Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

© The Author(s) 2020.[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.This research was supported by projects funded by the Spanish Ministry of Science and Innovation (AGL2016-75935-C2-R) and the Community of Madrid (PLATESA2-CM-P2018/BAA-4370). MF and RC were funded by UCM-Santander/2017 pre-doctoral grants, and PLATESA2 post-doctoral grants, respectively. CG was funded by DGAPA, National Autonomous University of Mexico (UNAM). RC, EC and LO are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint Programme.Peer reviewe

Influencia de la variabilidad en la virulencia de diferentes aislados de Toxoplasma gondii sobre las lesiones de encéfalos fetales ovinos

Trabajo presentado a la: XXXII Reunión de la Sociedad Española de Anatomía Patológica Veterinaria (SEAPV). 1 octubre. Congreso virtual.Peer reviewe

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep

Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1-105 tachyzoites intraperitoneally or with 25-2, 000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep

Survey of selected pathogens in free-ranging pinnipeds in Uruguay

Marine mammals, regarded as sentinels of aquatic ecosystem health, are exposed to different pathogens and parasites under natural conditions. We surveyed live South American fur seals Arctocephalus australis and South American sea lions Otaria flavescens in Uruguay for Leptospira spp., canine distemper virus (CDV), Mycobacterium spp., Toxoplasma gondii, and Neospora caninum. Samples were collected from 2007 to 2013. The seroprevalence of Leptospira spp. was 37.6% positive, 50.9% negative, and 11.5% suspect for A. australis (n = 61) while for O. flavescens (n = 12) it was 67% positive, 25% negative, and 8% suspect. CDV RNA was not detected in any of the analyzed samples. Most animals tested seropositive to tuberculosis antigens by WiZo ELISA (A. australis: 29/30; O. flavescens: 20/20); reactivity varied with a novel ELISA test (antigens MPB70, MPB83, ESAT6 and MPB59). Seroprevalence against N. caninum and T. gondii was 6.7 and 13.3% positive for O. flavescens and 0 and 2.2% positive for A. australis respectively. To evaluate possible sources of infection for pinnipeds, wild rats Rattus rattus and semi-feral cats Felis catus were also tested for Leptospira spp. and T. gondii respectively. Water samples tested for Leptospira revealed saprofitic L. bioflexa. Pathogenic Leptospira were detected in the kidneys of 2 rats, and cats tested positive for T. gondii (100%). These results represent a substantial contribution to the study of the health status of wild pinnipeds in Uruguay.CSIC (Council for Scientific Research) of the Universidad de la Republica.Peer reviewe

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection

19 páginas, 9 figuras, 1 tabla.Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring.This work was supported by the Spanish Ministry of Economy and Competi‑ tiveness (AGL2013-44694-R and AGL2016-75935-C2-1-R) and the Community of Madrid (PLATESA2-CM P2018/BAA-4370). Laura Jiménez-Pelayo was fnan‑ cially supported by a fellowship from the University Complutense of Madrid (including two research stays in 2017 and 2018) and Marta García-Sánchez was fnancially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-070723). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Early Neospora caninum infection dynamics in cattle after inoculation at mid-gestation with high (Nc-Spain7)- or low (Nc-Spain1H)-virulence isolates

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2013-44694-R) and the Community of Madrid (PLATESA2-CM P2018/BAA-4370). Laura Jiménez-Pelayo was financially supported by a fellowship from the Complutense University of Madrid and Marta García-Sánchez was financially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-070723). Patricia Vázquez had a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Alicia Román-Trufero was supported by a FPI-INIA fellowship from the Spanish National Institute for Agriculture and Food Research and Technology (INIA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe