Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti.

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Seroprevalence of Toxoplasma gondii in outdoor dogs and cats in Bangkok, Thailand.

The aim of this study was to estimate the seroprevalence and risk factors associated with Toxoplasma gondii exposure in dogs and cats from Bangkok, Thailand. Blood samples from 318 dogs and 321 cats were tested for T. gondii antibodies by modified agglutination test (cut-off 1:25). Additionally, 18 dogs and 20 cats were longitudinally sampled for T. gondii antibodies during the same study period, between June and July 2019. The overall seroprevalence in dogs and cats was 7.9% (25/318; 95% CI 4.9­10.8%) and 18.7% (95% CI 14.4­23.0%), respectively. For dogs, risk factors identified were being a mixed-breed animal and living totally outdoors, while increasing age was shown to be a risk factor for cats. Seroconversion was not detected and titres from positive animals remained constant over longitudinal study. The present study indicates that there is a prominent presence of T. gondii in urban and peri-urban areas of Bangkok, suggesting that outdoor dogs and cats should be considered as a possible risk factor for humans.

Besnoitiosis in donkeys: an emerging parasitic disease of equids in Italy.

Besnoitiosis is an emerging parasitic disease of equids. Italy is one of the few European countries where the circulation of Besnoitia spp. antibodies was demonstrated. In this study, a case of clinical besnoitiosis in two donkeys in northern Italy is reported. The two animals were clinically examined. Serum and blood samples were analyzed for the detection of Besnoitia spp. antibodies and for hematology, biochemistry, and enzyme activity, respectively. ITS-1 PCR and sequencing were carried out on DNA extracted from skin biopsies. Clinical examination revealed numerous scleral pearls in eyes of both animals; alopecia and hyperkeratosis with skin nodules in the region of the neck, hind leg, and on the pinnae were detected. No cysts were evidenced by endoscopy in respiratory and genital tracts. Both animals resulted seropositive to Besnoitia spp. antibodies by Western Blot. Hematology evidenced light anemia, leukocytosis with eosinophilia, and lymphocytosis; biochemistry and enzyme activity revealed hypoalbuminemia with decreased albumin/globulin ratio and elevated alkaline phosphatase values. Parasitic DNA extracted from skin biopsies of both donkeys demonstrated a homology of 100% with Besnoitia spp. This first clinical case of besnoitiosis in two donkeys in Italy both confirms the circulation of Besnoitia spp. in Italian equids and demonstrates that the distribution area of equine besnoitiosis in Europe could be wider than expected. Further studies are needed to infer its relevance, in relation to seroprevalence and clinical disease, and to identify the species of Besnoitia infecting donkeys. Besnoitiosis may be a neglected disease of donkeys in Europe: an early and accurate diagnosis is fundamental to implement adequate control measures to prevent a "silent" spread of Besnoitia spp. infection in equids populations.

Immune response to Neospora caninum live tachyzoites in prepubertal female calves.

The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

Absence of antibodies specific to Besnoitia spp. in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic.

Besnoitia besnoiti and B. caprae, which infect bovids (cattle and antelopes) and goats, respectively, are responsible for besnoitiosis, a chronic and debilitating disease. Bovine besnoitiosis is considered to be a reemerging disease in Central and Western Europe. In addition, infection by Besnoitia spp. has been reported in reindeer from Sweden and Finland. Recently, the parasite was also detected in roe deer and red deer from Spain, where an interconnection between the domestic and sylvatic cycles of B. besnoiti has been presumed. In contrast, caprine besnoitiosis seems to be enzootic to Kenya and Iran. The presence of Besnoitia spp. in small domestic ruminants has never been explored in Europe, and the role that these species might play in the epidemiology of bovine besnoitiosis, as intermediate hosts or reservoirs of B. besnoiti, remains unknown. Herein, the first serosurvey conducted in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic is described. Convenience sampling was conducted of 1943 sheep and 342 goats close to cattle from the Pyrenees and Central Spain that were infected with endemic Besnoitia spp. Serum samples were first analyzed by ELISA and then by confirmatory Western blot. Specific antibodies were not found in any sampled animal. Thus, sheep are unlikely to play a role in the epidemiology of bovine besnoitiosis, at least in the sampled areas. A larger serosurvey is necessary to determine whether goats might be a putative reservoir. To confirm the results of this study, sheep and goats should be further studied in other European countries and regions where their numbers are high and where bovine besnoitiosis is spreading.

Serological dynamics and risk factors of Besnoitia besnoiti infection in breeding bulls from an endemically infected purebred beef herd.

Bovine besnoitiosis has been deemed a re-emerging disease in Western Europe and considered endemic in Spain, Portugal, France and in some areas of Northern Italy. This report refers to an infection outbreak in a purebred beef herd from Northern Italy involving a large number of bulls. In October 2013, 544 animals were serologically tested with an in-house ELISA followed by a confirmatory Western blot to evaluate Besnoitia besnoiti seroprevalence. A year later, 461 animals were then serologically re-tested together with imported animals (n = 268). Overall, 812 animals were involved in the study. Histology and immunohistochemistry were performed on skin biopsies of suspected animals and several tissue samples from a slaughtered bull. In the first sampling, 100 animals were seropositive (18.4%); in the second sampling, prevalence increased up to 36.5%, with incidence calculated at 39.6%. The risk factor analysis revealed that the infection was associated with age (OR = 1.007) and sex, with males presenting a greater risk (OR = 2.006). In fact, prevalence values in bulls increased from 29.6 to 56.7%, with an incidence of infection of 53.3%. Moreover, mating with a seropositive bull enhanced infection risk for a seronegative cow (OR = 1.678). Clinical signs typical of bovine besnoitiosis were found in seven seropositive animals, with confirmation of B. besnoiti through histology and immunohistochemistry. The study outcomes confirm that bovine besnoitiosis is a disease with serious economic impact on beef cattle breeding, particularly on bulls in service. Good management practises such as clinical monitoring and serological testing of imported animals should be implemented to control its occurrence.

Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

Virulence 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.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

Neospora caninum infection as a cause of reproductive failure in a sheep flock.

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.

Toxoplasma gondii and Neospora caninum seroprevalences in domestic South American camelids of the Peruvian Andes.

The objective of this study was to investigate the presence of Toxoplasma gondii- and Neospora caninum-specific antibodies in domestic South American camelids (SAC) (llamas and alpacas) from the Peruvian Andes through a cross-sectional study. A wide panel of serum samples collected from 1,845 llamas and 2,874 alpacas from the two main SAC production areas of Peru was selected. Immunofluorescence antibody technique was employed to detect and titrate specific anti-T. gondii and anti-N. caninum immunoglobulins G in serum samples. The association between T. gondii and N. caninum seroprevalence and the geographical origin (Central and South Peruvian Andes) was evaluated. Anti-T. gondii antibodies were found in 460 (24.9 %) llamas and 706 (24.6 %) alpacas, whereas anti-N. caninum antibodies were detected in 153 (8.3 %) llamas and 425 (14.8 %) alpacas. Toxoplasma gondii infection was strongly associated with the South Peruvian Andes where moderate climate conditions, larger human population, compared to the Central region, and the presence of wildlife definitive hosts could favor horizontal transmission to SAC. In contrast, N. caninum infection was not associated with the geographical region. These results indicate that T. gondii and N. caninum infections are highly and moderately widespread, respectively, in both species of domestic SAC studied in the sampled areas and appropriate control measures should be undertaken to reduce the prevalence of both parasitic infections.

A systematic review and meta-analysis of the validation of serological methods for detecting anti-Toxoplasma gondii antibodies in humans and animals.

Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms "gold standard" and "reference test" and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.

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.

Specific antibody responses against Neospora caninum recombinant rNcGRA7, rNcSAG4, rNcBSR4 and rNcSRS9 proteins are correlated with virulence in mice.

The 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.

Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

Mice congenitally infected with low-to-moderate virulence Neospora caninum isolates exhibited clinical reactivation during the mating period without transmission to the next generation.

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.

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.

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.