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

Abdelbaky HH, Fereig RM, Nishikawa Y. Identification of the antigenic region of Neospora caninum dense granule protein 7 using ELISA. Parasitol Int. 2018;67(6):675-8. http://doi.org/10.1016/j.parint.2018.06.012. PMid:29959092.

Barros LD, Bogado ALG, Furlan D, de Melo Jardim A, Okano W, da Silva LC, Pereira CES, Bronkhorst DE, Cardim ST, Garcia JL. Effects of Neospora caninum on reproductive parameters in dairy cows from a closed herd in Brazil. Vet Parasitol Reg Stud Rep. 2021;23:100524. http://doi.org/10.1016/j.vprsr.2020.100524. PMid:33678379.

Barylyuk K, Koreny L, Ke H, Butterworth S, Crook OM, Lassadi I, Gupta V, Tromer E, Mourier T, Stevens TJ, Breckels LM, Pain A, Lilley KS, Waller RF. A comprehensive subcellular Atlas of the Toxoplasma proteome via hyperLOPIT provides spatial context for protein functions. Cell Host Microbe. 2020;28(5):752-66.e9. http://doi.org/10.1016/j.chom.2020.09.011. PMid:33053376.

Blackman MJ, Bannister LH. Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation. Mol Biochem Parasitol. 2001;117(1):11-25. http://doi.org/10.1016/S0166-6851(01)00328-0. PMid:11551628.

Debache K, Guionaud C, Alaeddine F, Mevissen M, Hemphill A. Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites. Int J Parasitol. 2008;38(12):1455-63. http://doi.org/10.1016/j.ijpara.2008.04.001. PMid:18495126.

Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev. 2007;20(2):323-67. http://doi.org/10.1128/CMR.00031-06. PMid:17428888.

Dubey JP, Schares G. Neosporosis in animals: the last five years. Vet Parasitol. 2011;180(1-2):90-108. http://doi.org/10.1016/j.vetpar.2011.05.031. PMid:21704458.

Dubremetz JF, Ferreira E, Dissous C. Isolation and partial characterization of rhoptries and micronemes from Eimeria nieschulzi zoites (Sporozoa, Coccidia). Parasitol Res. 1989;75(6):449-54. http://doi.org/10.1007/BF00930971. PMid:2755931.

Etzion Z, Murray MC, Perkins ME. Isolation and characterization of rhoptries of Plasmodium falciparum. Mol Biochem Parasitol. 1991;47(1):51-61. http://doi.org/10.1016/0166-6851(91)90147-X. PMid:1857385.

Garcia JL, Gennari SM, Navarro IT, Machado RZ, Sinhorini IL. Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom. Exp Parasitol. 2004;108(1-2):40-6. http://doi.org/10.1016/j.exppara.2004.07.002. PMid:15491547.

Gondim LFP, Mineo JR, Schares G. Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti. Parasitology. 2017;144(7):851-68. http://doi.org/10.1017/S0031182017000063. PMid:28241894.

Horcajo P, Coronado M, Pastor-Fernández I, Collantes-Fernández E, Rico-San Román L, Reyes-Palomares A, Ortega-Mora L-M. Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis. Front Immunol. 2023;14:1198609. http://doi.org/10.3389/fimmu.2023.1198609. PMid:37520552.

Howe DK, Crawford AC, Lindsay D, Sibley LD. The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infect Immun. 1998;66(11):5322-8. http://doi.org/10.1128/IAI.66.11.5322-5328.1998 PMid:9784539.

Junqueira LCU, Salles LMM. Artefatos de microscopia eletrônica aplicada a material biológico. Cien Cult. 1975;27:461-471.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5. http://doi.org/10.1038/227680a0. PMid:5432063.

Lamarque MH, Papoin J, Finizio A-L, Lentini G, Pfaff AW, Candolfi E, Dubremetz J-F, Lebrun M. Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii. Moreno SN. PLoS One. 2012;7(3):e32457. http://doi.org/10.1371/journal.pone.0032457. PMid:22427839.

Lei Y, Birch D, Davey M, Ellis JT. Subcellular fractionation and molecular characterization of the pellicle and plasmalemma of Neospora caninum. Parasitology. 2005;131(Pt 4):467-75. http://doi.org/10.1017/S003118200500805X. PMid:16174411.

Luft JH. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961;9(2):409-14. http://doi.org/10.1083/jcb.9.2.409. PMid:13764136.

Ma L, Liu J, Li M, Fu Y, Zhang X, Liu Q. Rhoptry protein 5 (ROP5) is a key virulence factor in Neospora caninum. Front Microbiol. 2017;8:370. http://doi.org/10.3389/fmicb.2017.00370. PMid:28326073.

Machado RZ, McElwain TF, Suarez CE, Hines SA, Palmer GH. Babesia bigemina: isolation and characterization of merozoite rhoptries. Exp Parasitol. 1993;77(3):315-25. http://doi.org/10.1006/expr.1993.1089. PMid:8224087.

Marugán-Hernández V, Álvarez-García G, Tomley F, Hemphill A, Regidor-Cerrillo J, Ortega-Mora LM. Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions. J Proteomics. 2011;74(5):629-42. http://doi.org/10.1016/j.jprot.2011.02.004. PMid:21315855.

Pastor-Fernández I, Regidor-Cerrillo J, Jiménez-Ruiz E, Álvarez-Garciá G, Marugán-Hernández V, Hemphill A, Ortega-Mora LM. Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle. Parasitology. 2016;143(1):97-113. http://doi.org/10.1017/S0031182015001511. PMid:26521890.

Reichel MP, Ayanegui-Alcérreca, MA, Gondim LFP, Ellis JT. What is the global economic impact of Neospora caninum in cattle: the billion dollar question. Int J Parasitol. 2013;43(2):133-42. http://doi.org/10.1016/j.ijpara.2012.10.022. PMid:23246675.

Reynolds ES. The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J Cell Biol. 1963;17(1):208-12. http://doi.org/10.1083/jcb.17.1.208. PMid:13986422.

Rico-San Román L, Horcajo P, Regidor-Cerrillo J, Fernández-Escobar M, Collantes-Fernández E, Gutiérrez-Blázquez D, Hernáez-Sánchez ML, Saeij JPJ, Ortega-Mora LM. Comparative tachyzoite proteome analyses among six Neospora caninum isolates with different virulence. Int J Parasitol. 2020;50(5):377-88. http://doi.org/10.1016/j.ijpara.2020.02.003. PMid:32360428.

Sinnott FA, Monte LG, Collares TF, Silveira RM, Borsuk S. Review on the immunological and molecular diagnosis of neosporosis (years 2011-2016). Vet Parasitol. 2017;239:19-25. http://doi.org/10.1016/j.vetpar.2017.04.008. PMid:28495191.

Trees AJ, Davison HC, Innes EA, Wastling JM. Towards evaluating the economic impact of bovine neosporosis. Int J Parasitol. 1999;29(8):1195-200. http://doi.org/10.1016/S0020-7519(99)00093-4. PMid:10576571.

Watson ML. Staining of tissue sections for electron microscopy with heavy metals. J Cell Biol. 1958;4(6):727-30. http://doi.org/10.1083/jcb.4.6.727.

Yoshimoto M, Otsuki T, Itagaki K, Kato T, Kohsaka T, Matsumoto Y, Ike K, Park EY. Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice. J Biosci Bioeng. 2015;120(6):715-9. http://doi.org/10.1016/j.jbiosc.2015.04.002. PMid:25935502.

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Published

2024-08-12

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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. 7];61:e219874. Available from: https://revistas.usp.br/bjvras/article/view/219874