Isolation and immunological characterization of rhoptries from Neospora caninum

Authors

  • Thais Agostinho Martins Universidade de Maringá. Faculdade de Medicina Veterinária
  • Luiz Daniel De Barros Universidade Federal de Lavras
  • Beatriz De Souza Lima Nino Universidade Estadual de Londrina
  • Juliana Correa Bernardes Universidade Positivo. Faculdade de Medicina Veterinária
  • Ana Clécia Dos Santos Silva Universidade Estadual de Londrina
  • Ana Flávia Minutti Universidade Federal de Lavras
  • Sergio Tosi Cardim Universidade Norte do Paraná. Faculdade de Medicina Veterinária
  • Milena Patzer Rose Universidade Estadual de Londrina
  • Valentina Martinez Universidade Estadual de Londrina
  • João Luis Garcia Universidade Estadual de Londrina https://orcid.org/0000-0003-1826-7582

DOI:

https://doi.org/10.11606/issn.1678-4456.bjvras.2024.219874

Keywords:

Neospora caninum, Neosporosis, Organelles, Rhoptries, Fractionation

Abstract

Neospora caninum is a parasite that causes considerable loss in cattle production. Understanding its infection mechanisms is essential to comprehend its impact on herds. Cell fractionation and characterization of the fractions could contribute to the selection of proteins and organelles with immunogenic characteristics. Thus, the present study aimed to isolate rhoptry and subcellular fractions of N. caninum using a sucrose gradient and characterize their immunogenicity in mice. Tachyzoites of the Nc-1 strain were cultured in VERO cells, fractionated by glass beads, and ultracentrifuged in a sucrose gradient ranging from 0.25–1.8 M. The fractions were characterized using transmission electron microscopy. Each fraction was inoculated with Quil-A adjuvant (20 μg) into mice to produce polyclonal antibodies for immunofluorescence cell culture and Western blotting. Ultracentrifugation resulted in three distinct fractions (F1, F2, and F3) and a pellet. Fraction one (F1) at 1.0 M concentration contained parasite membranes, F2 at 1.4 M contained rhoptry and conoid, F3 at 1.6 M contained mitochondria, and the pellet at 1.8 M fraction contained cell debris. The four fractions exhibited the same bands with molecular weights of 50, 51, 52, and 62 kDa. Only F2 showed rhoptry structures and a 54 kDa protein resembling NcROP2. This study successfully separated subcellular fractions of N. caninum through processing and ultracentrifugation, identified rhoptry structures, and determined specific protein weights of each fraction.

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References

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Published

2024-08-12

Issue

Vol. 61 (2024)

Section

FULL ARTICLE

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How to Cite

1.
Martins TA, Barros LDD, Nino BDSL, Bernardes JC, Silva ACDS, Minutti AF, et al. Isolation and immunological characterization of rhoptries from Neospora caninum. Braz. J. Vet. Res. Anim. Sci. [Internet]. 2024 Aug. 12 [cited 2024 Dec. 3];61:e219874. Available from: https://revistas.usp.br/bjvras/article/view/219874