23 jul 2021 - 14:12 CET

Genetic and phenotypic characterization of Toxoplasma gondii isolates obtained from sheep and Iberian pigs in Spain

Mercedes Fernández-Escobar, Doctoral Thesis (2021)

PhD supervisors: Luis Miguel Ortega-Mora, Esther Collantes-Fernández, Rafael Calero-Bernal

ABSTRACT

Toxoplasma gondii is an apicomplexan parasite globally distributed with a heteroxenous life cycle that virtually comprises all homoeothermic animals, including humans, as intermediate hosts and felids as definitive hosts. The zoonotic, abortifacient, and foodborne nature of the parasite makes toxoplasmosis a relevant public and animal health concern worldwide.

A comprehensive research effort on T. gondii biology along with the rapid development of molecular techniques suitable for strains genotyping over the last decades, led to the initial description of a widely clonal European and North American T. gondii genetic population dominated by three main clonal genetic types (I, II, and III), in contrast to an extremely diverse South American population. However, the information available from Europe is limited, with frequent methodological deficiencies and important sampling disparities among regions. Briefly, the available European literature evidences a clear predominance of type II strains (comprising around 80% of samples) coexisting with much less abundant type III and recombinant strains or mixed infections, as well as minor proportions of type I and imported genotypes. In the specific case of Spain, the majority of the scarce investigations dealt with direct genotyping from clinical samples, with the subsequent limitations to classify the strains and the impossibility of extending its characterization.

The virulence of T. gondii strains has been conventionally determined by mortality in laboratory mice, with type I isolates traditionally classified as highly virulent (100% lethality regardless of the dose), and types II and III considered intermediate (99-30% lethality) and non-virulent (<30% lethality) in a dose-dependent manner, respectively. Nonetheless, just as T. gondii population genetic structure is now known to go beyond this simple clonality, with up to 16 haplogroups described worldwide and an enormous diversity existing in South America, the traditional virulence strains classification is up for debate. In addition to the scarce information on circulating genotypes in Spain, the knowledge about the phenotypic characteristics of such strains is virtually nil.

With this background, the general objective of the present Doctoral Thesis was to obtain a representative panel of isolates from Spanish livestock, namely sheep and Iberian pigs, that might enable us to study the genetic and phenotypic diversity of circulating strains in such farm animals by implementing molecular, in vivo and in vitro methodologies. In the first specific objective proposed, isolation procedures were implemented on ovine abortion-derived tissues occurred all over our country and submitted for T. gondii diagnosis, as well as on myocardial tissues from chronically infected adult sheep slaughtered for human consumption (Sub-objective 1.1). A total of 31 T. gondii DNA-positive foetal brains and 50 adult heart tissues associated with high antibody ELISA titres, were selected for bioassay in mice. Thereby, 10 isolates (TgShSp2-10 and TgShSp18) were obtained from five different abortion outbreaks, and 20 isolates (TgShSp11-17 and TgShSp19-31) were obtained from slaughtered adults raised in five different locations. Together with the TgShSp1 isolate available in our laboratory, the panel represented a significant cross-section of the T. gondii population infecting Spanish sheep flocks. Genetic characterization of the isolates (Sub-objective 1.2) was firstly carried out by the widely used PCR-RFLP method based on SAG1, SAG2 (5’- 3’ SAG2, and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico markers. As a result, 90.3% (28/31) of the isolates were determined to present a genotype II PRU variant (ToxoDB #3), 6.5% (2/31) of them corresponded to a clonal genotype III (ToxoDB #2) and finally, only 3.2% (1/31) presented a clonal genotype II (ToxoDB #1). Thanks to the great effort on sample collection from ovine abortion outbreaks and the implementation of the PCR-RFLP method on all DNA-positive clinical samples (abortion-derived tissues and myocardial sample digests, n = 151), a more complex genetic diversity was observed, revealing co-infection events and demonstrating the selection of certain strains during bioassay experiments. Furthermore, PCR-DNA sequencing of fragments of the SAG3, GRA6 and GRA7 polymorphic genes, led to the confirmation of RFLP results and to the detection of a widespread single-nucleotide polymorphism at the SAG3 locus across the Spanish population, which had already been described in sheep French isolates, suggesting a common origin in livestock from both neighbouring countries.

Subsequently, and regarding the phenotypic characterization of some of the T. gondii isolates recently obtained from sheep (Sub-objective 1.3), a comprehensive virulence assessment in mice procedure including lethal (cumulative mortality) and non-lethal parameters (i.e., cumulative morbidity, as well as tropism, parasite burden and histopathological lesions in different tissues), was designed and further implemented on 10 selected isolates. Moreover, as a valuable complement for the in vivo virulence evaluation, an ovine trophoblast cell line (AH-1) was chosen for the characterization of in vitro invasion rates and proliferation kinetics of six selected isolates. Thus, most type II isolates possessed non-virulent characteristics, although important intra-genotype differences were observed. The TgShSp16 isolate (#3) stood out with 21% cumulative mortality rate and a significant enhanced ability to disseminate in vivo to the brain, despite low-intermediate in vitro invasion and proliferation rates. On the other hand, the type III (#2) TgShSp24 isolate presented similar lethality and parasite burdens in mice, as well as the highest invasion rate along with a tachyzoite production at 72 hpi nine to three times higher than that of the rest of the isolates in AH-1 cells. Finally, the CS3/ROP18/ROP5 allelic combination of isolates included in phenotypic characterization assays was investigated, resulting in a profile II/2/2 in those isolates with a RFLP-clonal genotype II or PRU variant, and in a profile III/3/3 in those isolates with a RFLP-clonal genotype III.

In the second specific objective, heart tissues from adult Iberian pigs collected at slaughterhouse were subjected to isolation procedures (Sub-objective 2.1). Thus, 15 myocardial tissues from animals with high antibody ELISA titres and reared in different locations of southwestern Spain were bioassayed in mice. As a result, five isolates (TgPigSp1-5) were obtained. Genetic characterization of the isolates (Sub-objective 2.2) by PCR-RFLP resulted in 60% (3/5) of the isolates presenting a genotype II PRU variant (ToxoDB #3) and 40% (2/5) corresponding to a clonal genotype III (ToxoDB #2). Aiming phenotypic characterization of T. gondii isolates obtained from Iberian pigs belonging to the two prevalent genotypes in the population (Sub-objective 2.3), the above-mentioned virulence in mice assay was implemented on TgPigSp1 (type III, #2) and TgPigSp4 (type II PRU variant, #3) isolates. Accordingly, the TgPigSp1 isolate was moderately/highly virulent in mice with notable cumulative mortality (87.5%) and morbidity (100%) rates, whereas TgPigSp4 was non-virulent (0% lethality) and triggered only mild clinical signs in 42.1% of seropositive mice. Parasite burdens and histopathological lesions found in mouse tissues supported the significant differences between both strains.

In short, in the different investigations that comprise this Doctoral Thesis, a representative panel of circulating isolates in sheep and Iberian pigs in Spain has been obtained, covering a wide part of the country and especially leading regions in sheep and Iberian pigs breeding. The genetic characterization findings agreed with the available European genotyping data on T. gondii strains infecting domestic animals and highlight the specific predominance of the genotype II PRU variant within the haplogroup 2 and the noteworthy prevalence of type III strains. Concerning the virulence assessment, type III isolates presented the most virulent profile among the strains evaluated, although it should be noted the large intragenotype phenotypic variability within type II and type III genotypes in the T. gondii Spanish population. These findings directly contradict former classifications that considered type III strains as the least virulent among the three T. gondii clonal types and demonstrate that current widely used genetic characterization methods are not informative enough to sort out Toxoplasma population virulence. The CS3/ROP18/ROP5 allelic combination of selected isolates showed no strong link with the degree of virulence shown, providing additional evidence that other genetic factors must be involved in the virulence of T. gondii in mice. The limitations of current genotyping methods have become apparent in present studies, emphasizing the need to implement next-generation tools (e.g., whole-genome sequencing) that may allow us to obtain much more detailed, accurate, and resolutive genetic information of Spanish T. gondii isolates, which in turn may help to explain the wide phenotypic variability observed.

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