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.

Search for antibodies against Trichinella in two synanthropic Procyonidae species from southeast Mexico: white-nosed coatis (Nasua narica) and raccoons (Procyon lotor).

Trichinella is a nematode that are spread by the consumption of parasitized meat. Carnivora, a mammalian order, serve as key hosts for this parasite. However, evidence of Trichinella in wildlife from the Neotropics is extremely scarce, with reports documenting its presence only for five carnivore species: two Felidae, one Otariidae and two Mustelidae. Other widely distributed species that are consumed as bushmeat, such as Procyonidae, have not been studied in this context. A long-term study was performed for antibodies against Trichinella in coatis (Nasua narica) and common raccoons (Procyon lotor) in southeastern Mexico. Between the summer of 2009 to the winter 2013, a total of 291 coati samples and 125 raccoon samples were collected from a tropical green area located within an urban zone. An Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the excretory and secretory products of Trichinella spiralis muscle larva. ELISA-positive samples were further confirmed by Western Blot analysis. Results showed no evidence of antibodies during the first two years of study. However, in 2011, a sudden appearance of anti-Trichinella occurred. The seroprevalence reached its highest peak of 43% for coatis during winter 2013 and 53% for raccoons in summer 2013. This is the first study that provides evidence of Trichinella circulation within a neotropical procyonid community.

Congenital Toxoplasmosis Diagnosis: Current Approaches and New Insights.

PURPOSE: The aim of this study is to describe and discuss current disadvantages in congenital toxoplasmosis (CT) diagnosis, and what can be improved or changed through new perspectives and technological advances. METHODS: We used Pubmed, Cochrane, and EBSCO databases to research publications from 10 years to date describing current diagnostic methods for CT. The keywords used for this Mini-Review were Toxoplasma gondii, congenital toxoplasmosis, diagnosis, and prospects using Boolean operators such as AND, OR, identifying scientific publications highlighting the importance of implementing new diagnostic methods. RESULTS: Current diagnosis methods have several disadvantages, i.e., time-consuming, low sensitivity or specificity, and non-cost effective, that bring up the necessity of improving or developing new approaches. Recombinant proteins can help improve specificity by generating tests that use circulating strains in a specific geographical region, SAG1 and BAG1, as they are expressed during a particular stage of the disease (acute or chronic, respectively), for its use in serological diagnoses, such as capture ELISA and immunochromatography. Point of Care (POC) tests are methods performed at the patient care site, which leads to rapid patient treatment; despite the advantages, several improvements and perspectives are necessary to be implemented globally. CONCLUSIONS: Although already established diagnosis methods for CT may be sufficient in some regions, there is still a persistent demand to develop tests with higher throughput, cost, and time reduction in developing countries, where prevalence is high. New approaches in CT diagnosis, such as recombinant proteins, capture ELISA, immunochromatography, and POC tests methods, can increase performance in terms of specificity and sensitivity simplifying diagnostic tests' requirements.

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', alt. SAG2, 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.

Host-pathogen interactions mediated by extracellular vesicles in Toxoplasma gondii infection during pregnancy.

Molecular communication between a pathogen and its host is crucial for a successful interplay. Extracellular vesicles (EVs) act as mediators for the delivery of molecular signals among pathogens or between pathogens and the host. Toxoplasma gondii (T. gondii), an intracellular parasite with a worldwide presence, produces EVs itself, or induces the secretion of EVs from infected host cells potentially having capacities to modulate the host immune response. T. gondii infection is particularly important during pregnancy. Depending on the gestational age at the time of infection, the parasite can be transmitted through the placenta to the fetus, causing clinical complications such as jaundice, hepatosplenomegaly, chorioretinitis, cranioencephalic abnormalities, or even death. T. gondii infection is related to a pro-inflammatory immune response in both mother and fetus, which may enhance parasite transmission, but the implication of EV signaling in this process remains unclear. In this review, we summarize the current knowledge on EV release from T. gondii and its human host cells in regard to the immunological consequences and the passage through the placenta.

Acute Disseminated Toxoplasmosis in Two Specimens of Macropus rufogriseus Caused by a Genotype so far Exclusive to South America.

Macropods are included among the species considered highly susceptible to Toxoplasma gondii infection. Clinically, it is difficult to distinguish between acute toxoplasmosis due to primary infection and reactivation of chronic latent infection in susceptible species until pathologic studies are performed. Here, we described the clinical cases and lesions found in two deceased Bennett's wallabies (Macropus rufogriseus) with a presumptive diagnosis of toxoplasmosis, as well as the genetic characterization of the T. gondii isolates obtained from these specimens. Both animals presented acute infection lesions in the lungs, liver, spleen and lymph nodes associated to T. gondii infection. Histopathology and immunohistochemistry also demonstrated tissue cysts of different sizes, indicating that the wallabies were previously infected with this parasite. Two isolates were obtained, one from each specimen and the molecular characterization was done; both isolates were the ToxoDB #116 genotype. This is the first study that reports the isolation of this particular genotype outside South America, and given the histopathological findings, it could be considered virulent for this species. The dynamics of infection that T. gondii is causing in definitive and intermediate hosts in a region allows us to know the risks to which the animals and humans that live in the area are exposed, and in the future to implement a preventive medicine plan against this parasite.

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 6.25 ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65 cycles 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.

SAG3 Toxoplasma gondii cloning reveals unexpected fivefold infection in the blood of feral cats in the Mexican Caribbean.

BACKGROUND: Currently, more than 300 genotypes of Toxoplasma gondii (T. gondii) have been described throughout the world, demonstrating its wide genetic diversity. The SAG3 locus is one of the genes included in the genotyping panel of this parasite. It is associated with its virulence since it participates during the invasion process of the host cells. Therefore, cloning, sequencing, and bioinformatic analysis were used to deepen the understanding of the SAG3 locus genetic diversity of T. gondii in blood samples from feral cats. RESULTS: Six different SAG3 sequences were detected, five of which were detected in one feline. Three sequences were first reported here; one of them was an intragenic recombinant. In the cladogram, four out of ten SAG3 sequences did not share nodes with others reported worldwide. CONCLUSIONS: Cloning and sequencing of samples with more than one restriction pattern by PCR-RFLP were very helpful tools to demonstrate the presence of more than three genotypes of T. gondii in the blood of feral cats from southeastern Mexico. This suggests a potential mixed infection of multiple T. gondii strains and high genetic diversity of the parasites in felines in this tropical region of Mexico.

Toxoplasma gondii infection in European mouflons (Ovis musimon) and captive wild felines from Puebla, México.

The presence of Toxoplasma gondii in zoos is cause of alert because many susceptible species kept in captivity die of clinical toxoplasmosis. Moreover, excretion of T. gondii oocysts by infected captive wild felines into the facilities could pose a risk to workers. Herbivores in wild collections can serve as sentinels of local transmission, since they get infected by the consumption of oocysts present in ground or water. Both herbivores and felids may reveal the parasite variants which are circulating in the region. We determined the seroprevalence of T. gondii in European mouflons (n = 55) and wild felines (n = 15) from a private zoological collection located in the Eastern region of México, as well as the incidence in 41 of the mouflons using ELISA. The prevalence of T. gondii in mouflons was 14.5% (n = 55) and 17.1% (n = 41) in 2011 and 19.5% in 2012. The estimated incidence was 9.8%-12.2%. In wild felines the frequency was 80%. Four sero-positive animals (two mouflons and the two oldest African lions) were euthanized. Histopathology, conventional PCR (for B1 and SeqRep529 loci) and molecular characterization were carried out. All euthanized animals were positive to T. gondii by PCR. We identified a triple infection (I + II + III) in the brain of a mouflon. In conclusion, a high infective pressure of T. gondii in the collection was found, supported by changes in its prevalence in European mouflons. A high prevalence of infection in wild felines was determined. At least four genotypes of T. gondii are present in herbivores and carnivores, and one mouflon had a mixed infection.

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.