Genetic diversity of Toxoplasma gondii isolates obtained from free-living wild birds rescued in Southeastern Brazil.

Recent studies carried out in Brazil have shown that strains from the same Toxoplasma gondii genotype can infect humans, domestic animals (dogs and cats) and animals slaughtered for human consumption (pigs, sheep, goats, and chickens), suggesting a common infection route. However, little is known about the importance of free-living wild birds within this epidemiological context. The objective of this work was to isolate, genotype, and evaluate the virulence for mice of new isolates of T. gondii obtained from free-living wild birds from the state of Minas Gerais, Southeastern Brazil. From August 2016 to June 2017, T. gondii was isolated from the hearts and brains collected from 6 out of 45 free-living wild birds, namely, a roadside hawk (Rupornis magnirostris), a campo flicker (Colaptes campestris), a southern caracara (Caracara plancus) and a tropical screech-owl (Megascops choliba), all rescued in Belo Horizonte. One isolate was obtained from a toco toucan (Ramphastos toco), rescued in Cristiano Otoni, and another was obtained from southern caracara, rescued in Santa Luzia. Five different genotypes were identified by PCR-RFLP. A unique genotype was shared in two different isolates obtained from a southern caracara and a toco toucan. This genotype has never been previously described in any other host or place. Three isolates were classified as of intermediary virulence and three isolates as avirulent for mice. The combined analysis of alleles ROP18/ROP5 (a serine/threonine kinase, and a polymorphic pseudokinase, respectively) was effective in determining the virulence of five of all the isolates with the exception of that from R. magnirostris. Atypical isolates of T. gondii obtained from free-living wild birds rescued in the state of Minas Gerais share the same genotypes of strains that infect humans, domestic animals, and animals slaughtered for human consumption.

Association of ROP18 and ROP5 was efficient as a marker of virulence in atypical isolates of Toxoplasma gondii obtained from pigs and goats in Piauí, Brazil.

Recent studies indicate that proteins GRA15, ROP5, ROP16, ROP17, and ROP18 of Toxoplasma gondii are involved in the process of interaction, cellular invasion, and immune response of the host. Among these proteins, alleles of the polymorphic ROP18 and ROP5 seem to be directly associated with T. gondii virulence in mice. The purpose of this work was to isolate and genotype T. gondii from pig, goat, and sheep slaughtered for human consumption in the state of Piauí, Northeastern Brazil and relate the variability of genes that express virulence proteins of the parasite to virulence in mice. T. gondii was isolated from 16 pigs and 9 goats. The parasite was not isolated from sheep samples. Eleven different genotypes were identified using PCR-RFLP. A unique genotype not yet described in any other host and or anywhere else was common to three pig isolates. Eighteen isolates (72%) were characterized as avirulent, four (16%) as intermediate virulence and three (12%) as virulent to mice. The combined analysis of ROP18 and ROP5 in the isolates studied in Piauí, showed four different allele associations: 4/3 (virulent strains), 3/3, 3/1, and 2/3 (avirulent strains). The association 2/3 was not previously described in the literature. Our results indicated that GRA15, ROP16, and ROP17 alleles were not associated with T. gondii virulence in mice. Pigs and goats raised and slaughtered for human consumption in the state of Piauí are infected with isolates of T. gondii presenting different genotypes. We concluded that the virulence protein ROP18, analyzed alone or in combination with ROP5, was effective in determining virulence for mice for the new isolates of T. gondii.

Genotyping of Toxoplasma gondii Directly from Human and Animal Biological Samples: From Partial Genotypes to a New Genotype.

Genotyping of Toxoplasma gondii is traditionally performed using DNA obtained from tachyzoites after isolation by bioassay in mice. In this study, genotyping of T. gondii was performed by multiplex nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP) in DNA obtained from the lungs of experimentally infected mice, the hearts of naturally infected free-range chickens, and human blood samples of newborns with congenital toxoplasmosis. The efficiency of Mn-PCR varied according to the marker. We obtained complete genotypes of all of the mice lung samples. In chickens, total or partial genotyping was performed on all of the 15 samples. Two complete genotypes were obtained, including one identified for the first time, and another previously described in different hosts including dogs, cats, and humans. In blood from infants, partial genotypes were obtained in 8 of the 12 samples. Mouse bioassay is the most efficient method to obtain DNA from T. gondii , but direct tissue genotyping enhances the likelihood of obtaining molecular information on T. gondii and is an effective tool as a complement to isolation in mice. In this study, we genotyped Toxoplasma gondii directly from human (blood samples of newborns with congenital toxoplasmosis) and free-range chickens (hearts) by Mn-PCR-RFLP. We present partial and complete genotypes and provide technical and scientific information about T. gondii genotyping methods.