for other animals and humans.

Experimental infection of sheep at mid-pregnancy with archetypal type II and type III Toxoplasma gondii isolates exhibited different phenotypic traits

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii-DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted.

Molecular characterization and epidemiology of Toxoplasma gondii isolates from free-range chickens in the southwest region of Goiás: new genotypes.

The purpose of this study was to isolate Toxoplasma gondii from tissues of free-range chickens in the southwestern region of Goiás, to detect and molecularly characterize the genetic material of the parasite, and to determine the seroprevalence of the protozoan parasite in these animals. A seroprevalence of T. gondii antibodies of 76% (19/25) was found among the chickens, while genetic material from their tissues was detected in 56% (14/25). A total of 14 isolates was obtained in the bioassay, ten of which were considered acute, eight were considered isolates of high virulence lethal to mice, and four of low virulence, considered non-lethal but with the ability to chronify the infection. Seven of the ten isolates showed significant morphometric differences from the RH strain, in terms of nucleus-complex-apical distance, length and width. Genotyping of the acute isolates was performed by RFLP-PCR, using 11 genetic markers: SAG1, SAG2 (3'SAG2 and 5'SAG2), alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and APICO. The results were compared and classified according to the genotypes listed on the ToxoDB Platform, where different profiles were observed indicating the presence of two known genotypes (#7 and #63) and five new genotypes (NEW 3, NEW4, NEW5, NEW6, NEW 7). The results showed high seroprevalence, isolation rate, molecular detection and genotypic variations of T. gondii in free-range chickens in the southwestern region of Goiás.

Effects of Ovine Monocyte-Derived Macrophage Infection by Recently Isolated Toxoplasma gondii Strains Showing Different Phenotypic Traits.

Ovine toxoplasmosis is one the most relevant reproductive diseases in sheep. The genetic variability among different Toxoplasma gondii isolates is known to be related to different degrees of virulence in mice and humans, but little is known regarding its potential effects in sheep. The aim of this study was to investigate the effect of genetic variability (types II (ToxoDB #1 and #3) and III (#2)) of six recently isolated strains that showed different phenotypic traits both in a normalized mouse model and in ovine trophoblasts, in ovine monocyte-derived macrophages and the subsequent transcript expression of cytokines and iNOS (inducible nitric oxide synthase). The type III isolate (TgShSp24) showed the highest rate of internalization, followed by the type II clonal isolate (TgShSp2), while the type II PRU isolates (TgShSp1, TgShSp3, TgShSp11 and TgShSp16) showed the lowest rates. The type II PRU strains, isolated from abortions, exhibited higher levels of anti-inflammatory cytokines and iNOS than those obtained from the myocardium of chronically infected sheep (type II PRU strains and type III), which had higher levels of pro-inflammatory cytokines. The present results show the existence of significant intra- and inter-genotypic differences in the parasite-macrophage relationship that need to be confirmed in in vivo experiments.