Yanina Paola Hecker

Macrophages (MØs) are pivotal immune cells exhibiting significant plasticity that has been widely studied in human and murine models. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are key regulators of macrophage differentiation from monocytes. In this study, we comprehensively investigated the immunophenotypic, functional, and transcriptomic profiles of ovine MØs differentiated with GM-CSF (GM-oMØs) or M-CSF (M-oMØs) to provide a more nuanced understanding of their activation states. After 7 days, GM-oMØs displayed a smaller, more varied morphology with lower cell yields compared to the larger, uniformly amoeboid M-oMØs. Immunophenotypically, M-oMØs showed significantly higher CD163 expression, consistent with human M-MØs, while CLEC5A was uninformative for differentiation. Transcriptomic analysis, complemented by qPCR and ELISA, revealed clearly distinct profiles, with GM-oMØs exhibiting a pronounced pro-inflammatory phenotype and showing significantly higher expression of 408 genes, mostly associated with interferon and inflammatory response pathways, a feature that aligns with the functional and phenotypic characteristics of human and mouse GM-MØ. Conversely, M-oMØs displayed a regulatory and anti-inflammatory profile, marked by a significantly higher expression of IL-10 and a set of 248 genes involved in cellular homeostasis. Notably, LPS stimulation dramatically shifted the M-oMØ phenotype toward a pro-inflammatory state, unequivocally demonstrating their substantial plasticity, and mirroring human M-CSF-polarized monocytes. Our findings fundamentally challenge the prevailing M1/M2 simplification in ovine macrophage biology and provide a robust foundation for selecting appropriate in vitro macrophage models for future investigations into ovine host defense and disease pathogenesis. This study demonstrated that M-oMØs exhibit greater plasticity, making them more suitable for pathogen-host interaction studies. Unlike GM macrophages, which already have a defined phenotype, M-oMØs more accurately reflect the dynamic immune response induced by a pathogen in the host.

Congenital toxoplasmosis in humans and in some mammalian species, such as small ruminants, is a well-known cause of abortion and foetal 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. The rates of congenital infection and foetal damage in sheep seem to mimic the situation in human toxoplasmosis more closely than those in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg exhibited therapeutic plasma levels for 23 days and no systemic or pregnancy-related toxicity was observed. In sheep experimentally infected at 90 days of pregnancy with a T. gondii oocyst dose which was lethal for all foetuses, 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, higher IFNγ levels 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.

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.

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.

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.

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.