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

Abstract

The present study aimed to isolate and perform molecular and phenotypic characterization of Toxoplasma gondii strains infecting Iberian pigs bred under semi-free conditions and destined for human consumption. Blood and heart tissue samples from 361 fattening pigs from 10 various herds selected in the main areas of Iberian pig production were collected at a slaughterhouse; the sera were tested for anti-T. gondii antibodies using a commercial indirect ELISA kit, and a mouse bioassay was carried out using heart muscle of seropositive individual representatives from each geographical location. Seventy-nine (21.9%) of the 361 animals tested positive for anti-T. gondii antibodies according to the serology test. Fifteen samples of myocardial tissue were subjected to bioassay and 5 isolates (TgPigSp1 to TgPigSp5) were obtained. The isolates were characterized by using 11 PCR-RFLP genetic markers; three isolates had a ToxoDB #3 genotype (3/5) and two isolates had a ToxoDB #2 genotype (2/5). The TgPigSp1 and TgPigSp4 isolates were selected for virulence in mice characterization as instances of each different RFLP-genotype found. The TgPigSp1 isolate (#2 genotype) was virulent in mice with notable cumulative mortality (87.5%) and morbidity rates (100%); the TgPigSp4 (#3) was nonvirulent and triggered mild clinical signs in 42.1% of seropositive mice. Infection dynamics and organ distribution of both isolates were analyzed; the data revealed significant differences, including substantially higher parasite load in the lung during the acute phase of infection, in mice infected with TgPigSp1 than in the case of TgPigSp4 (median parasite load 7.6 vs. 0 zoites/mg, respectively; p < 0.05). Furthermore, degrees of severity of detected histopathological lesions appeared to be related to higher parasite burdens. Taking into account the unexpectedly high mortality rate and parasite load associated with the clonal genotype III, which is traditionally considered nonvirulent in mice, the need for further investigation and characterization of the T. gondii strains circulating in any host in Europe is emphasized.

In vivo and in vitro models show unexpected degrees of virulence among Toxoplasma gondii type II and III isolates from sheep

“…However, this simplistic criterion may hide important differences not only in lethality but also in the severity of the clinical outcome [ 45 ]. Along with cumulative mortality rates, the virulence degree has also been evaluated by morbidity, parasite burdens and pathological lesions detected in different tissues (e.g., central nervous system) [ 12 ], and by other nonlethal infection parameters, such as weight loss, anti- T. gondii IgG antibodies and haptoglobin levels in serum, cystogenic capacity, or even animal behavioural changes [ 31 , 45 – 48 ]. Variations in specific virulence features have already been demonstrated among strains presenting different genotypes, as well as within some belonging to the same genetic type determined by RFLP-based methods [ 8 , 31 , 45 , 49 , 50 ].…”

“…Toxoplasma gondii antibody detection by IFAT and DNA extraction, detection and quantification in assay B were implemented exactly as in reference [ 12 ]. In both assays A and B, mouse Toxoplasma infections were confirmed by IFAT (titer > 1:25) or by brain or lung tissue imprints.…”

“…After washing with PBS (× 3), wells were treated with 3% BSA in PBS for 30 min at room temperature (RT) to block nonspecific antibody binding. Then, parasites were stained using positive murine serum samples from previous experimental infections (1:200 in a 0.3% BSA/PBS solution) [ 12 , 15 ] as a primary antibody (1 h, 37 °C) and a 1:1000 dilution (in PBS) of goat anti-mouse IgG conjugated to Alexa Fluor ® 488 (green, Thermo Fisher Scientific, Waltham, MA, USA) as a secondary antibody (1 h, RT). The nuclei were stained by washing the cells with a solution of 1:10 000 DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; Invitrogen™) in PBS.…”

“…Nonetheless, the studies were strongly biased by the fact that isolates included were mainly from human patients and domestic animals originating from France and the USA, along with the implementation of monolocus typing strategies promoting the misidentification of atypical and recombinant strains circulating [ 8 ]. Two decades of scientific effort in isolation and both molecular and phenotypic characterization of the parasite led to the discovery of a much more complex reality involving a population structure with at least 16 haplogroups worldwide [ 9 – 11 ] and a virulence degree classification under debate [ 12 ]. Regarding strains that circulate in European and Spanish sheep livestock, there are several genotyping studies that demonstrate the total predominance of type II strains, which coexist with small percentages of type III and recombinant strains [ 13 – 15 ].…”

Molecular Detection of Toxoplasma gondii and Neospora caninum in Domestic Ducks in Hunan Province, China

“…Eight reference T. gondii strains (GT1, PTG, CTG, MAS, TgCgCa1, TgCatBr5, TgCatBr64, and TgRsCr1) were included as controls as reported in previous studies ( 33 , 34 ). The genotype was determined by comparing its multilocus pattern to the pattern of all genotypes present in ToxoDB ( http://toxodb.org/toxo/ ) ( 35 ).…”