RESUMEN
Protozoan parasite Neospora caninum causes abortion in infected cattle while others remain asymptomatic. Host immunity plays a critical role in the outcome of bovine neosporosis. Despite extensive research, there is a critical gap in therapeutic and preventive measures, and no effective vaccines are available. Both beef and dairy cattle can suffer from N. caninum-induced abortions, but cumulative evidence suggests a breed susceptibility being higher in dairy compared with beef breeds. It has been established that the response to N. caninum infection primarily involves a cell-mediated immune response (CMIR) regulated by T-helper type 1 (Th1) cells and specific cytokines. The delayed-type hypersensitivity (DTH) skin test has been used to measure the ability of livestock to generate CMIR, in the context of breeding for disease resistance and as a method for diagnosis of several diseases. In this study, we evaluated the immune response triggered by an N. caninum-induced DTH skin test between Holstein - a dairy breed intensively selected- and Argentinean Creole heifers - a beef breed with minimal genetic selection- to assess differences in CMIR following experimental N. caninum infection. The immune response, measured through skinfold thickness and histological and immune molecular analysis, revealed variations between the breeds. Our study found an increased CMIR in Argentinean Creole heifers compared to Holstein heifers. Differential gene expression of key cytokines was observed at the DTH skin test site. Argentinean Creole heifers exhibited elevated IFN-γ, IL-12, IL-10, and IL-4, while Holstein heifers only showed higher expression of IL-17. This finding could underscore genetic diversity in response to neosporosis, which could be used in breeding cattle strategies for disease resistance in cattle populations.
Asunto(s)
Enfermedades de los Bovinos , Coccidiosis , Inmunidad Celular , Neospora , Animales , Bovinos , Neospora/inmunología , Coccidiosis/veterinaria , Coccidiosis/inmunología , Coccidiosis/parasitología , Femenino , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/parasitología , Enfermedades de los Bovinos/genética , Citocinas/genética , Citocinas/inmunología , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/veterinariaRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos , Coccidiosis , Ciervos , Enfermedades de los Perros , Enfermedades de las Cabras , Neospora , Enfermedades de las Ovejas , Embarazo , Femenino , Animales , Perros , Bovinos , Ovinos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/prevención & control , Coccidiosis/epidemiología , Coccidiosis/veterinaria , Estudios Seroepidemiológicos , Argentina/epidemiología , Anticuerpos Antiprotozoarios , Cabras , Zorros , Búfalos , Enfermedades de los Perros/epidemiologíaRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos , Neospora , Animales , Argentina/epidemiología , Bovinos , Enfermedades de los Bovinos/diagnóstico , Granjas , LecheRESUMEN
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.
Asunto(s)
Bovinos/inmunología , Calostro/inmunología , Regulación del Desarrollo de la Expresión Génica/inmunología , Inmunidad Humoral , Inmunidad Innata , Neospora/inmunología , Receptores Toll-Like/metabolismo , Animales , Anticuerpos Antiprotozoarios/inmunología , Bovinos/embriología , Bovinos/metabolismo , Bovinos/parasitología , Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/parasitología , Femenino , Inmunoglobulina G/inmunología , Interleucina-10/genética , Interleucina-10/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Embarazo , Proteínas/genética , Proteínas/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptores Toll-Like/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Cordón Umbilical/metabolismoRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos , Coccidiosis , Hipersensibilidad Tardía/parasitología , Pruebas Cutáneas/veterinaria , Animales , Anticuerpos Antiprotozoarios/inmunología , Argentina , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/parasitología , Coccidiosis/diagnóstico , Coccidiosis/veterinaria , Femenino , Hipersensibilidad Tardía/inmunología , Neospora/inmunologíaRESUMEN
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.
Asunto(s)
Aborto Veterinario/epidemiología , Enfermedades de los Bovinos/epidemiología , Coccidiosis/veterinaria , Neospora/aislamiento & purificación , Aborto Veterinario/parasitología , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Coccidiosis/epidemiología , Coccidiosis/parasitología , Industria Lechera , Ecuador/epidemiología , Femenino , Prevalencia , Estudios SeroepidemiológicosRESUMEN
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 (nâ¯=â¯4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (nâ¯=â¯4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (nâ¯=â¯4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (nâ¯=â¯4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (nâ¯=â¯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.
Asunto(s)
Coccidiosis/inmunología , Feto/inmunología , Inmunización/veterinaria , Neospora/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Encéfalo/citología , Encéfalo/inmunología , Bovinos , Enfermedades de los Bovinos/inmunología , Femenino , Feto/citología , Inmunohistoquímica , Pulmón/citología , Pulmón/inmunología , EmbarazoRESUMEN
The aim of this study was to describe the lectin-binding pattern in the placentas of cows infected experimentally with Neospora caninum. Four cows were inoculated intravenously with 1 × 108 tachyzoites of the NC-1 strain of N. caninum at 150 ± 7 days of pregnancy. Two control cows were administered a placebo. An indirect fluorescence antibody test (IFAT) was performed on serum samples obtained before and after the inoculation. The cows were killed at 30 and 37 days post inoculation. Samples of placenta were taken for histopathology and lectin histochemistry. Fetal tissues and fluids were collected for histopathology and IFAT, respectively. All infected cows had high antibody titres. All fetuses had characteristic histopathological lesions, including non-suppurative meningoencephalitis, myocarditis, hepatitis and myositis, suggesting N. caninum infection. Only two infected fetuses developed specific antibodies. Mild non-suppurative inflammatory infiltrates were recorded in the placentae. Differences in the lectin-binding pattern were observed between infected animals and controls in the glycocalyx (CON-A and WGA) and apical cytoplasm (RCA-I and CON-A) of the trophoblastic cells; giant trophoblastic cells (CON-A and DBA); glycocalyx (PNA, WGA) and apical cytoplasm (CON-A, WGA, PNA, DBA and RCA-I) of endometrial cells; trophoblast of the interplacentomal region (WGA); endothelium (CON-A, SBA, RCA-1 and WGA); and finally, mesenchyme (CON-A, RCA-1, SBA, PNA and DBA). These findings indicate that there is a distinctive pattern of lectin binding in the placenta of cattle infected with N. caninum. The direct effect of the presence of the protozoa as well as the altered expression of cytokines could explain these changes in the maternofetal interface.
Asunto(s)
Enfermedades de los Bovinos/metabolismo , Coccidiosis/veterinaria , Lectinas/análisis , Placenta/metabolismo , Animales , Bovinos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Neospora , Placenta/microbiología , EmbarazoRESUMEN
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.
Asunto(s)
Feto Abortado/parasitología , Enfermedades de las Ovejas/diagnóstico , Mortinato/veterinaria , Toxoplasmosis Animal/diagnóstico , Aborto Veterinario/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Argentina , Estudios de Casos y Controles , ADN Protozoario/análisis , Encefalitis/parasitología , Femenino , Feto/parasitología , Hepatitis/parasitología , Miocarditis/parasitología , Reacción en Cadena de la Polimerasa , Embarazo , Complicaciones Parasitarias del Embarazo/veterinaria , Ovinos , Enfermedades de las Ovejas/parasitología , Toxoplasma/aislamiento & purificaciónRESUMEN
The aim of this study was to evaluate the frequencies of Neospora caninum horizontal and vertical transmissions in beef cow-calf operations under three different extensive management systems: group A: 0.75 head per hectare pasturing on natural grass; group B: 1.1 head per hectare on natural grass and improved cultured pastures; and group C: 2 head per hectare on natural grass, improved cultured pasture and whole corn silage. Serum samples from 72 multiparous cows assigned to each beef cow-calf operations were obtained every 3 months during 2 years. A group of 30 replacement heifers from each group were tested similarly since they were 10-21 months old. Twenty four, 20 and 34 calves from groups A, B and C respectively, were bled before colostrum intake and again 6 months later. The samples were analyzed by indirect fluorescence antibody test (IFAT) for detection of total IgG against N. caninum at a serological titre ≥ 200 for multiparous cows and replacement heifers, and a serological titre ≥ 25 for calves. Serum samples from seropositive cows were assessed by ELISA to evaluate the avidity of their specific antibodies. There were no differences in the proportion of seropositive cows from groups A, B and C at the beginning of the trial (p>0.05). Interestingly, the lowest serological titres in seropositive cows from all groups were observed during the first trimester (p<0.05). Although seropositive cows had medium to high avidity antibodies, suggesting chronic infection; seroconversion associated with low antibody avidity was found in 2, 3 and 3 seropositive cows from groups A, B and C. All replacement heifers remained seronegative. No abortions were recorded but 2, 1, and 2 calves from groups A, B and C were seropositive before colostrum intake, respectively. Seropositive calves born from cows having intermediate or high avidity remained with the same serostatus at 6 months of age. Even under varying extensive management conditions, both N. caninum horizontal and vertical transmission methods do occur in beef cow-calf operations.
Asunto(s)
Crianza de Animales Domésticos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/transmisión , Coccidiosis/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Argentina , Bovinos , Coccidiosis/epidemiología , Coccidiosis/transmisión , Transmisión de Enfermedad Infecciosa/veterinaria , Femenino , Inmunoglobulina G/sangre , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Neospora , Embarazo , Distribución Aleatoria , Estudios SeroepidemiológicosRESUMEN
Neospora caninum is one of the most important causes of bovine abortion, but isolation of live parasites from infected tissue is difficult. The aims of the present study were to obtain new isolates of N. caninum from congenitally infected asymptomatic newborn cattle in Argentina and to perform characterization by multilocus-microsatellite analysis. Five clinically normal born calves, with demonstrable N. caninum antibodies in precolostrum serum by indirect fluorescent antibody test, were euthanized and their brain samples were processed for histopathological, immunohistochemical, polymerase chain reaction (PCR) analysis, and for bioassay in γ-interferon knockout (GKO) mice. Although N. caninum DNA was detected in brain from all the calves by PCR, viable N. caninum was isolated in GKO mice from only one calf. Neospora caninum tachyzoites of this Argentinean isolate, designated NC-Argentina LP1, were propagated in VERO cell cultures seeded with tachyzoites from the infected GKO mice tissues. Multilocus-microsatellite typing on DNA derived from cell cultured tachyzoites revealed a unique genetic pattern, different from reported isolates. This is the first bovine isolation and genetic characterization of N. caninum in Argentina.
Asunto(s)
Enfermedades de los Bovinos/parasitología , Coccidiosis/veterinaria , Neospora/aislamiento & purificación , Animales , Animales Recién Nacidos , Anticuerpos Antiprotozoarios/sangre , Argentina/epidemiología , Bioensayo/veterinaria , Encéfalo/parasitología , Bovinos , Enfermedades de los Bovinos/epidemiología , Coccidiosis/epidemiología , Coccidiosis/parasitología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Inmunohistoquímica/veterinaria , Masculino , Ratones , Ratones Noqueados , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus/veterinaria , Neospora/genética , Neospora/inmunología , Reacción en Cadena de la Polimerasa , Estudios SeroepidemiológicosRESUMEN
The aim of this study was to investigate how Neospora caninum serostatus may be affected by variables such as host species (water buffaloes or cattle) and age in animals cohabiting in the same ranch. A convenience cross-sectional study was performed on four ranches in the Northeast of Argentina, where water buffalo are cohabitating with beef cattle. Blood samples were collected from 1350 female water buffaloes (Bubalus bubalis) and 880 female beef cattle (Bos taurus and Bos indicus crossbreeds) from four ranches. Calving and weaning percentages at herd level for each ranch were also recorded. N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) (reciprocal antibody titers ≥ 100). Serological results were classified into 2 categories (0: negative; 1: positive). A logistic regression model was used to describe the relationship between N. caninum serostatus and specie (water buffalo or cattle), age or ranch and their interactions. Likelihood ratio tests were used to assess the significance of the model and their terms. Odds ratios were estimated and 95% profile likelihood (LR) and Wald confidence intervals (CI) obtained. Overall, specific antibody titers were found in 43.3% (584/1350) of water buffaloes and 28.6% (252/880) of cattle. Seropositive water buffaloes and cattle were observed on all ranches. Age was statistically significant (p=0.01) with an overall estimate of logit (log odds) of age of 0.03 for both species. This indicates that for every one year increase in age, the expected change in log odds of being seropositive increased by 0.03. On three of four ranches a water buffalo was 4.48, 1.54 and 2.25 times more likely to be seropositive than cattle for animals of the same age. The N. caninum serostatus was affected by age in the first place, but also by species on at least three of the four ranches. Calving and weaning percentages were higher in water buffaloes than in beef cattle (p<0.05). Even though the low pathogenicity that N. caninum seems to have in water buffaloes, this study reinforces the importance of this specie as maintenance of the disease.
Asunto(s)
Búfalos/parasitología , Enfermedades de los Bovinos/epidemiología , Coccidiosis/veterinaria , Neospora/inmunología , Factores de Edad , Animales , Anticuerpos Antiprotozoarios/sangre , Argentina/epidemiología , Bovinos , Coccidiosis/epidemiología , Estudios Transversales , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Modelos Logísticos , Factores de Riesgo , Estudios Seroepidemiológicos , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos/inmunología , Coccidiosis/veterinaria , Células Asesinas Naturales/patología , Neospora , Placenta/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/patología , Animales , Búfalos , Bovinos , Enfermedades de los Bovinos/patología , Coccidiosis/inmunología , Femenino , Células Asesinas Naturales/inmunología , Placenta/patología , Embarazo , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
The aim of the present study was to compare the immunogenicity and protective efficacy of live tachyzoites and native antigen extract obtained from the NC-6 Argentina strain against vertical transmission of Neospora caninum, following experimental challenge in pregnant heifers with the NC-1 strain. Sixteen pregnant heifers were divided in 4 groups of 4 animals, each receiving different inoculation before mating: group A animals were intravenously (iv) inoculated with 6.25×10(7) live tachyzoites of the NC-6 strain, group B heifers were inoculated twice subcutaneously (sc) with N. caninum native antigen extract formulated with ISCOMs, group C heifers were sc injected with sterile phosphate-buffered saline (PBS) and group D heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv challenged with the NC-1 strain at 70 days of gestation. Serum and heparinized blood samples were collected eight times on weeks 0, 2, 3, 5, 9, 13, 16 and 17 post-inoculation. Dams were slaughtered at the 17th week of experiment (104 days of pregnancy) and placental and fetal tissue samples were collected. Specific antibody responses in heifers were tested by indirect enzyme linked immunosorbent assay (iELISA). The cellular immune response in dams was assessed by quantifying IFN-γ production and the percentages of T-cells (CD4(+), CD8(+) and γδ(+)) and monocytes in peripheral blood mononuclear cells (PBMC). Fetal fluids and tissue samples were tested using the indirect fluorescence antibody test, western blot, histopathology, immunohistochemistry and nested-PCR. A significant increase in N. caninum antibody response was detected in heifers of groups A and B from week 3 after inoculation (P<0.001). IFN-γ production was similar in groups A and B at week 13 (P>0.05). All fetuses were viable at necropsy. Specific IgG against N. caninum was detected in 1/4 fetal fluids recovered from groups A, C and D heifers and 3/4 fetal fluids from group B. Transplacental transmission could be determined in one fetus from group A and three fetuses from group B by nPCR. All fetuses from groups C and D were positive by nPCR. It is noteworthy that dams with higher CD4(+)/CD8(+) ratios in PBMC, regardless of the experimental group, had lower pathology scores. The results of this study confirm that inoculation with live parasites pre-mating may provide at least partial protection against vertical transmission of N. caninum following challenge in heifers at early gestation.
Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Bovinos/prevención & control , Coccidiosis/veterinaria , Neospora/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Coccidiosis/prevención & control , Femenino , EmbarazoRESUMEN
Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.
Asunto(s)
Coccidiosis/patología , Coccidiosis/parasitología , Enfermedades Fetales/parasitología , Neospora/patogenicidad , Complicaciones Parasitarias del Embarazo/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Bovinos , Coccidiosis/inmunología , ADN Protozoario/genética , Modelos Animales de Enfermedad , Femenino , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Repeticiones de Microsatélite , Neospora/clasificación , Neospora/genética , Neospora/aislamiento & purificación , EmbarazoRESUMEN
Mice immunized with a soluble extract of Neospora caninum tachyzoites (sNcAg) formulated with Providean-AVEC, an aqueous soy-based adjuvant, are fully protected from N. caninum multiplication. Here we evaluated the dose-dependent immunogenicity of this vaccine formulation in cattle. Cattle (N=3 per group) were immunized with two applications (30 days apart) of formulations containing Providean-AVEC and different payloads of sNcAg (100, 50 and 10 µg), that were five to fifty times lower than the only reported study using this same antigen in cattle. Kinetics and magnitude of the vaccine-induced immune responses were dose-dependent. Cattle immunized with 100 µg-sNcAg elicited high-avidity specific antibodies 3 weeks after the primary vaccination while those that received 50 µg of antigen had maximum levels of specific high-avidity antibodies 5 days after the day 30 boost. Vaccination with 10 µg of sNcAg induced comparable antibody responses after 2 weeks post re-vaccination. IgG1 was the predominant isotype in all vaccinated animals. Maximum systemic IFN-γ levels were measured in cattle immunized with 50 and 100 µg-sNcAg (14 ± 2.8 ng/ml). CD4(+)-T cells from vaccinated animals proliferated after sNcAg stimulation in vitro, producing IFN-γ. Recall IFN-γ responses mediated by CD4(+)-T cells were detected up to 140 days post vaccination. Formulations containing Providean-AVEC and 50 µg of sNcAg stimulated broad cellular and humoral immune responses against N. caninum in cattle. The profile and magnitude of the immune response elicited by this vaccine can be modified by the antigen-dose and vaccination schedule. This is the first dose-response study performed in cattle using sNcAg as antigen.
Asunto(s)
Enfermedades de los Bovinos/prevención & control , Coccidiosis/veterinaria , Lecitinas/química , Neospora/inmunología , Vacunas Antiprotozoos/inmunología , beta-Glucanos/química , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Bovinos , Coccidiosis/prevención & control , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Pruebas SerológicasRESUMEN
The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1 × 10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1 × 10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.
Asunto(s)
Aborto Veterinario/parasitología , Enfermedades de los Bovinos/parasitología , Coccidiosis/veterinaria , Muerte Fetal/veterinaria , Neospora/clasificación , Placenta/patología , Animales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Bovinos , Enfermedades de los Bovinos/patología , Coccidiosis/parasitología , Coccidiosis/patología , ADN Protozoario/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Muerte Fetal/parasitología , Técnica del Anticuerpo Fluorescente Indirecta , Interferón gamma , Repeticiones de Microsatélite , Placenta/parasitología , EmbarazoRESUMEN
The aim of this study was to characterize the pathogenesis of Neospora caninum in experimentally inoculated pregnant water buffalo (Bubalus bubalis). Twelve Mediterranean female water buffaloes ranging in age from 4 to 14 years old and seronegative to N. caninum by indirect fluorescent antibody test (IFAT) were involved. Ten females were intravenously inoculated with 10(8) tachyzoites of NC-1 strain at 70 (n=3) or 90 (n=7) days of pregnancy (dp). Two control animals were inoculated with placebo at 70 and 90 dp, respectively. Serum samples were obtained weekly following inoculation to the end of the experiment. Three animals inoculated at 70 dp were slaughtered at 28 days post inoculation (dpi), three animals inoculated at 90 dp were slaughtered at 28 dpi and the remaining four animals inoculated at 90 dp were slaughtered at 42 dpi. Fetal fluids from cavities and tissue samples were recovered for IFAT and histopathology, immunohistochemistry and PCR, respectively. Genomic DNA from fetal tissues was used for parasite DNA detection and microsatellite genotyping in order to confirm the NC-1 specific-infection. Dams developed specific antibodies one week after the inoculation and serological titers did not decrease significantly to the end of the experiment. No abortions were recorded during the experimental time; however, one fetus from a dam inoculated at 70 dp was not viable at necropsy. Specific antibodies were detected in only two fetuses from dams inoculated at 90 dp that were slaughtered at 42 dpi. No macroscopic changes in the placentas and organs of viable fetuses were observed. Nonsuppurative placentitis was a common microscopic observation in Neospora-inoculated specimens. Microscopic fetal lesions included nonsuppurative peribronchiolar interstitial pneumonia, epicarditis and myocarditis, interstitial nephritis, myositis and periportal hepatitis. Positive IHC results were obtained in two fetuses from dams inoculated at 70 dp and slaughtered at 28 dpi. N. caninum DNA was detected in placentas and fetuses from all inoculated animals. The pattern of amplified microsatellites from placental and fetal tissues resembled the NC-1 strain. Water buffaloes, like cattle, are susceptible to experimental inoculation with N. caninum at early pregnancy.
Asunto(s)
Búfalos , Coccidiosis/veterinaria , Neospora/fisiología , Complicaciones Parasitarias del Embarazo/veterinaria , Animales , Coccidiosis/parasitología , Coccidiosis/patología , Femenino , Feto/parasitología , Feto/patología , Embarazo , Complicaciones Parasitarias del Embarazo/parasitología , Complicaciones Parasitarias del Embarazo/patologíaRESUMEN
The aim of this study was to incorporate antigens from Mannheimia haemolytica culture supernatant, and an immune modulatory molecule, recombinant bovine C3d (rBoC3d), into immune stimulating complexes (ISCOMs) using neutravidin-biotin interaction. Biotinylated ISCOM matrix was generated using a commercial kit. The biotinylated ISCOM matrix was incubated with neutravidin and then centrifuged in a sucrose density gradient. The rBoC3d was expressed as an in vivo biotinylated protein and with a c-Myc tag (EQKLISEEDL) engineered to facilitate detection. The neutravidin-coated ISCOM matrix was incubated with biotinylated antigens from M. haemolytica culture supernatants and rBoC3d. To test the association among the neutravidin-coated ISCOM matrix, biotinylated antigens and rBoC3d, an analytical sucrose density gradient (10-40%, w/w) was performed. The experimental formulations were run in SDS-PAGE gels under reducing conditions. For Western immunoblot analysis, polyclonal bovine antiavidin, monoclonal anti-c-Myc, monoclonal antileukotoxin, and anti-GS60 antibodies were used to detect the presence of neutravidin, rBoC3d, leukotoxin, and GS60 antigens, respectively. By taking advantage of the biotin-neutravidin interaction, not only leukotoxin but also the recombinant immunomodulatory molecule, rBoC3d, was incorporated into ISCOM particles.
Asunto(s)
Antígenos Bacterianos/inmunología , Avidina/metabolismo , Biotina/metabolismo , Complemento C3d/inmunología , Medios de Cultivo/química , ISCOMs/inmunología , Mannheimia haemolytica/inmunología , Animales , Antígenos Bacterianos/aislamiento & purificación , Biotecnología , Bovinos , Mannheimia haemolytica/citología , Proteínas Recombinantes/inmunologíaRESUMEN
The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.