Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. in brain samples of dogs with and without neurological signs in Southern Brazil (2008-2023)
DOI:
https://doi.org/10.5433/1679-0359.2025v46n4p1051Keywords:
Apicomplexa, Histopathology, Molecular diagnosis, PCR, RFLP.Abstract
Encephalitis can be associated with protozoa of the phylum Apicomplexa. Sarcocystis spp., Toxoplasma gondii, and Neospora caninum share morphological similarities that complicate differentiation in histopathology, leading to inaccurate diagnoses without complementary techniques. Clinical manifestations often associate with host immunosuppression secondary to other diseases. This study examined brain samples from dogs undergoing necropsy in Rio Grande do Sul, Brazil, from 2008 to 2023, using two groups: dogs with neurological signs caused by protozoa (G1) and dogs without neurological signs (G2). G1 aimed to verify if protozoa identified histopathologically matched molecular techniques. G2 observed the frequency of protozoa detection using PCR in dogs without neurological signs. A total of 230 samples were analyzed: 11 paraffin-embedded in G1 and 219 frozen in G2. DNA extraction used commercial kits, and all samples underwent PCR for the 18S rRNA gene. G1 samples also underwent Restriction Fragment Length Polymorphism (RFLP) analysis. Protozoal DNA was detected in six samples (6/11) from G1 with varying molecular identifications. In G2, 82 samples (82/219; 37.45%) were PCR-positive. These findings highlight the importance of molecular techniques for accurate etiological confirmation and diagnosis, extending epidemiological knowledge and emphasizing Toxoplasma gondii, Neospora. caninum, and Sarcocystis spp. as differential diagnoses for central nervous system diseases in dogs.
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Aguiar, D. M., Amude, A. M., Santos, L. G. F., Ribeiro, M. G., Ueno, T. E. H., Megid, J., Paes, A. C., Alfieri, A. F., Alfieri, A. A. & Gennari, S. M. (2012). Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 64 (1), 221-224. doi: 10.1590/S0102-09352012000100032
Ajzenberg, D., Year, H., Marty, P., Paris, L., Dalle, F., Menotti, J., Aubert, D., Franck J., Bessières, M.H., Quinio, D., Pelloux, H., Delhaes, L., Desbois, N., Thulliez, P., Robert-Gangneux, F., Kauffmann-Lacroix, C., Pujol, S., Rabodonirin, M., Bougnoux, M-E., Cuisenier, B., ...Villena, I. (2009). Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. Journal of Infectious Diseases, 199(8), 1155-1167. doi: 10.1086/597477
Arruda, I. F., Millar, P. R., Barbosa, A. S., Abbout, L. C. S., Reis, I. C., Moreira, A. S. C., Guimarães, M. P. P., & Amendoeira, M. R. R. (2021). Toxoplasma gondii in domiciled dogs and cats in urban areas of Brazil: risk factors and spatial distribution. Parasite Journal, 28(56), 1-12. doi: 10.1051/parasite/2021049
Barker, D. C. (1989). Molecular approaches to DNA diagnosis. Molecular approaches to DNA diagnosis. Parasitology, 99(1), 125-146. doi: 10.1017/s0031182000083463
Barber, J. S., Payne-Johnson & Trees, A. J. (1996). Distribution of Neospora caninum within the central nervous system and other tissues of six dogs with clinical neosporosis. Journal of Small Animal Practice, 37(12), 568-574. doi: 10.1111/j.1748-5827.1996.tb02332.x
Behmer, O. A., Colasante, G., & Silva, R. P. (1976). Manual de técnicas histológicas adaptadas. São Paulo.
Bjerkas, I., Mohn, D. F., & Presthus, J. (1984). Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Zeitschrift fur Parasitenkd, 70(2), 271-274. doi: 10.1007/BF00942230
Blanchard, N., Dunay, I. R., & Schuter, D. (2015). Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system. Parasite Immunology, 37(3), 150-158. doi: 10.1111/pim.12173
Brandt, H. R. C., Arnone, M., Valente, N. Y. S., Criado, P. R. C., & Sotto, M. N. (2007). Small vessel cutaneous vasculitis: etiology, pathogenesis, classification and diagnostic criteria - Part I. Anais Brasileiros de Dermatologia, 82(5), 387-406.
Cantile, C., & Youssef, S. (2016). Nervous system. Jubb, Kennedy & Palmer's Pathology of Domestic Animals, 1(20), 250-406. doi: 10.1016/B978-0-7020-5317-7.00004-7
Caramalac, S. M., Caramalac, S. M., Castilho, P. M., Lucas, J. I., Minutti, A. F., Garcia, J. L., Barros, L. D., & Palumbo, M. I. P. (2021). Seroprevalence of Toxoplasma gondii, Neospora caninum, and Leishmania spp. in hunting dogs from Mato Grosso do Sul, Brazil. Ciência Rural, 51(5), 1-5. doi: 10.1590/0103-8478cr20200533
Caramalac, S. M., Caramalac, S. M., Leite, B. S. N. N. C., Castilho, P. M., Minutti, A. F., Martins, T. A., Garcia, J. L., Barros, L. D., Romão, F. G., & Palumbo, M. I. P. (2023). Anti-Toxoplasma gondii and anti-Neospora caninum antibodies in dogs with and without neurological signs. Ciência Rural, 53(9), 1-5. doi: 10.1590/0103-8478cr20220133
Chaves, R. O., Beckmann, D. V., Santos, R. P., Aiello, G., Andrades, A. O., Baumhardt, R., Silveira, L. B., & Mazzanti, A. (2014). Neurological diseases in dogs examined at the Veterinary Teaching Hospital of the Federal University of Santa Maria, RS: 1.184 cases (2006-2013). Pesquisa Veterinária Brasileira, 34(10), 996-1001. doi: 10.1590/S0100-736X2014001000012
Cooley, A. J., Barr, B., & Rejmanek, D. (2007). Sarcocystis neurona encephalitis in a Dog. Veterinary Pathology, 44(6), 956-961. doi: 10.1354/vp.44-6-956
Cooley, A. J., Clemmons, R. M., & Gross, T. L. (1987). Heartworm disease manifested by encephalomyelitis and myositis in a dog. Journal of the American Veterinary Medical Association, 190(4), 431-432.
Didiano, A., Monti, P., Taeymans, O., & Cherubini, G. B. (2020). Canine central nervous system neosporosis: clinical, laboratory and diagnostic imaging findings in six dogs. Veterinary Record Case Reports, 8(1), 1-7. doi: 10.1136/vetreccr-2019-000905
Dubey, J. P., & Lappin, M. R. (2005). Toxoplasmosis and neosporosis. In Greene, C. E. (Ed.), Infectious Diseases of Dogs and Cats (pp. 754-775). St. Louis, MO.
Dubey, J. P., & Speer, C. A. (1991). Sarcocystis canis n. sp. (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs. The Journal of Parasitology, 77(4), 522-527. doi: 10.2307/3283155
Dubey, J. P., Black, S. S., Verma, S. K., Calero-Bernal, R., Morris, E., Hanson, M. A., & Cooley, A. J. (2014). Sarcocystis neurona schizonts-associated encephalitis, chorioretinitis, and myositis in a two-month-old dog simulating toxoplasmosis, and presence of mature sarcocysts in muscles. Veterinary Parasitology, 202(3-4), 194-200. doi: 10.1016/j.vetpar.2014.02.055.s
Dubey, J.P., Lindsay, D.S. & Speer C.A. (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews, 11(2), 267–299. doi: 10.1128/cmr.11.2.267
Elbert, J. A., Yau, W., & Rissi, D. R. (2022). Neuroinflammatory diseases of the central nervous system of dogs: a retrospective study of 207 cases (2008-2019). The Canadian Veterinary Journal, 63(2), 178-186. PMID: 35110776
Ferreira, M. A. J. da F. (2003). Utilização das técnicas de marcadores moleculares na genética de populações, na genética quantitativa e no melhoramento de plantas. (Documentos, 1). EMBRAPA Roraima.
Ferreira, A. M., Vitor, R. W., Gazzinelli, R. T. & Melo, M. N. (2006) Genetic analysis of natural recombinant Brazilian Toxoplasma gondii strains by multilocus PCR‑RFLP. Infection, Genetics and Evolution, 6(1), 22–31. doi: 10.1016/j.meegid.2004.12.004
Fitzgerald, S. D., Janovitz, E. B., Kazacos, K. R., Dubey, J. P., & Murphy, D. A. (1993). Sarcocystosis with involvement of the central nervous system in lambs. The Journal of Veterinary Diagnostic Investigation, 5(2), 291-296. doi: 10.1177/10406387930050023
Frade, M. T. S., Ferreira, J. S., Nascimento, M. J. R., Aquino, V. V. F., Macêdo, I. L., Carneiro, R. S., Souza, A. P., & Dantas, A. F. M. (2018). Center nervous system disorders diagnosed in dogs. Pesquisa Veterinária Brasileira, 38(5), 935-948. doi: 10.1590/1678-5150-PVB-5100
Freitas, B. R. de, Rosa, G. da, Roman, I. J., Cunha, R. C., Gressler, L. T., Cargnelutti, J. F., & Vogel, F. S. F. V. (2023). Molecular detection of Toxoplasma gondii, Neospora caninum and Sarcocystis spp in tissues of Sus scrofa slaughtered in southern Brazil. Brazilian Journal of Veterinary Parasitology, 32(3), 1-11. doi: 10.1590/S1984-29612023048
Gaitero, L., Añor, S., Montoliu, P., Zamora, A., & Pumarola, M. (2006). Detection of Neospora caninum tachyzoites in canine cerebrospinal fluid. Journal of Veterinary Internal Medicine, 20(2), 410-414. doi: 10.1111/j.1939-1676.2006.tb02877.x
Gerhold, R., Newman, S. J., Grunenwald, C. M., Crews, A., Hodshon, A., & Su, C. (2014). Acute onset of encephalomyelitis with atypical lesions associated with dual infection of Sarcocystis neurona and Toxoplasma gondii in a dog. Veterinary Parasitology, 205(3-4), 697-701. doi: 10.1016/j.vetpar.2014.09.008.
Giraldi, J. H., Bracarense, A. P. F. R. L., Vidotto, O., Tudury, E. A., Navarro, I. T., & Batista, T. N. (2002). Serology and histopathology of Toxoplasma gondii and Neospora caninum in dogs with neurologic disorders. Semina: Ciências Agrárias, 23(1), 9-14. doi: 10.5433/1679-0359.2002v23n1p9
Gondim, L. F. P. (2006). Neospora caninum in wildlife. Trends in Parasitology, 22(6), 247-252. doi: 10.1016/j.pt.2006.03.008
Graham, A. K., Fong, C., Naqvi, A., & Lu, J. Q. (2021). Toxoplasmosis of the central nervous system: manifestations vary with immune responses. The Journal of the Neurological Sciences, 420, 117223. doi: 10.1016/j.jns.2020.117223
Hajj-Ali, H. A., & Calabrese, L. H. (2014). Diagnosis and classification of central nervous system vasculitis. The Journal of Autoimmunity, 48-49, 149-152. doi: 10.1016/j.jaut.2014.01.007
Headley, S. A., Alfieri, A. A., Fritzen, J. T. T., Garcia, J. L., Weissenböck, H., Silva, A. P. da, Bodnar, L., Okano, W., & Alfieri, A. F. (2013). Concomitant canine distemper, infectious canine hepatitis, canine parvoviral enteritis, canine infectious tracheobronchitis, and toxoplasmosis in a puppy. The Journal of Veterinary Diagnostic Investigation, 25(1), 129-135. doi: 10.1177/10406387124713
Henderson, J. M., Dies, K. H., Haines, D. M., Higgs, G. W., & Ayroud, M. (1997). Neurological symptoms associated with sarcocystosis in adult sheep. The Canadian Veterinary Journal, 38(3), 168-170.
Kubo, M., Kawachi, T., Murakami, M., Kubo, M., Tokuhiro, S., Agatsuma, T., Ito, K., Okano, T., Asano, M., Fukushi, H., Nagataki, M., Sakai, H., & Yanai, T. (2010). Meningoencephalitis associated with Sarcocystis spp. in a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus). Journal of Comparative Pathology, 143(2-3), 185-189. doi: 10.1016/j.jcpa.2009.12.014
Langoni, H., Matteuci, G., Medici, B., Camossi., L. G., Richini-Pereira, V. B., & Silva, R. C. da. (2012). Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders. Revista da Sociedade Brasileira de Medicina Tropical, 45(3), 365-368. doi: 10.1590/S0037-86822012000300016
Lotti, T. M., Comacchi, C., & Ghersetich, I. (1999). Cutaneous necrotizing vasculitis. Relation to systemic disease. Advances in Experimental Medicine and Biology, 455, 115-125.
Lüder, C. G. K., & Rahman, T. (2017). Impact of the host on Toxoplasma stage differentiation. Microbial Cell, 4(7), 203-211. doi: 10.15698/mic2017.07.579
McAllister, M. M., Dubey. J. P., Lindsay, D. S., Jolley, W. R., Wills, R. A., & McGuire, A. (1998). Rapid communication: dogs are definitive hosts of Neospora caninum. Internacional Journal Parasitology, 28(9), 1473-1479. doi: 10.1016/S0020-7519(98)00138-6
Miller, M. A., Crosbie, P. R., Sverlow, K., Hanni, K., Barr, B. C., Kock, N., Murray, M. J., Lowenstine, L. J., & Conrad, P. A. (2001). Isolation and characterization of Sarcocystis from brain tissue of a free-living southern sea otter (Enhydra lutris nereis) with fatal meningoencephalitis. Parasitology Research, 87(3), 252-257. doi: 10.1007/s004360000340
Moretti, L. d’Arc., Ueno, T. E. H., Ribeiro, M. G. M., Aguiar, D. M., Paes, A. C., Pezerico, S. B., & Silva, A. V. (2002). Toxoplasmosis in distemper virus infected dogs. Semina Ciências Agrárias, 23(1), 85-91. doi: 10.5433/1679‑0359.2002v23n1p85
Morita, T. C. A. B., Trés, G. F. S., Criado, R. F. J., Sotto, M. N., & Criado, P. R. (2020). Atualização em vasculites: visão geral e pontos relevantes dermatológicos para o diagnóstico clínico e histopatológico - Parte I. Anais Brasileiros de Dermatologia, 95(3), 355-371. doi: 10.1016/j.abdp.2020.01.004
Neupane, S., Wellehan, J. F. X., Childerss, A. L., Snook, E. R., & Porter, B. F. (2023). Meningoencephalitis caused by concurrent infection with canine distemper virus and a unique Sarcocystis sp. in a gray fox. The Journal of Veterinary Diagnostic Investigation, 35(4), 444-448. doi: 10.1177/10406387231169768
Oliveira, C. M. C., Oliveira, P. B., Albuquerque, G. R., & Gondim, L. F. (2020). Serologic reactivity of canine sera to Sarcocystis neurona and Sarcocystis cruzi antigens. Veterinary Parasitology: Regional Studies and Reports, 22, 100429. doi: 10.1016/j.vprsr.2020.100439
Patitucci, A. N., Alley, M. R., Jones, B. R., & Charleston, W. A. (1996). Protozoal encephalomyelitis of dogs involving Neospora caninum and Toxoplasma gondii in New Zealand. The New Zealand Veterinary Journal, 45(6), 231-235. doi: 10.1080/00480169.1997.36035
Perkins, S. L., & Kjeldsberg, C. R. (1993). Immunophenotyping of lymphomas and leukemias in paraffin- embedded tissues. American Journal of Clinical Pathology, 99(4), 362-373. doi: 10.1093/ajcp/99.4.362
Ravi, M., Patel, J., Pybus, M., Coleman, J. K., Childress, A. L., & Wellehan, J. F. X., Jr. (2015). Meningoencephalitis associated with disseminated sarcocystosis in a free-ranging moose (Alces alces) calf. The Canadian Veterinary Journal, 56(8), 872-875.
Robson, K., & Smith, P. M. (2011). Cryptococcal meningoencephalitis in a dog. Veterinary Record, 168(20), 538. doi: 10.1136/vr.d438
Rodrigues, J. Y., Almeida, A. B. P. F. de, Sorte, E. C. B., Gasparetto, N. D., Cruz, F. A. C. S. da, & Souza, V. R. F. (2016). Seroprevalence of Toxoplasma gondii in dogs of riverside communities of Mato Grosso Pantanal, Brazil. Brazilian Journal of Veterinary Parasitology, 25(4), 531-535. doi: 10.1590/S1984-29612016067
Scorsato, A. P., & Telles, J. E. Q. (2011). Factors that affect the quality of DNA extracted from biological samples stored in paraffin blocks. Jornal Brasileiro de Patologia e Medicina Laboratorial, 47(5), 541-548. doi: 10.1590/S1676-24442011000500008
Shannon, L. D., Lindsay, S. A., Krockenberg, M., Phalen, D., & Šlapeta, J. (2015). A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. The International Journal for Parasitology: Parasites and Wildlife, 4(2), 216-238. doi: 10.1016/j.ijppaw.2015.04.002
Silva, A. V. da, Pezerico, B. S., Lima, V. Y. de, Moretti, L. D., Pinheiro, J. P., Tanaka, E. M., Ribeiro, G. R., & Langoni, H. (2005). Genotyping of Toxoplasma gondii strains isolated from dogs with neurological sign. Veterinary Parasitology, 127(1), 23-27. doi: 10.1016/j.vetpar.2004.08.020
Silva, R. C. da, Chunlei, S. U., & Langoni, L. (2009). First identification of Sarcocystis tenella (Railliet, 1886) Moulé, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil. Veterinary Parasitology, 165(3-4), 332-336. doi: 10.1016/j.vetpar.2009.07.016
Silva, V. L. B., Pacheco, T. A., Melo, A. L. T., Aguiar, D. M., Pacheco, R. C., & Ramos, D. G. S. (2022). Epidemiological aspects of the occurrence of anti-Sarcocystis spp. anti-Toxoplasma gondii antibodies in dogs in the Northern region of Brazil. Brazilian Journal of Veterinary Science, 29(4), 185-188. doi: 10.4322/rbcv.2022.033
Skariah, S., McIntyre, M. C., & Mordue, D. G. (2010). Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion. Parasitology Research, 107(2), 253-260. doi: 10.1007/s00436-010-1899-6
Speer, C. A., Dubey, J. P., McAllister, M. M., & Blixt, J. A. (1999). Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii. The International Journal for Parasitology, 29(10), 1509-1519. doi: 10.1016/s0020-7519(99)00132
Su, C., Shwab, E. K., Zhu, X. Q., & Dubey, J. P. (2010). Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology, 137(1), 1-11. doi: 10.1017/S0031182009991065
Tavares, R. G., Staggemeier, R., Borges, A. L. P., Rodrigues, M. T., Castelan, L. A., Vasconcelos, J., Anschau, M. E., & Spalding, S. M. (2011). Molecular techniques for the study and diagnosis of parasite infection. Journal of Venomous Animals and Toxins Including Tropical Diseases, 17(3), 239-248. doi: 10.1590/S1678-91992011000300003
Toscan, G., Camillo, G., Weber, A., Oliveira, C. S., Gonçalves, P. B. D., Sangioni, L. A., & Vogel, F. S. F. (2012). Detection of nucleic acids in tissues of gerbils submitted to acute infection with Neospora caninum. Ciência Rural, 42(10), 1865-1871. doi: 10.1590/S0103-84782012005000079
Uggla, A., Dubey, J. P., Lundmark, G., & Olson, P. (1989). Encephalomyelitis and myositis in a boxer puppy due to a Neospora-like infection. Veterinary Parasitology, 32(2-3), 255-260. doi: 10.1016/0304-4017(89)90125-8
Vashisht, K., Lichtensteiger, C. A., Miller, L. A., Gondim, L. F. P., & McAllister, M. M. (2005). Naturally occuring Sarcocystis neurona-like infection in a dog with myositis. Veterinary Parasitology, 133(1), 19-25. doi: 10.1016/j.vetpar.2005.04.042
Yamage, M., Flechtner, O., & Gottstein, B. (1996). Neospora caninum: specific oligonucleotide primers for the detection of brain "Cyst" DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). The Journal of Parasitology, 82(2), 272-279. doi: 10.2307/3284160
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