Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
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RESEARCH
Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks Mercedes Fernández‑Escobar1, Rafael Calero‑Bernal1*, Julio Benavides2, Javier Regidor‑Cerrillo3, María Cristina Guerrero‑Molina1, Daniel Gutiérrez‑Expósito2, Esther Collantes‑Fernández1 and Luis Miguel Ortega‑Mora1*
Abstract Background: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diver‑ sity among the T. gondii strains circulating in ovine livestock in Spain. Methods: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at para‑ site isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCRRFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. Results: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pat‑ tern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. Conclusions: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity. Keywords: Toxoplasma gondii, Sheep, Abortion, Isolates, Genotyping, Sequencing, Population structure, Spain Background Toxoplasma gondii (Apicomplexa) is known as one of the main causes of ovine reproductive failure, causing significant economic losses to the sheep industry *Correspondence: [email protected]; [email protected] 1 SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain Full list of author information is available a
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