Search

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is currently recognized as one of the major causes of acute human hepatitis worldwide. In Europe, the increasing number of hepatitis E cases is mainly associated with the consumption of animal food products or contact with infected animals. Dogs and cats have been suggested as a zoonotic source of HEV infection. The aim of this study was to assess Orthohepevirus circulation, including HEV-A, HEV-B and HEV-C species, in sympatric urban cats and dogs in southern Spain. Between 2017 and 2020, blood samples were collected from 144 stray cats and 152 dogs, both strays and pets. The presence of antibodies against HEV were tested using a double-antigen sandwich ELISA and seropositive samples were further analysed by western blot. A RT-PCR was performed to detect RNA of Orthohepevirus species (HEV-A, HEV-B and HEV-C). A total of 19 (6.4%; 95%CI: 3.6-9.2) of the 296 animals tested showed anti-HEV antibodies by ELISA. Seropositivity was significantly higher in dogs (9.9%; 15/152; 95%CI: 5.1-14.6) than in cats (2.8%; 4/144; 95%CI: 0.1-5.5). Ten of the 18 ELISA-positive animals that could be further analysed by western blot, reacted against HEV-3 and/or HEV-C1 antigens, which suggest circulation of both genotypes in urban cats and dogs in the study area. However, HEV-A, HEV-B and HEV-C RNA were not detected in any of the tested sera. This is the first study to assess HEV circulation in both stray cats and dogs in Europe. Our results provide evidence of HEV exposure in sympatric urban cat and dog populations in southern Spain. Further studies are needed to determine the role of these species in the epidemiology of HEV.

Apicomplexan parasites, such as Toxoplasma gondii, possess unique organelles, cytoskeletal structures, signalling cascades, replicate by internal budding within a specialised compartment and actively invade and exit the host cell, to name a few aspects of the unique biology that characterise this phylum. Due to their huge phylogenetic distance from well established model organisms, such as opisthokonts, comparative genomics has a limited capacity to infer gene functions and conserved proteins can fulfil different roles in apicomplexans. Indeed, approximately 30% of all genes are annotated as hypothetical and many had a crucial role during the asexual life cycle in genome-wide screens. While the current CRISPR/Cas9-based screens allow the identification of fitness conferring genes, only little information about the respective functions can be obtained. To overcome this limitation, and to group genes of interest into functional groups, we established a conditional Cas9-system in T. gondii that allows phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we identified critical genes required for defined steps during the asexual life cycle. The detailed characterisation of two of these candidates revealed them to be critical for host cell egress and invasion and to act at different time points in the disassembly of the intravacuolar F-actin network. While the signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, a novel conoid protein (conoid gliding protein, CGP) acts late during egress and is required for the activation of gliding motility.

Apicomplexan parasites invade host cells in an active process, involving their ability to move by gliding motility and invasion. While the acto-myosin-system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions, such as how the force powering motility is generated. In many eukaryotes a secretory-endocytic cycle leads to recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility and generation of retrograde membrane flow. Here we demonstrate that endocytosis operates during gliding motility inToxoplasma gondiiand appears to be crucial for the establishment of retrograde membrane flow, since inhibition of endocytosis blocks retrograde flow and motility. We identified lysophosphatidic acid (LPA) as a potent stimulator of endocytosis and demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as nanogold particles. Furthermore, we show that surface proteins of the parasite are recycled during this process. Interestingly, the endocytic and secretory pathways of the parasite converge, and endocytosed material is subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings and we propose a novel model that reconciles parasite motility with observations in other eukaryotes: the fountain-flow-model for apicomplexan parasite motility.

SUMMARYThe dissection of apicomplexan biology has been highly influenced by the genetic tools available for manipulation of parasite DNA. Here, we describe different techniques available for the generation of conditional mutants. Comparison of the advantages and disadvantages of the three most commonly used regulation systems: the tetracycline inducible system, the regulation of protein stability and site-specific recombination are discussed. Using some previously described examples we explore some of the pitfalls involved in gene-function analysis using these systems that can lead to wrong or over-interpretation of phenotypes. We will also mention different options to standardize the application of these techniques for the characterization of gene function in high-throughput.

Endogenous transplacental transmission (EnTT) is the major transmission route of Neospora caninum in cattle. Thus, the development of a standardised experimental model of EnTT is needed for more appropriate testing of parasite biology and control strategies. A recent study reported up to 40–50% EnTT rates in chronically infected dams with either high or low-to-moderate virulence isolates, although low fertility rates were observed in dams inoculated with the high virulence isolate. Therefore, low-to-moderate virulence N. caninum isolates (Nc-Spain 3H; G1 and Nc-Spain 8; G2) that previously showed high TT rates versus low mortality and morbidity rates in a congenital mouse model were inoculated into BALB/c dams (first generation). The new approach followed in the present study aimed to start with a high number of congenitally infected mice (second generation), which allowed a more efficient EnTT from congenitally infected dams to their progeny (third generation). Interestingly, a reactivation of infection occurred in several congenitally infected non-pregnant females (second generation) from both infected groups. This fact was evidenced by neosporosis-associated clinical signs after mating accompanied by an increase of specific antibody levels (IgG1, IgG2a and specific antibodies against rNcGRA7) (P < 0.0001; one-way ANOVA). Moreover, a higher number of PCR-positive mice compared to pregnant females were observed (P < 0.05; Fisher’s exact test). These results support the hypothesis that only mice without clinical signs and with a low parasite burden in the brain became pregnant, which may explain the posterior failure in inducing EnTT from the second to the third generation. These findings confirm that this mouse model is not a suitable experimental EnTT model for testing the efficacy of drugs and vaccine candidates against EnTT. The employment of other putative suitable species with a similar placenta structure, such as small ruminants, should be taken into consideration.

SUMMARYThe intraspecific diversity of Neospora caninum is a determinant for in vivo parasite virulence and in vitro parasite behaviour. The relationship between isolate virulence and specific antibody responses against key parasite proteins has not been well characterized. The response kinetics and the differences in specific anti-rNcGRA7, -rNcSAG4, -rNcBSR4 and -rNcSRS9 antibody levels were analysed by recombinant protein-based ELISA in groups of mice inoculated with 10 different N. caninum isolates that differ in their virulence. The majority of the virulence parameters analysed correlated with the specific antibody levels against the 4 recombinant proteins. The antibodies developed against the highly immunogenic protein NcGRA7 were significantly higher in mice inoculated with high virulence isolates than in those inoculated with low-to-moderate virulence isolates in both non-pregnant and pregnant mouse models. Moreover, these levels were correlated with the anti-N. caninum IgG1 and IgG2a responses and the in vitro tachyzoite yield at 56 h. The antibodies directed against the bradyzoite-specific proteins were not detected in a non-pregnant mouse model. However, some seropositive mice were found in groups inoculated with high virulence isolates in a pregnant mouse model. NcGRA7 and NcSAG4 are proteins clearly correlated with virulence, and to a lesser extent NcBSR4 and NcSRS9 proteins. Moreover, antibodies to bradyzoite-specific proteins appear to also be related to virulence in mice. Further analyses should be performed in order to verify the usefulness of these proteins as predictive markers for virulence in an experimental bovine model of neosporosis.

Summary Nucleo‐cytoplasmic RNA export is an essential post‐transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans.

At present, there is no effective treatment or vaccine to prevent vertical transmission or abortion associated with Neospora caninum infection in cattle. Different vaccine formulations have been assayed, and live vaccines have shown the most promising results in terms of protection. Previously, transgenic N. caninum tachyzoites expressing the bradyzoite stage-specific NcSAG4 antigen in a constitutive manner (Nc-1 SAG4(c)) were obtained and showed a reduced persistence of parasite in inoculated mice. Thus, the present study evaluates the Nc-1 SAG4(c)1.1 and Nc-1 SAG4(c)2.1 transgenic strains and the Nc-1 wild-type (WT) strain to determine their protective efficacy against vertical transmission and cerebral neosporosis in mice. Consequently, dams were immunized twice with 5 × 10(5) tachyzoites of each strain and challenged with 2 × 10(6) tachyzoites of a heterologous and virulent isolate at 7-10 days of gestation. The Nc-1 SAG4(c)1.1 strain offered less protection than the other transgenic strain (Nc-1 SAG4(c)2.1) or their ancestor (Nc-1 WT). Indeed, 40%, 7% and 5.6% of the postnatal deaths corresponded to pups from dams vaccinated with Nc-1 SAG4(c)1.1, Nc-1 SAG4(c)2.1 and Nc-1 (WT) strains, respectively. In comparison, the non-immunized challenge group had a 100% mortality rate. In addition, mice were protected against congenital transmission; vertical transmission rates were 45%, 11.1% and 10.8% in the Nc-1 SAG4(c)1.1, Nc-1 SAG4(c)2.1 and Nc-1 WT immunized groups, respectively, vs. 94.9% in the non-vaccinated infected group. However, this protection against the postnatal mortality and the vertical transmission was not associated with a consistent Th1 or Th2-type immune response. Nonetheless, the Nc-1 SAG4(c)2.1 strain appears to be the best candidate for use as a live vaccine, as evidenced by results demonstrating its high levels of protection against vertical transmission and its lower persistence in mice, making this transgenic strain safer than Nc-1 WT.

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

SUMMARYVirulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

In absence of powerful siRNA approaches, the functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple one-step approach that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI in Ku80 knockout and RH T. gondii tachyzoites. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this new approach. This new gene silencing tool kit allows protein tracking and functional studies simultaneously.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4 h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P < 0.0001). A correlation was found between the IRs and TY (ρ > 0.885, P < 0.033), as well as between the TY and both dam morbidity (ρ = 0.8452, P < 0.033) and pup mortality (ρ > 0.8117, P < 0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates. Fil: Dellarupe, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Universidad Complutense de Madrid; España Fil: Regidor Cerrillo, Javier. Universidad Complutense de Madrid; España Fil: Jiménez Ruiz, Elena. Universidad Complutense de Madrid; España Fil: Schares, Gereon. Greifswald-Isle of Riems; Alemania Fil: Unzaga, Juan Manuel. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Venturini, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Ortega Mora, Luis. Universidad Complutense de Madrid; España

We compared the clinical outcome and vertical transmission of six canine Neospora caninum isolates using a pregnant BALB/c model. Four of the isolates were obtained from oocysts of naturally infected dogs (Nc-Ger2, Nc-Ger3, Nc-Ger6 and Nc-6Arg) and two were from diseased dogs with neurological signs (Nc-Bahia and Nc-Liv). The dams were inoculated with 2×106 tachyzoites of each isolate at day 7 of pregnancy. Morbidity, mortality and the antibody responses were evaluated in both the dams and the offspring, as was parasite transmission to the progeny. The mortality rates varied from 100% in Nc-Bahia and Nc-Liv-infected pups to 19% or less for those infected with the isolates from oocysts. The vertical transmission rates varied from 9 to 53% for N. caninum from oocysts, compared with 100% for the Nc-Liv and Nc-Bahia isolates. All dams showed specific IgG responses against tachyzoite and rNc-GRA7 antigens, confirming Neospora infection. The highest IgG levels were detected in mice inoculated with the Nc-Liv and Nc-Bahia isolates. These results demonstrate marked differences in virulence between the N. caninum isolates obtained from oocysts and neurologically affected dogs. This variability could help us to explain the differences in the outcome of the infection in definitive and intermediate hosts. Fil: Dellarupe, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Complutense de Madrid; España. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Regidor Cerrillo, Javier. Universidad Complutense de Madrid; España Fil: Jimenez Ruiz, Elena. Universidad Complutense de Madrid; España Fil: Schares, Gereon. Friedrich Loeffler Institut; Alemania Fil: Unzaga, Juan Manuel. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Venturini, María Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ortega Mora, Luis M.. Universidad Complutense de Madrid; España

SUMMARYEndogenous transplacental transmission (EnTT) of Neospora caninum is the most common route of infection in cattle and occurs as a consequence of a reactivation of N. caninum infection that may lead to abortion or to the birth of congenitally infected calves. The reactivation of N. caninum infection was studied during the gestation of chronically infected dams and, for the first time, in their congenitally infected pups. BALB/c mice were infected with Nc-Spain 7 (Group 1) or Nc-Spain 3H (Group 2), high virulence isolates and low-to-moderate virulence isolates, respectively. The mice were mated after 90 days post-infection, and the morbidity, mortality, vertical transmission and humoral immune responses were recorded for 2 consecutive generations. In the first generation, higher morbidity and mortality rates were observed in G1 before mating than in G2 (P N. caninum infection were induced in chronically infected mice and decreased in subsequent generations regardless of the isolate employed in the inoculations. Thus, further studies are needed to improve this reactivation mouse model.

The protective efficacy of vaccination with Neospora caninum recombinant antigens was evaluated in Balb/c pregnant and non-pregnant mouse models of neosporosis. A major immunodominant dense granule protein (NcGRA7) and three bradyzoite-specific surface antigens (NcSAG4, NcBSR4 and NcSRS9) were expressed in Escherichia coli and encapsulated within poly-e-caprolactone (PCL) nanoparticles for the first time. Good efficiencies of entrapment (greater than 50%) were obtained for all encapsulated proteins. Moreover, antigenicity was unaffected after formulation. Afterwards, separate groups of mice were immunised with the nanoparticles and were then challenged with N. caninum tachyzoites. High IgG1 and IgG2a antibody levels of anti-N. caninum and specific antibodies directed against recombinant proteins were developed by all of the immunised groups. Mice previously inoculated with encapsulated rNcGRA7 produced significant levels of IFN-γ. However, in general, a low production of IFN-γ was detected. This may indicate a failure in the complete liberation of antigens after immunisation or an incorrect balance of the Th1/Th2 response to combat acute neosporosis during pregnancy. In fact, high morbidity and mortality rates were observed in dams. Moreover, vertical transmission was not prevented, and high neonatal mortality rates occurred similarly among the groups. Despite the global absence of efficacy, the study reveals some results of positive efficacy regarding dams and pups' survival and parasite presence for NcSRS9 recombinant protein. Furthermore, vaccination with rNcGRA7 encapsulated alone or combined with rNcSAG4 resulted in a slight decrease of parasite presence in non-pregnant mice. These promising results are further discussed to suggest new approaches that may be more suitable to test vaccine formulations based on bradyzoite stage-specific proteins.

The characteristics of Balantidium from ostriches (Struthio camelus) are similar to those of Balantidium coli; however, the species Balantidium struthionis was proposed on the basis of the host species (ostriches) and the shape of the macronucleus (with a deep depression in one side). In the present work, we have performed morphological and genetic comparisons between isolates of Balantidium from ostriches and B. coli from pigs to determine the specific status of B. struthionis. The morphological characteristics of the trophozoites of Balantidium from ostriches were reviewed in 100 trophozoites from two isolates. The macronucleus' shape of the ostrich Balantidium was highly variable, thus the use of this criterion for diagnostic purposes is not reliable. Besides, very few trophozoites showed a deep depression in their macronucleus and almost all the trophozoites conform to the description of B. coli. The complete sequence of the DNA coding for the 18s-rRNA-ITS1-5.8s-rRNA-ITS2 regions were obtained by PCR from five pig and five ostrich isolates. The sequences corresponding to the 18s and 5.8s-rRNA genes were identical for the ostrich and pig isolates. Two clearly different genotypes were found in the analysis of the ITS1 and ITS2 regions of the pig isolates; the genotype A was identified in all isolates, while the genotype B was found in only two of them. Their sequences show clear differences from that published corresponding to a B. coli gorilla isolate, which we will consider as a different genotype, C. In our opinion, these different B. coli genotypes reflect the genetic variability of this organism, but further studies would be necessary to determine if it could have practical importance. The polymorphism of the ITS regions have been also found in the ostrich isolates. The same genotypes A and B have been identified, although not as mixed infections. The morphological characteristics and the genetic results suggest that the species name B. struthionis is a synonym of B. coli; however, until experimental infections are carried out to determine if the parasite is transmissible between pigs and ostriches, it would be preferable to tentatively designate it as B. coli-like.