Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

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 diversity 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 parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP 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 pattern, 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.

Isolation, Genotyping, and Mouse Virulence Characterization of Toxoplasma gondii From Free Ranging Iberian Pigs

“…Our CS3 typing results disagree with previous Brazilian and Chinese studies reporting high mortality rates associated with the type I or II alleles of the CS3 gene and low or null rates associated with the type III alleles (16,39,40). However, opposite results have been reported in studies from Brazil (41,42) and Spain (14). Clearly, additional investigations are required to determine the role of this locus and its interaction with related well-known virulence factors, such as ROP5 and ROP18 (43), in Toxoplasma virulence in mice and other hosts.…”

“…Genetic characterization based on PCR-RFLP identified 2 genotypes, ToxoDB #3 (type II PRU-variant) and #2 (clonal type III), predominantly identified in the European domestic livestock (reviewed in (14)), wildlife (23) and humans (24)(25)(26). In Europe, the available data on genotyping of T. gondii in infected pigs is scarce; recently, an study (27) summarized the major facts, including the presence of all three clonal lineage alleles with an apparent predominance of type II; however, it should be noted that in most of studies, only one or a few molecular markers was used thus limiting the resolution of the results.…”

“…Mice were observed daily, and clinical signs were scored. Procedures of confirmation of mouse infection and in vitro isolation in cell culture were carried out as described in reference (14).…”

“…Serum samples from mice, which were humanely euthanized, presented sudden death, or reached the end of the experiment at 6 weeks post-inoculation, were collected and stored at −20 • C until anti-T. gondii antibodies detection by IFAT to confirm the infection. Alternative procedures to evidence animal infection were carried out as described in reference (14).…”

“…Amplification was performed according to a standard protocol ( In addition, due to the unexpected mortality rates observed, we conducted nested PCR for CS3 locus amplification (16) over Toxoplasma-positive DNA samples from lung tissues of mice infected by TgPigSp1 (n = 3) and TgPigSp4 (n = 4) isolates (sacrificed at 7 or 30 dpi, respectively) for inocula genotype confirmation. PCR products were subjected to Sanger sequencing and obtained sequences were analyzed as described previously (14). CS3 sequences alignment to previously obtained TgPigSp1 (MW132600), TgPigSp4 (MW132601) and other reference strains (TgRH, MW151245; TgMe49, MW151246; TgNED, MW151247) sequences was carried out using the Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/).…”