Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

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.

Morphometric study of encephalic lesions in aborted bovine fetuses naturally infected by two subpopulations of Neospora caninum.

Neospora caninum is a major reproductive disease in cattle worldwide. In the Argentinian Humid Pampa, the seroprevalence, incidence of abortions, and economic losses due to neosporosis are considerably higher in dairy than in beef cattle. Despite this, we recently demonstrated that N. caninum subpopulations are indistinctly distributed in both dairy and beef production systems. The association between genotypic characteristics defined by microsatellite analysis and the virulence of the different strains-particularly with regard to the severity and extension of histological lesions-is largely unknown. Herein, we used a morphometric approach to analyze encephalic lesions in 62 bovine fetuses spontaneously infected by N. caninum. Morphometric parameters (average size of focal lesions, number of foci/cm2 and the percentage of the section affected by lesions) were compared according to the N. caninum subpopulations found in our previous microsatellite genotyping analysis, animal biotype (beef versus dairy), and fetal age (second stage of gestation versus third stage). The average size of the lesions differed significantly among fetuses with different gestational ages; however, no significant differences among animal biotypes or genotypic patterns were found. Further research into the genetic, molecular, and husbandry factors that could account for this greater impact in Argentinian dairy herds is needed.

Microsatellite genotyping reveals extensive genetic diversity in bovine Neospora caninum from the humid Pampa region in Argentina.

Neospora caninum is an apicomplexan protozoan and a major cause of abortion in cattle worldwide. In the Argentinian Humid Pampa, bovine neosporosis causes severe economic losses. Despite this, information on the genetic structure of N. caninum in this region is limited. Therefore, this study aimed to genetically characterize N. caninum isolates associated with bovine abortion in the Humid Pampa region. For this purpose, spontaneous bovine fetal tissues submitted for diagnosis to the Veterinary Diagnostic Service at INTA Balcarce during 2008-2019 were assessed by PCR, indirect fluorescent antibody test (IFAT), and histologic analysis. PCR-positive samples were tested by multilocus microsatellite genotyping (MLGs) using 9 microsatellite markers. Thirty-one different genotypes were identified from 32 samples with at least seven markers. Argentinian MLGs were grouped into two clonal clusters when analyzed using eBURST network and principal coordinate analysis. No segregation based on the year of collection, animal biotype, or geographic origin was observed. In addition, the presence of linkage disequilibrium supported the clonal propagation of Argentinian MLGs. One Argentinian subpopulation was associated with isolates from Spain, Uruguay, Brazil, and Mexico, and the other one was linked to isolates from Scotland, Spain, and Germany. These findings reveal the presence of two clonal subpopulations of N. caninum in the Humid Pampa.

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.

A serosurvey of selected cystogenic coccidia in Spanish equids: first detection of anti-Besnoitia spp. specific antibodies in Europe.

BACKGROUND: Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. RESULTS: Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. CONCLUSIONS: This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.

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.

Experimental ruminant models for bovine neosporosis: what is known and what is needed.

At present, bovine neosporosis is an important worldwide concern because of its wide geographic distribution and economic impact. Abortion is the main clinical sign of bovine neosporosis in both dairy and beef cattle. Ruminant challenge models are critical to evaluate potential vaccine candidates to help tackle bovine neosporosis and to study pathogenesis and host responses to infection. Several research groups have developed ruminant models of Neospora caninum infection independently of others, resulting in a high degree of variability due to the use of different species of animals, breeds, strains/isolates of N. caninum, doses, routes and times of inoculation. Standardization is greatly needed to advance research in a more collaborative, timely and efficient manner. In the absence of widely accepted international guidelines, this manuscript serves to summarize and discuss the different models and parameters currently in use. Parameters essential for the development of non-pregnant and pregnant ruminant models are outlined and the main knowledge gaps are identified. This information could act as the basis to develop a consensus for international standard guidelines for ruminant models of neosporosis that would be helpful for researchers in this field worldwide.

Clinical outcome and vertical transmission variability among canine Neospora caninum isolates in a pregnant mouse model of infection.

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.

Control options for Neospora caninum--is there anything new or are we going backwards?

Recent work has highlighted and enumerated the economic annual losses due to Neospora caninum abortions worldwide, which should provide strong motivation for the control of bovine neosporosis. However, with the recent withdrawal from sale of the only commercially available vaccine, control options for N. caninum have become more restricted. While researchers continue to work on developing alternative efficacious vaccines, what are the control options presently available for the cattle industries? At the practical level, recommendations for 'Test-and-cull', or 'not breeding from seropositive dams' stand diametrically opposed to alternative options put forward that suggest a primary producer is better advised to keep those cows in the herd that are already seropositive, i.e., assumed to be chronically infected, and indeed those that have already aborted once. Treatment with a coccidiostat has been recommended as the only economically viable option, yet no such treatment has gained official, regulatory approval. Dogs are central to the life cycle of N. caninum and have repeatedly been associated with infection and abortions in cattle by epidemiological studies. Knowledge and understanding of that pivotal role should be able to be put to use in control programmes. The present review canvasses the relevant literature for evidence for control options for N. caninum (some of them proven, many not) and assesses them in the light of the authors' knowledge and experience with control of N. caninum.

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.

Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice.

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum ß-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 µM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 µM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 µM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 µM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers.

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

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.

Reactivation and Foetal Infection in Pregnant Heifers Infected with Neospora caninum Live Tachyzoites at Prepubertal Age.

Neospora caninum is recognised for causing cattle abortion, provoking severe economic losses in the livestock industry worldwide. The aim of the present study was to evaluate the reactivation and foetal infection in pregnant heifers inoculated with live N. caninum tachyzoites before puberty. A total of 15 30-month-old pregnant heifers were allocated into four groups: animals inoculated with live tachyzoites of NC-Argentina LP1 isolate before puberty and challenged with live tachyzoites of NC-1 strain at 210 days of gestation (DG) (Group A); animals mock inoculated before puberty and challenged with NC-1 strain at 210 DG (Group B), animals inoculated before puberty but not subsequently challenged (Group C); and noninfected and nonchallenged animals (Group D). The results of this study showed that 100% of animals infected before puberty (Groups A and C) suffered reactivation of the infection at the seventh month of gestation. In addition, in three and two calves from Groups A and C, respectively, congenital infection was confirmed. Interestingly, we provide evidence that the use of live N. caninum tachyzoites in young animals as a strategy to induce protection is neither safe nor effective.

Prevalence of Bovine Genital Campylobacteriosis, Associated Risk Factors and Spatial Distribution in Spanish Beef Cattle Based on Veterinary Laboratory Database Records.

Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease that causes early reproductive failure in natural breeding cattle that are managed extensively. The aim of this study was to assess the BGC prevalence in Spain from 2011 to 2019 using data collected cross-sectionally from the diagnostic reports issued by the SALUVET veterinary diagnostic laboratory from a total of 5,182 breeding bulls from 1,950 herds managed under "dehesa" systems (large herds within fenced pastures and all-year breeding season) or mountain systems (smaller herds with seasonal breeding management and grazing in communal mountain pastures). Infection was detected by PCR in 7.7 and 12.2% of the bulls and herds tested, respectively. The "dehesa" herd management system (OR = 2.078, P = < 0.001, 95% CI = 1.55-1.77), bovine trichomonosis status of the herd (OR = 1.606, P = 0.004, 95% CI = 1.15-2.22), and bulls ≥3 years old (OR = 1.392, P = 0.04, 95% CI = 1.01-1.92) were identified as risk factors associated with Campylobacter fetus venerealis infection. We also studied the high-risk areas for circulation of the infection in extensive beef cattle herds in Spain, showing four significant clusters in "dehesa" areas in the south-western provinces of the country and a fifth cluster located in a mountain area in northern Spain. The results obtained in the present study indicate that BGC is endemic and widely distributed in Spanish beef herds. Specifically, "dehesa" herds are at greater risk for introduction of Cfv based on relatively high local prevalence of the infection and the use of specific management practices.

Direct economic losses of Toxoplasma gondii abortion outbreaks in two Spanish sheep flocks.

This study estimates the economic losses due to outbreaks of toxoplasma abortions in a dairy (1928 sheep) and a meat (700 sheep) flock in Spain raised under intensive and semi-extensive management conditions, respectively. In both flocks, sheep were divided into multiple groups to synchronise reproduction. The outbreaks resulted in abortion rates in individual lots of 12.6% (30/239) in the dairy flock and 33.3% (70/210) in the meat flock. Toxoplasma gondii was definitively diagnosed in most submitted cases and the only abortifacient pathogen identified despite extensive investigation. Upon completion of lambing and lactation, veterinarians and farmers completed a questionnaire to gather the data to determine the direct economic impact. The calculated total direct economic losses were €5154.5 (€171.8/abortion) in the dairy flock and €4456 (€63.6/abortion) in the meat flock. Results suggest that flock size, production system, abortion rate and control measures are the key factors influencing economic losses, which vary greatly between individual flocks.