Neosporosis in Argentina: Past, present and future perspectives.

Neosporosis, caused by the protozoan Neospora caninum, was first diagnosed in Argentinean cattle in the 90's. With a national bovine stock of approximately 53 million head, the cattle industry is socially and economically relevant. Severe economic losses have been estimated at US$ 33 and 12 million annually in dairy and beef cattle, respectively. Approximately 9% of bovine abortions in the Buenos Aires province are caused by N. caninum. In 2001, the first isolation of N. caninum oocysts from feces of a naturally infected dog was performed in Argentina and named as NC-6 Argentina. Further strains were isolated from cattle (NC-Argentina LP1, NC-Argentina LP2) and axis deer (Axis axis, NC-Axis). Epidemiological studies revealed a high distribution of Neospora-infections not only in dairy but also in beef cattle, with seroprevalence rates of 16.6-88.8% and 0-73%, respectively. Several experimental infection studies in cattle have been carried out, as well as attempts to develop effective vaccines to avoid Neospora-abortions and transmission. However, no vaccine has proven successful for its use in daily practice. Reduction of seroprevalence, vertical transmission and Neospora-related abortions have been achieved in dairy farms by the use of selective breeding strategies and embryo transfer. Neospora-infections have been also detected in goats, sheep, deer, water buffaloes (Bubalus bubalis) and gray foxes (Lycalopex griseus). Moreover, Neospora-related reproductive losses were reported in small ruminants and deer species and could be more frequent than previously thought. Even though diagnostic methods have been improved during the last decades, control of neosporosis is still not optimal. The development of new strategies including new antiprotozoal drugs and vaccines is highly needed. This paper reviews the information from the previous 28 years of research of N. caninum in Argentina, including seroprevalence and epidemiological studies, available diagnostic techniques, experimental reproduction, immunization strategies, isolations and control measures in domestic and non-domestic animals from Argentina.

Frequency of Neospora caninum-specific antibodies in bulk milk from dairy farms from Mar y Sierras Dairy Basin, Argentina.

The aim of this study was to describe the prevalence of Neospora caninum based on the detection of specific antibodies in bulk tank milk (BTM) from dairy cattle farms in the Mar y Sierras Basin by using an enzyme-linked immunosorbent assay (ELISA). A total of 98 BTM samples from 49 dairy farms were collected during autumn and spring of 2019. Additionally, 147 paired individual milk and serum samples were collected from two dairy farms to assess the prevalence within-herd by ELISA and indirect fluorescent antibody test (IFAT, serum samples). Additionally, 12 individual serum samples were also assessed to test the agreement between IFAT and ELISA (total serum samples 159). Noteworthy, 100 and 91.84% of 49 dairy farms were positive in the BTM in autumn and spring, respectively. For the within-herd individual samples, a good agreement between serum and milk results was obtained for ELISA and IFAT (0.86-0.90). This is the first study in Argentina in which milk samples were tested to determine the N. caninum infection status at herd and within-herd levels in dairy farms, providing a base for further research.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Bovine neosporosis in dairy cattle from the southern highlands of Ecuador.

The aim of this study was to describe bovine neosporosis in dairy cattle from the Sierra region, Ecuador. A case-control study was performed on 841 dairy cattle from 5 dairy herds. The overall seroprevalence was 23.4% having significant association between abortion and seropositivity (p < .05). Additionally, 46 fetuses were recovered from a local slaughterhouse to evaluate the frequency of vertical transmission. Seventeen and 3 fetuses were positive by PCR and had compatible histopathological lesions, respectively. N. caninum infection must be considered as a relevant cause of reproductive losses in Ecuador.

Spongy Myelinopathy in Newborn Beef Calves Associated with Consumption of Corn Infected with Stenocarpella maydis.

Stillbirth and perinatal mortality with neurological signs and lesions were diagnosed in two calves following ingestion by their dams of corn infected with Stenocarpella maydis during the third trimester of gestation. Grossly, the brain and spinal cord were unremarkable. Microscopically, diffuse severe status spongiosis of the white matter was detected in the cerebral hemispheres, brainstem, spinal cord and cerebellum. To the best of our knowledge this is the first pathological description of congenital disease in calves associated with the consumption of S. maydis-infected corn; the findings resemble those reported for the naturally occurring and experimentally induced disease in lambs.

Phenotypic characterization of immune cells in fetal tissues of cattle immunized and challenged with Neospora caninum.

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70-90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.

First case report of Toxoplasma gondii-induced abortions and stillbirths in sheep in Argentina.

The aim of this study is to report an episode of reproductive losses due to toxoplasmosis in a sheep flock in Argentina. A total of 15 abortions and 9 stillbirths were recorded in a flock of 190 Texel ewes. The affected ewes were more likely to be seropositive for Toxoplasma gondii (15/24) than ewes that delivered normal lambs (5/34, OR=9.6, 95%CI=2.7-34.0, p=0.0004). A pair of aborted twins was recovered for diagnostic investigation. One of these fetuses and its dam were seropositive for T. gondii. Histological examination of the two fetuses revealed non-suppurative myocarditis and epicarditis, portal hepatitis and multifocal necrotizing encephalitis with protozoal cysts in the brain. T. gondii was detected intralesionally by immunohistochemistry in one fetus and by PCR in both. Further investigations are necessary to evaluate the economic losses due to T. gondii in the Argentinean ovine industry.

Immunization with inactivated antigens of Neospora caninum induces toll-like receptors 3, 7, 8 and 9 in maternal-fetal interface of infected pregnant heifers.

Neospora caninum is an obligate parasite and a major cause of abortion in cattle. Pregnancy failures appear to be associated with weak innate defences on the maternal-fetal interface during infection with N. caninum. Herein, we studied the gene expression of Toll-like receptors (TLRs) in pregnant heifers immunized with different vaccine formulations against N. caninum before mating and then challenged the heifers with live N. caninum on day 70 of gestation. TLR7 and TLR8 expression was upregulated in the placental caruncle of infected-pregnant heifers previously exposed to live N. caninum as immunogen. However, TLR7 and 8 expression in both placenta and caruncle as well as, TLR3 and 9 expression in caruncle were upregulated when heifers were previously immunized with inactivated soluble whole antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. All dams were carrying viable fetuses when they were culled at day 104 of gestation. Upregulation of TLR7 and IFNγ expression was detected in fetal spleen when their mothers where previously vaccinated with soluble antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. These studies demonstrate that soluble or recombinant NcSAG1, NcHSP20 and NcGRA7 antigens induce key TLRs expression at the maternal-fetal interface, probably triggering damaging inflammatory cellular immune responses associated with abortion. Previous infection with N. caninum seems to attenuate the innate immune response at the maternal-fetal interface, which could favour pregnancy maintenance and perpetuation of the disease. This finding represents novel information on how N. caninum vaccination and infection modulate TLRs expression at the placenta and fetal spleen, the possible role in the pregnancy outcomes and transplacental transmission of the protozoa.

Toll-like receptors 3, 7 and 8 are upregulated in the placental caruncle and fetal spleen of Neospora caninum experimentally infected cattle.

Innate immune responses at the maternal-fetal interface are key in the pathogenesis of Neospora caninum, an obligate parasite that causes abortion in cattle. Herein, we determined the gene expression of endosomal Toll-like receptors (TLRs) in the placenta and fetuses from both non-infected pregnant heifers and pregnant heifers intravenously challenged with live tachyzoites of N. caninum on day 70 of gestation. On day 104 of pregnancy, mRNA expression of TLRs 3 and 8, as well as that of TLRs 7 and 9, was high in the spleen of fetuses from N. caninum-infected heifers. Gene expression levels of endosomal TLRs were also detectable in the placenta and the maternal caruncle from infected heifers, being TLRs 3, 7 and 8 particularly upregulated, mostly in the caruncle. Basal TLR levels were higher in fetal spleen than in placental tissues. This study provides novel information on how innate TLR responses are induced at the maternal-fetal interface of cattle in response to intracellular N. caninum.

Characterization of immune cell infiltration in the placentome of water buffaloes (Bubalus bubalis) infected with neospora caninum during pregnancy.

Neospora caninum infection in cattle stimulates host immune responses, which may be responsible for placental damage leading to abortion. Susceptibility of water buffaloes (Bubalus bubalis) to neosporosis is not well understood, although vertical transmission and fetal death have been documented. The aim of this study was to characterize the immune response in the placentome of water buffalo following experimental infection in early gestation with the Nc-1 strain of N. caninum. Placentomes were examined by immunohistochemistry using antibodies specific for T-cell subsets, natural killer cells and CD79(αcy) cells. Placental inflammation was characterized by the infiltration of CD3(+) and CD4(+) T cells and T cells expressing the γδ T-cell receptor. The distribution of these cellular subsets in buffalo placentomes was similar to that previously described in cattle infected with N. caninum in early gestation, but the lesions were milder, which may explain the lower number of abortions observed in this species after infection.