A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

Transcriptomics of Besnoitia besnoiti-Infected Fibroblasts Reveals Hallmarks of Early Fibrosis and Cancer Progression.

Endothelial injury, inflammatory infiltrate and fibrosis are the predominant lesions in the testis of bulls with besnoitiosis that may result in sterility. Moreover, fibroblasts, which are key players in fibrosis, are parasite target cells in a Besnoitia besnoiti chronic infection. This study aimed to decipher the molecular basis that underlies a drift toward fibrosis during the disease progression. Transcriptomic analysis was developed at two times post-infection (p.i.), representative of invasion (12 h p.i.) and intracellular proliferation (32 h p.i.), in primary bovine aorta fibroblasts infected with B. besnoiti tachyzoites. Once the enriched host pathways were identified, we studied the expression of selected differentially expressed genes (DEGs) in the scrotal skin of sterile infected bulls. Functional enrichment analyses of DEGs revealed shared hallmarks of cancer and early fibrosis. Biomarkers of inflammation, angiogenesis, cancer, and MAPK signaling stood out at 12 h p.i. At 32 h p.i., again MAPK and cancer pathways were enriched together with the PI3K-AKT pathway related to cell proliferation. Some DEGs were also regulated in the skin samples of naturally infected bulls (PLAUR, TGFß1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.

An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.

A questionnaire-based survey in Spain provides relevant information to improve the control of ovine coccidiosis.

Ovine coccidiosis is a widespread intestinal parasitic disease caused by Eimeria spp. Lambs are infected by the ingestion of sporulated oocysts, experiencing diarrhea and low growth rates. Control should be based on measures to reduce infection pressure and stress on the animals as well as on appropriate diagnosis and strategic treatment. To obtain information on how control measures are implemented in the ovine sector in Spain, a questionnaire-based survey was completed in 2022 by 154 veterinarians and 173 farmers working in this sector. Coccidiosis was highlighted as a relevant disease by 34% of the respondents. The period of greatest risk seemed to differ between production systems, being mainly early after weaning (7-15 days after weaning) in meat flocks and feedlots and later (1-2 months after weaning) in dairy flocks. The absence of cleaning and disinfection measures was identified as a risk factor by 51% of the veterinarians, with 22% mentioning overcrowding of animals and 22% indicating that coccidiosis has more incidence in flocks with large number of animals. The use of laboratory diagnosis methods (fecal oocyst count) was unusual in 70 and 84% of the veterinarians and farmers, respectively. Regarding control, dairy flocks usually housed a larger number of animals under intensive conditions, and they implemented more frequently control measures for coccidiosis than meat flocks. Anticoccidial drugs were used in 79% of the flocks, and in 74-82% of them, they were applied based on clinical criteria. Comparing protocols for anticoccidial treatment among different production systems, in meat flocks, anticoccidial drugs were applied more frequently when clinical signs were observed, and coccidiostats were used for less than 28 days compared to dairy flocks. These results highlight the need for improvement in the use of anticoccidial treatments adjusted to the new regulatory framework in the EU, which in turn will rationalize the use of antimicrobial compounds and may help to mitigate the impact of coccidiosis in flocks.

Anti-Toxoplasma gondii Antibodies in European Residents: A Systematic Review and Meta-Analysis of Studies Published between 2000 and 2020.

Toxoplasmosis has a major impact on animal and public health. Information regarding the seroprevalence of human Toxoplasma gondii infections from a European perspective has not yet been compiled to date. Thus, the present review summarized available resident data from the period 2000-2020. The overall seroprevalence of anti-T. gondii IgG was 32.1%, with great variability between countries (n = 30). The subgroup analysis identified different pooled prevalence data depending on the geographic area (p < 0.0001), target population (p = 0.0147), and serological diagnosis assays used (p = 0.0059). A high heterogeneity (I2 = 100%, p < 0.001; Q = 3.5e+05, d.f. = 135, p < 0.001) and degree of publication bias (Egger's test = 6.14, p < 0.001) were observed among the 134 studies considered. The occurrence of anti-T. gondii IgM, which was reported in 64.7% of studies, reached a pooled seroprevalence of 0.6%. In addition, among the eight main risk factors identified, "contact with soil", "consumption of undercooked beef", and "intake of unwashed vegetables" were the most significantly associated with infections. The fact that one-third of the European population has been exposed to T. gondii justifies extra efforts to harmonize surveillance systems and develop additional risk-factor analyses based on detailed source attribution assessment.

Single and combination treatment of Toxoplasma gondii infections with a bumped kinase inhibitor and artemisone in vitro and with artemiside in experimentally infected mice.

In previous studies, the artemisinin derivatives artemisone, its pro-drug artemiside and the bumped-kinase inhibitor BKI-1748 were effective against T. gondii via different modes of action. This suggests that they may act synergistically resulting in improved efficacies in vitro and in vivo. To test this hypothesis, the compounds were applied alone and in combination to T. gondii infected human fibroblast host cells in order to determine their inhibition constants and effects on cellular ultrastructure. In addition, the efficacy of either single- or combined treatments were assessed in an acute TgShSp1-oocyst infection model based on CD1 outbred mice. Whereas the IC50 of the compounds in combination (42 nM) was close to the IC50 of BKI-1748 alone (46 nM) and half of the IC50 of artemisone alone (92 nM), the IC90 of the combination was half of the values found with the single compounds (138 nM vs. ca. 270 nM). Another indication for synergistic effects in vitro were distinct alterations of the cellular ultrastructure of tachyzoites observed in combination, but not with the single compounds. These promising results could not be reproduced in vivo. There was no decrease in number of T. gondii positive brains by either treatment. However, the levels of infection in these brains, i. e. the number of tachyzoites, was significantly decreased upon BKI-1748 treatment alone, and the combination with artemiside did not produce any further decrease. The treatment with artemiside alone had no significant effects. A vertical transmission model could not be established since artemiside strongly interfered with pregnancy and caused abortion. These results show that is difficult to extrapolate from promising in vitro results to the situation in vivo.

Bovine infectious abortion: a systematic review and meta-analysis.

The aim of the present systematic review and meta-analysis was to identify the main infectious agents related to bovine abortion worldwide in the period between 2000 and 2022. First, we investigated the global prevalence of infectious agents related to bovine abortion. For this analysis, only 27 articles detected of a wide panel of agents were included. The random effects model revealed that the estimated prevalence of the abortifacient agents in bovine abortion was 45.7%. The heterogeneity among studies was high, but Egger's test showed that there was no publication bias, even though the total number of samples analyzed in these articles was variable. There was no significant effect of the year of the study publication on the estimated prevalence, although an increasing trend was observed over time, possibly due to the implementation of new diagnostic techniques. Then, we analyzed the prevalence of the main transmissible agents in bovine abortion. For this analysis, 76 studies that analyzed 19,070 cases were included. Some infectious agent was detected in 7,319 specimens, and a final diagnosis was reached in 3,977 of these, when both the infectious agent and compatible histopathological changes were detected. We found that Neospora caninum was the most detected agent (22.2%), followed by opportunistic bacteria (21.4%), Chlamydiaceae family (10.9%) and Coxiella burnetii (9.5%). Regarding viral agents, bovine herpes virus type 1 and bovine viral diarrhea displayed similar prevalence rates (approximately 5%). After considering the description of specific histopathological changes, our analyzes showed that N. caninum was a confirmed cause of abortion in 16.7% of the analyzed cases, followed by opportunistic bacteria (12.6%) and Chlamydia spp. (6.8%); however, C. burnetii was only confirmed as a cause of abortion in 1.1% of the cases. For all agents, the heterogeneity among studies was high, and the subgroup analyzes discarded the diagnostic method as the cause of such heterogeneity. This study provides knowledge about the global prevalence of the different infectious agents related to bovine abortion, the most coming of which is N. caninum. In addition, this review reveals the existing deficiencies in the diagnosis of bovine abortion that must be addressed in the future.

A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance.

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.

Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens.

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Optimization of the most widely used serological tests for a harmonized diagnosis of Toxoplasma gondii infection in domestic pigs.

The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63-0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30-0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Fatal toxoplasmosis in a captive squirrel monkey (Saimiri boliviensis) in Portugal.

New World monkeys are especially vulnerable to develop severe clinical manifestations and succumb to acute toxoplasmosis. This study aimed to describe the histopathological findings and genotypic characterization of the Toxoplasma gondii strain involved in a lethal case occurring in a zoo-housed black-capped squirrel monkey (Saimiri boliviensis) in Portugal. Cyst-like structures suggestive of Sarcocystidae parasites and acute injuries in liver and brain were observed by light microscopy examination. By immunohistochemistry, calprotectin, T. gondii antigen and Iba1 antigen had a positive signaling in lung, liver and brain tissues. Toxoplasma gondii B1, ITS1 and 529 repetitive element fragments amplifications together with the genotyping of 13 microsatellite markers confirmed a systemic T. gondii infection linked to a non-clonal type II strain. This description is consistent to the majority T. gondii strains circulating in Europe.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages.

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.

Short-term culture adaptation of Toxoplasma gondii archetypal II and III field isolates affects cystogenic capabilities and modifies virulence in mice.

Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (TgShSp1 (Genotype ToxoDB#3), TgShSp2 (#1), TgShSp3 (#3) and TgShSp16 (#3)) and type III (#2) isolates (TgShSp24 and TgPigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p10) to 50 (p50), and isolate virulence at p10 versus p50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25-30 passages. The TgShSp1, TgShSp16 and TgShSp24 isolates failed to generate spontaneously formed mature cysts at p50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p50 with events of exacerbation, increasing cumulative morbidity for TgShSp2 and TgShSp3 isolates and mortality for TgShSp24 and TgPigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for TgShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the TgShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence.

In Vitro versus in Mice: Efficacy and Safety of Decoquinate and Quinoline-O-Carbamate Derivatives against Experimental Infection with Neospora caninum Tachyzoites.

The effects of decoquinate (DCQ) and three O-quinoline-carbamate-derivatives were investigated using human foreskin fibroblasts (HFF) infected with Neospora caninum tachyzoites. These compounds exhibited half-maximal proliferation inhibition (IC50s) from 1.7 (RMB060) to 60 nM (RMB055). Conversely, when applied at 5 (DCQ, RMB054) or 10µM (RMB055, RMB060), HFF viability was not affected. Treatments of infected cell cultures at 0.5µM altered the ultrastructure of the parasite mitochondrion and cytoplasm within 24 h, most pronounced for RMB060, and DCQ, RMB054 and RMB060 did not impair the viability of splenocytes from naïve mice. Long-term treatments of N. caninum-infected HFF monolayers with 0.5µM of each compound showed that only exposure to RMB060 over a period of six consecutive days had a parasiticidal effect, while the other compounds were not able to kill all tachyzoites in vitro. Thus, DCQ and RMB060 were comparatively assessed in the pregnant neosporosis mouse model. The oral application of these compounds suspended in corn oil at 10 mg/kg/day for 5 d resulted in a decreased fertility rate and litter size in the DCQ group, whereas reproductive parameters were not altered by RMB060 treatment. However, both compounds failed to protect mice from cerebral infection and did not prevent vertical transmission/pup mortality. Thus, despite the promising in vitro efficacy and safety characteristics of DCQ and DCQ-derivatives, proof of concept for activity against neosporosis could not be demonstrated in the murine model.

Experimental infection of sheep at mid-pregnancy with archetypal type II and type III Toxoplasma gondii isolates exhibited different phenotypic traits.

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii-DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted.

A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs.

Introduction: Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available. Methods: Herein, the first comparative study of six "in house" and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT-qPCR results) as a reference. Four "in house" tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera). Results and discussion: ELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.