Research Article
Volume 17 Issue 2 - 2021
Molecular Detection of Toxoplasma gondii Cyst Formation in Fresh or Paraffin-Embedded Tissues of Three Chronic Strains
María A Vethencourt1,2*, Gloriana Loaiza2, Cristal López2, José E Bolaños1, Idalia Valerio1,3, Laura Valerio1 and Misael Chinchilla1
1Basic Research Laboratory, University of Medical Sciences, San Jose, Costa Rica
2Faculty of Microbiology, University of Medical Sciences, San Jose, Costa Rica
3Faculty of Medicine, University of Medical Sciences, San Jose, Costa Rica
*Corresponding Author: María A Vethencourt, Basic Research Laboratory, University of Medical Sciences, San Jose, Costa Rica.
Received: November 11, 2020; Published: January 27, 2021




Abstract

The purpose of this study was to compare molecular detection of T. gondii cyst formation in fresh or paraffin-embedded tissues of three chronic strains. Molecular detection was carried out using a nested PCR that detects the B1 gene of T. gondii. Parasite DNA was extracted from fresh tissue and formalin-fixed and paraffin-embedded (FFEP) tissues and from CD1 strain mice (Swiss) infected with three chronic strains. Ten (10) mice (5 female and 5 male) were orally infected with serial dilutions of brain tissue cysts from previously infected mice. The chronic strains of T. gondii came from a domestic cat (Felis catus, TFC-1; A), from Leopardus wiedii (TLW-1; B) and from white mice fed with Bos taurus beef (TBT-1; D). The FFEP tissues of 10 mice that survived after oral infection with different dilutions of tissue cysts, were used respectively. The presence of tachyzoites (Tq) or tissue cysts (Q) was estimated per mm3 of tissue analyzed. Three 10 um sections of the FFEP tissues were used for DNA extraction with two manual protocols (salt and ammonium acetate) and with a commercial one, using silica columns. DNA was detected with the nested PCR that amplified the T. gondii B1 gene, after its standardization. In this work, it was found that standardized nested PCR could amplify at least 0.1 fg/µL of DNA. Only with the commercial methodology was it possible to detect the parasite DNA, after applying a nested PCR for the T. gondii B1 gene, although the quality of the DNA after extraction was similar with the three extraction protocols. In unfixed tissues, the highest molecular detection of the parasite occurred in the brain and skeletal muscle of mice infected with chronic strains A and B. The molecular detection in FFEP tissues follows, like the kinetic tests, a maximum detection curve that depends on the inoculum. A higher molecular detection was observed in the tissues at lower doses of the inoculum, which was related to a higher reading of cysts per mm3 of tissue and with a lower virulence, previously studied. It is concluded that the molecular detection of the T. gondii B1 gene, with nested PCR of FFEP tissues, was 10 times more sensitive than the histological detection of cysts by microscopy. In addition, the molecular and histological detection of cysts of a chronic strain of T. gondii were inversely proportional to the virulence of the previously characterized strain.

Keywords: Toxoplasma gondii; Chronic Strains; Molecular Detection; FFEP; Cyst Formation; Costa Rica

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Citation: María A Vethencourt., et al. “Molecular Detection of Toxoplasma gondii Cyst Formation in Fresh or Paraffin-Embedded Tissues of Three Chronic Strains”. EC Microbiology 17.2 (2021): 231-246.

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