Blastocystis sp. subtype diversity in wild mesocarnivores from Spain

Authors: CALERO-BERNAL, RAFAEL - Complutense University Of Madrid (UCM); Santin-Duran, Monica; MALONEY, JENNY - US Department Of Agriculture (USDA); MARTIN-PEREZ, MANUEL - Universidad De Extremadura; HABELA, MIGUEL ANGEL - Universidad De Extremadura; FERNANDEZ-GARCIA, JOSE - Universidad De Extremadura; FIGUEIREDO, ANA - University Of Aveiro (UA); NAJERA, FERNANDO - Centro De Investigaciones Cientificas Y Tecnologicas De Extremadura; PALACIOS, MARIA - Centro De Investigaciones Cientificas Y Tecnologicas De Extremadura; MATEO, MARTA - Alfonso X El Sabio University; BALSEIRO, ANA - Agriculture Research And Development Service (SERIDA); BARRAL, MARTA - Neiker-Instituto Vasco De Investigacion Y Desarrollo Arario; LIMA BARBERO, JOSE FRANCISCO - University Of Castilla-La Mancha(UCLM); KOSTER, PAMELA - Instituto De Salud Carlos Iii; CARMENA, DAVID - Instituto De Salud Carlos Iii

Submitted to: Journal of Eukaryotic Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2019
Publication Date: 11/26/2019
Citation: Calero-Bernal, R., Santin, M., Maloney, J.G., Martin-Perez, M., Habela, M., Fernandez-Garcia, J.L., Figueiredo, A., Najera, F., Palacios, M.J., Mateo, M., Balseiro, A., Barral, M., Lima Barbero, J., Koster, P.C., Carmena, D. 2019. Blastocystis sp. subtype diversity in wild mesocarnivores from Spain. Journal of Eukaryotic Microbiology. 67(2):273-278.

Interpretive Summary: Blastocystis sp. is probably the most common enteric parasite in humans globally. Transmission is fecal-oral through ingestion of cyst-contaminated water or food. Blastocystis infections have been linked with gastrointestinal symptoms and extra-intestinal disorders (hives), but Blastocystis carriage could be also asymptomatic. In addition to humans, Blastocystis is frequently reported in a wide range of animals, including domestic and wild, raising concern on the importance of animal reservoirs in the epidemiology of this parasite. Little information is currently available on the occurrence of Blastocystis in wildlife animal populations. To fill this gap a study to investigate the role of wild carnivores as potential natural reservoirs of human blastocystosis was conducted. A total of 380 fecal specimens from host species belonging to the Canidae (n = 187), Erinaceidae (n = 2), Felidae (n = 25), Herpestidae (n = 11), Mustelidae (n = 133), Procyonidae (n = 11), and Viverridae (n = 11) families were collected in Spain. Detection of Blastocystis sp. was achieved by molecular methods. Overall, Blastocystis was confirmed in 1.6% (6/380) of the specimens analyzed. Positives included four foxes, a common genet, and a European polecat. Next generation sequencing showed higher sensitivity and revealed a higher genetic diversity that Sanger sequencing. Overall, molecular characterization identified five Blastocystis subtypes in this population, ST1, ST2, ST4, ST7, and ST14. ST14 was found in a fox and a European polecat, ST7 in a fox and a common genet, and the other two foxes presented mixed infections with ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Blastocystis ST1, ST2, ST3, ST4, and sporadically ST7, are the most frequently subtypes found in both symptomatic and asymptomatic Spanish human populations. Our data suggest that wild mesocarnivore species could act as carriers of zoonotic Blastocystis subtypes. and as a source of human infection and environmental contamination. This research provides beneficial information to other scientists, veterinarians and public health agencies on the prevalence and subtypes of Blastocystis that infect wild carnivores.

Technical Abstract: Little information is currently available on the occurrence and molecular diversity of the heterokont eukaryote Blastocystis sp. in wildlife animal populations. To fill this gap a study to investigate the role of free-living mesocarnivores as potential natural reservoirs of human blastocystosis was conducted. A total of 380 faecal specimens from host species belonging to the Canidae (n = 187), Erinaceidae (n = 2), Felidae (n = 25), Herpestidae (n = 11), Mustelidae (n = 133), Procyonidae (n = 11), and Viverridae (n = 11) families were collected in Spain. Detection of Blastocystis sp. was achieved by PCR amplification of a fragment of the small subunit (ssu) rDNA gene using two different sets of primers and subtype (ST) identification was performed by Sanger and next generation amplicon sequencing (NGS). Overall, Blastocystis sp. was confirmed by PCR and sequencing in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by the first set of primers and Sanger. Using the second set of primers and NGS, five specimens from 4 foxes and a European polecat were successfully subtyped. NGS data revealed a higher genetic diversity: ST14 was found in a fox and a European polecat, ST7 in a fox, and the other two foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Blastocystis ST1, ST2, ST3, ST4, and sporadically ST7, have previously been described in both symptomatic and asymptomatic Spanish human populations. Our data suggest that wild mesocarnivore species could act as carriers of zoonotic Blastocystis subtypes, although the extent and directionality of these infections (zoonotic or anthroponotic) remains to be determined in future surveys.