A comparative pattern of lectin-binding in the endometrial glands of the uterus and placenta of healthy buffaloes and bovines at early gestation

Water buffalo (Bubalus bubalis) and domestic cattle (Bos taurus) are closely related species. However, embryo transfer interspecies has been attempted without any success. The failure in hybrid embryo-implantation is associated with the glycocode in the maternal-fetal interface. Glycosylation patterns have been studied in different species of ruminants; however, in B. bubalis, only the binucleated cells (BNC) have been analyzed. This glycocode is essential for a successful embryo-implantation and can be defined by Lectin-Histochemistry (LHC). The aim of this study is to compare the glycosylation pattern of placenta and uterus in water buffaloes and cattle by LHC. Tissue samples of placenta and uterus from pregnant Mediterranean female water buffaloes (Buf1) and Angus cows (Bov1) were analyzed. All animals were euthanized at 98 days of gestation. LHC was carried out using twelve lectins (Con A, LCA, PSA, sWGA, PHA-e, SBA, UEA-1, WGA, RCA-1, PNA, DBA, BSA-1). The intensity of lectin binding was semiquantitatively scored using a scale of 0 (negative) to 3 (strongly positive). Difference between species was found in trophoblast layer by PSA, SWGA, PNA and BSA-1, in BNC, and in the mononuclear cells by LCA, PSA, PHA-e, DBA, BSA-1, PNA. In utero, differences in the apical cellular membrane and the secretion of glands were identified by DBA and RCA-1, and in the cytoplasm of those glandular epithelial cells by PHA-e, BSA-1, WGA, and SBA. In both species, BNC presented a strong positive reaction with DBA and SBA, a moderate response by LCA, PHA-e, BSA-1 and PNA lectin, and a low reaction by PSA, UEA-1, SWGA, WGA, Con A and RCA-1. The results found in this study suggest that although both species are closely related, glycosylation patterns in the placenta and uterus are different, thus providing a possible reason for embryo transfer not being possible between these species.EEA MercedesFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Konrad, Jose Luis. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Moore, Prando Dadin. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Sala, Juan Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Lischinsky, Lilian Haydee. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Barbeito, Claudio G. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina

First report of congenital Neospora caninum encephalomyelitis in two newborn calves in the Argentinean Pampas

Bovine neosporosis is a parasitic disease of major concern in cattle industry worldwide. Although abortion is the more frequent clinical presentation, sporadic congenital clinical bovine neosporosis has been scarcely reported in newborn calves. Neospora caninum encephalomyelitis was diagnosed in two of 314 newborn calves submitted for necropsy and laboratory analysis between 2000 and 2014. The two new born calves had limb dysfunction, loss of conscious proprioception and ataxia. Protozoal multifocal nonsuppurative encephalomyelitis was observed and diagnosis was confirmed using serology, immunohistochemistry and PCR. This is the first report of clinical congenital neosporosis in calves from Argentina.Fil: Micheloud, Juan Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Salta; ArgentinaFil: Moore, Prando Dadin. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Canal, Ana Maria. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Lischinsky, Lilian Haydee. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Hecker, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Canton, German Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odriozola, Ernesto Raul. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odeon, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation

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 × 108 tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1 × 108 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.EEA MercedesFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Moore, Prando Dadin. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Cano, Dora Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Lischinsky, Lilian Haydee. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Regidor-Cerrillo, Javier. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias. Departamento de Salud Animal. SALUVET; EspañaFil: Alvarez-García, Gema. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias. Departamento de Salud Animal. SALUVET; EspañaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Bacigalupe, Diana. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Laboratorio de Inmunoparasitología; ArgentinaFil: Ortega-Mora, Luis M. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias. Departamento de Salud Animal. SALUVET; EspañaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Experimental inoculation of Neospora caninum in pregnant water buffalo

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 108 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.EEA MercedesFil: Konrad, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias. Cátedra de Teriogenología; ArgentinaFil: Moore, Prando Dadin. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crudeli, Gustavo Angel. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias. Cátedra de Teriogenología; ArgentinaFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Cano, Dora Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Lischinsky, Lilian Haydee. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Regidor-Cerrillo, Javier. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias. Departamento de Salud Animal. SALUVET; EspañaFil: Odeon, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Ortega-Mora, Luis M. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias. Departamento de Salud Animal. SALUVET; EspañaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P 0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P > 0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation.EEA BalcarceFil: Hecker, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Cóceres, Verónica Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas. Laboratorio de Parasitología Molecular; ArgentinaFil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Jaramillo Ortiz, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Morrell, Eleonora Lidia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Verna, Andrea Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Ganuza, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas. Laboratorio de Parasitología Molecular; ArgentinaFil: Cano, Dora Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Lischinsky, Lilian Haydee. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas. Laboratorio de Parasitología Molecular; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Odeon, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Morein, Bror. Uppsala University. Department of Medical Sciences. Infectious Diseases; SueciaFil: Moore, Prando Dadin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin