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.

Identification of Sarcocystis spp. in synanthropic (Muridae) and wild (Cricetidae) rodents from Argentina.

The occurrence of Sarcocystis species was investigated in synanthropic (Muridae) and wild (Cricetidae) rodents from Argentina. Nine species were captured (n = 356). Sarcocysts were detected in muscles of 8.7% (31/356) and 3.7% (4/106) of the rodents by histopathology and direct microscopic observation, respectively. PCR-sequencing targeting the 18S rRNA, cox1, and ITS1 regions was performed on samples with positive histopathology. Four different 18S rRNA sequences or sequence groups with high intra-group identities (99.6-100%) were detected in Mus musculus, Oxymycterus rufus, Akodon azarae, and Necromys lasiurus. Eight sequences showed 99.5-99.7% identity with S. dispersa. Thirteen sequences showed low identity (95.3-96.4%) with other Sarcocystis spp. The obtained coxI sequences (n = 9) were almost identical to each other and showed a high similarity with S. strixi (99.2-99.5%) and S. lutrae (99.1%), despite the 18S rRNA sequences from the same samples suggested the occurrence of at least two species. This suggests that coxI may not show high variability in Sarcocystis spp. that use rodents as intermediate hosts. Six ITS1 sequences were obtained, showing high identity but low coverage with several Sarcocystis spp. Multilocus sequence typing and BLAST analysis did not lead to an accurate species identification. Possible reasons are the detection of new species or the limited molecular information available from previously described Sarcocystis spp. Phylogeny suggests that the detected Sarcocystis spp. may use raptor birds or snakes as definitive hosts. This study represents the first molecular identification of Sarcocystis spp. in naturally infected rodents of the Cricetidae and Muridae families in South America.

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.

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.

Spatial-temporal trends and economic losses associated with bovine abortifacients in central Argentina.

The aims of this work are, firstly, to provide the geolocalization of cases of bovine abortion with definitive diagnosis and, secondly, to estimate the economic losses due to the most frequent abortifacients diagnosed agents in cattle in Buenos Aires province, Argentina. The total beef and dairy cattle population at risk of abortion is 8,358,186 and 538,076, respectively. In beef cattle, the overall risk of abortion was estimated at 4.5% for all pregnancies, where 27.9% are due to Campylobacter fetus, Neospora caninum, Leptospira spp., Brucella abortus, and bovine viral diarrhea virus with economic losses of US$ 440 per abortion, being the annual loss to the beef industry of US$ 50,144,101. In dairy cattle, there was an 8.0% risk of suffering abortion, 26.1% produced by the same abortigenic agents. The economic losses were estimated at US$ 1,415 per abortion, which equals a total loss of US$ 17,298,498 for the dairy industry in the region. The results of this study show that infectious causes are highly prevalent in Buenos Aires province, and they caused severe economic impacts in the dairy and beef industries. Furthermore, changes in temporal trends of infectious abortion occurrence were detected, probably related to the inclusion of molecular diagnostic techniques with more sensitivity or different epidemiological or husbandry conditions in the region analyzed.

Parasitemia and Associated Immune Response in Pregnant and Non-Pregnant Beef Cows Naturally Infected With Neospora caninum.

The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (-Preg, n = 4), and seronegative non-pregnant cows (-Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg (p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio (p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting (p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg (p <0.05). Avidity correlated with total IgG and IgG2 (p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle.

Epidemic abortions due to Neospora caninum infection in farmed red deer (Cervus elaphus).

This study describes for the first time an abortion outbreak caused by Neospora caninum in farmed red deer. During a 5-year period, farmed hinds, naturally mated, were regularly ultrasound monitored to detect reproductive losses over their gestation. During the 4 years previous to the outbreak, abortion rates ranged from 4.7 to 8.6% (average 6.5%), and serology for indirect diagnosis of neosporosis and toxoplasmosis was performed. At the fifth year, the abortion rate increased to 25.3%. During this outbreak, three aborted foetuses and their placentas were recovered and submitted to laboratory for etiological diagnosis. Blood samples were collected from the 81 hinds at the end of the gestational period and the seropositivity rate for N. caninum, Toxoplasma gondii, Brucella abortus, bovine viral diarrhoea virus and bovine alphaherpesvirus type 1 was 66.7%, 67.9%, 0.0%, 8.6% and 0.0%, respectively. Neospora caninum-seropositive hinds (OR = 5.7, P = 0.0271) and hinds with high antibody titres to N. caninum (OR = 7.4, P = 0.0130) were more likely to abort than seronegative hinds. In addition, N. caninum seropositivity rate in the aborted hinds was higher (OR = 5.4, P = 0.033) than the non-aborted hinds. No association was found between T. gondii nor BVDV-seropositivity and abortions. Typical protozoal histopathologic findings (necrotizing non suppurative encephalitis, meningitis, myocarditis, hepatitis, among others) were observed in all foetuses. Neospora caninum was immunolabelled by immunohistochemistry in several tissues from two foetuses, and infection was also confirmed in the three foetuses by serology and/or DNA detection. No other abortifacient agent was detected in the foetuses. Their dams showed high N. caninum antibody titres (≥ 6400). Serologic evidence and epidemiological data recorded suggested a point-source of N. caninum infection before the occurrence of the outbreak, probably related with contaminated feedstuff with oocysts. Moreover, the intensive production system with a high stocking rate could be also considered a factor which might have increased the risk of horizontal N. caninum infection in this herd.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

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.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Neospora caninum and Toxoplasma gondii as causes of reproductive losses in commercial sheep flocks from Argentina.

The aim of this study is to provide preliminary data about the occurrence of Neospora caninum- and Toxoplasma gondii-related abortions and perinatal deaths in sheep from Argentina. Thirty ovine aborted foetuses and 33 perinatal deaths were submitted to the Veterinary Diagnostic Service at INTA EEA Balcarce (Argentina) during 2017-2019. A complete necropsy was performed on all specimens submitted, and foetal and placental tissues were examined. Foetal cavity fluids were collected for assessment of antibodies to N. caninum and T. gondii by indirect fluorescent antibody technique (IFAT). Placental and foetal tissue samples were collected for DNA extraction and histopathological analysis. The differential diagnosis with other causes of abortion was carried out. Of the sampled specimens, 20.63% (13/63) displayed evidence for N. caninum infection by IFAT and PCR, and in 61.5% (8/13) of the positive specimens the parasite was confirmed as the cause of abortion/perinatal death based on the presence of compatible histological lesions and/or positive immunohistochemistry test, positive PCR and/or positive IFAT, and no other infectious agents diagnosed. In contrast, T. gondii infection was confirmed in 9.52% (6/63) of the analysed specimens, but only in 2 lambs T. gondii was determined as the death cause. Neospora caninum and T. gondii co-infections were confirmed in 4 analysed specimens (2 aborted foetuses and 2 perinatal deaths). These results demonstrated that N. caninum is efficiently transmitted and a frequent cause of ovine reproductive failure in the commercial analysed flocks compared with T. gondii. Despite T. gondii congenital infection was detected in some specimens (6/63), it was confirmed as the cause of death in only two of them. Thus, and considering the limited availability of confirmed samples, we could not determine whether toxoplasmosis is a major problem in Argentinian sheep flocks or not. More studies on a greater number of specimens from different ovine production systems under different management conditions are necessary to assess the real impact of neosporosis and toxoplasmosis in Argentina.

MANα1-2MAN decorated liposomes enhance the immunogenicity induced by a DNA vaccine against BoHV-1.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, 'naked' DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis.

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.

Ovine Abortion by Neospora caninum: First Case Reported in Argentina.

PURPOSE: The aim of this study was to describe for the first time a natural case of ovine abortion associated with Neospora caninum in a flock with reproductive losses in Argentina. MATERIALS AND METHODS: The analyzed flock consisted of 256 Texel sheep, of which 134 had been mated. A single blood sample was obtained by jugular vein puncture from 220 ewes (116 adult ewes, 104 yearling ewes) and 93 lambs. Serum samples and fetal fluid were tested using the indirect fluorescence antibody test to detect antibodies against N. caninum and T. gondii. Fetal and placental tissues from aborted fetus were examined by standard gross pathology procedures and were tested using histopathology and immunohistochemistry. Moreover, DNA from fetal and placental tissues was isolated and a PCR assay to detect N. caninum, T. gondii and Chlamydia spp. was used. RESULTS AND DISCUSSION: The pregnancy rate was 89% (119/134), the abortion rate was 8.4% (10/119) whereas the perinatal mortality rate was 15% (16/109). Out of 116 adult ewes sampled, 34.48% presented anti-N. caninum antibodies. Ten ewes had aborted, and one fetus was directly submitted to the diagnostic laboratory for further processing. Antibodies against N. caninum were detected in fetal fluid and in the aborted dam. Histopathological analysis in fetal tissues showed multifocal lymphohistiocytic glossitis, diffuse mild lymphohistiocytic endocarditis, pericarditis and focally extensive myocarditis. Severe multifocal necrotizing placentitis and diffuse mild lymphohistiocytic placentitis with the presence of lymphohistiocytic vasculitis were observed in placenta. N. caninum was immunolabeled in the placenta and fetal tongue. In addition, N. caninum DNA was detected in placenta, central neural system, lung and heart of the aborted fetus. There was no evidence of other infectious abortifacients in the aborted fetus. CONCLUSION: The present study described for the first time an ovine abortion caused by N. caninum in Argentina. Further investigations at a larger scale are required to establish the role of N. caninum as an important cause of reproductive losses in sheep flocks from the region.

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.

Delayed type hypersensitivity induced by intradermal inoculation of a Neospora caninum tachyzoite antigen in previously exposed cattle.

The aim of this study was to evaluate delayed type hypersensitivity (DTH) induced by the intradermal inoculation of a Neospora caninum tachyzoite soluble lysate in cattle previously exposed with the protozoa. Four experimental groups were selected according to the prior exposure to N. caninum antigen. All cows were intradermally injected with a N. caninum tachyzoite soluble lysate and skinfold thickness growth at the inoculation sites was measured at 0, 24, 48, 72 and 96 h post inoculation (hpi). Additionally, specific antibodies and IFN-γ production were assessed. Cows experimentally infected with live N. caninum tachyzoites and cows naturally exposed to N. caninum developed skin reactions compatible with DTH between 24 and 96 hpi (p < 0.05). Moreover, cows inoculated with an experimental N. caninum vaccine and cows without evidence of exposure to N. caninum did not show a significant increase in skin thickness (p > 0.05). Furthermore, serological status of the animals was not modified due to the intradermal inoculation. The highest IFN-γ production was observed at 15 days after intradermal inoculation (p < 0.05). Therefore, these results suggest that cattle previously exposed to N. caninum develop a reaction compatible with DTH which could be useful as in vivo cell mediated immunity parameter for assessed bovine neosporosis.

Controlling Endemic Neospora caninum-Related Abortions in a Dairy Herd From Argentina.

After diagnosis of endemic abortions due to neosporosis in a commercial dairy farm, routes of Neospora caninum-transmission were evaluated in order to choose the best strategy for reducing its seroprevalence and related abortions. Fifty two dam-calf pairs were bled at parturition. Additionally, 22 female calves were also sampled at regular 3 month intervals until 18-22 months. N. caninum specific antibodies were assayed by IFAT. Serum samples were tested at a dilution 1:25 for calves before colostrum intake and heifers before mating and 1:100 for multiparous cows. Only serum samples from IFAT seropositive cattle involved in the evaluation of the routes of transmission were assessed by a commercial IgG avidity ELISA. Seropositive cows or heifers were artificially inseminated with semen from Hereford bulls. The progenies from these female animals were sent to a feed lot to produce meat. Different generalized linear models (GLM) were used to study the relationship between abortion, age category, and serostatus. Seropositive heifers were more likely to have a record of abortion (OR 2.7; 95% CI 1.6-4.7). Vertical transmission frequency was 55.5% (5 seropositive calves/9 seropositive cows). Horizontal transmission was 22.7% (5 female calves seroconverted at least one time/22 females calves sampled during 24 months) and these 5 female calves had low avidity. In heifers, both seroprevalence and abortion rates decreased from 22.1 and 8.4% of 475 in 2009 to 6.1 and 4.3% of 578 in 2015, respectively (p < 0.01). Over 5 years, N. caninum-seroprevalence and the related abortions in heifers decreased after the control strategy was assessed.

Evaluation of frequency of antibodies against Toxoplasma gondii,Neospora caninum and Sarcocystis spp. and transmission routes in sheep from Humid Pampa, Argentina.

The aim of this study was to describe the frequency of ovine specific antibodies to Toxoplasma gondii, Neospora caninum and Sarcocystis spp. and to estimate different transmission routes of these infections. One hundred and thirty Texel sheep and their 117 Texel lambs were included in the study. Serum samples were tested for antibodies to T. gondii, N. caninum and Sarcocystis spp. using IFAT. Toxoplasma gondii seroprevalence was 10.00% in sheep (IC95%: 4.80-15.20%), being higher in adult sheep (≥12 year) than in younger sheep (OR 1.30; 95% CI, 1.10-1.50). N. caninum and Sarcocystis spp. seroprevalences were 1.54% (IC95%: 0.00-5.70) and 72.09% (IC95%: 67.70-82.70), respectively, with no association between age and seropositivity in sheep (P>0.05). T. gondii seroprevalence in lambs was 4.27% (IC95%: 0.61-7.94). No association between T. gondii serological status in sheep and their lambs was detected (P = 0.07). Two T. gondii and Sarcocystis spp. seropositive lambs were euthanized and T. gondii and Sarcocystis spp. DNA was detected by PCR in their tissues. In conclusion, the increase of T. gondii seropositivity in relationship with sheep age and the lack of association between sheep-lamb serological status, suggest that horizontal infection is the main transmission route in this flock as reported before. Due to the low number of N. caninum-seropositive ewes no assumptions can be done about the impact of this parasite in this flock. According with previous reports, the main transmission route for Sarcocystis spp. in this species in the present study was horizontal.

Pampas fox (Lycalopex gymnocercus) new intermediate host of Sarcocystis svanai (Apicomplexa: Sarcocystidae).

Several Sarcocystis spp. have carnivores as definitive host and sarcocysts are common in muscles of herbivores (intermediate host). However, sarcocysts have been found in muscles of wild and domestic carnivores suggesting they are intermediate host for some Sarcocystis spp. Here, we report mature sarcocysts in the muscles of Pampas fox (Lycalopex gymnocercus). A total of 36 free-living foxes were analyzed. Different skeletal muscles were assessed by microscopic and molecular methods. Cysts and/or DNA of Sarcocystis sp. were detected in 61.1% (22/36) foxes. Histopathology revealed the presence of sarcocysts in 52.8% (19/36) foxes. The tongue and masseter were the muscles more frequently infected. Of all the samples processed by homogenization of pooled muscles of each animal, 45.4% (10/22) evidenced muscle cysts and 68.2% (15/22) resulted positives by PCR. Individual cysts obtained from the ten positive samples in direct microscopic examination were all positive by PCR. Five amplicons from individual cysts from different samples were selected for sequencing together with four PCR products obtained from the pooled muscles. All nine sequences shared a high identity among them (99.8-100%) and showed the highest identity by BLAST (99%) with a S. svanai sequence (KM362428) from a North American dog. By transmission electron microscopy, the sarcocyst wall was thin (<1µm), had minute undulations, with tiny evaginations and without evident villar protrusions. The cyst wall type is referred as "type 1". Sarcocystis svanai infects L. gymnocercus with a high prevalence and the presence of mature sarcocysts suggests the role of the Pampas fox as natural intermediate host. The definitive host of S. svanai remains unknown.