The bovine viral diarrhea virus (BVDV) infection control should be based on elimination of persistently infected animals and on immunization through vaccination, to prevent fetal infection. However, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. This study evaluated the humoral immune response against homologous and heterologous strains of 7 inactivated BVDV vaccines, in bovines and two experimental models (ovine and guinea pig) which might be used to test candidate vaccines. Vaccines formulated with BVDV Singer, Oregon, NADL, and multivalent, induced seroconversion in the three animal models studied, reaching antibody titres higher than 2. Vaccine containing 125C -genotype 2- only induced a low antibody response in ovine, while VS-115 NCP vaccine was not immunogenic. Furthermore, bovine sera at 60 dpv presented homologous as well as heterologous antibody response, indicating a high degree of cross-reactivity among the strains studied. However, when bovine sera were tested against the Argentine field strain 00-693, they showed the lowest levels of cross-reactivity, suggesting the need of continued surveillance to identify and characterize emerging field BVDV strains. Finally, optimal correlations between bovine-ovine and bovine-guinea pig models were observed, indicating that two alternative species could replace bovines when testing the immunogenicity of BVDV candidate vaccines.

El control del virus de la diarrea viral bovina (VDVB) se basa en la eliminación de animales persistentemente infectados, y la inmunización de hembras para prevenir infecciones fetales. La eficiencia de estas vacunas es variable por su baja inmunogenicidad. Se evaluó la respuesta inmune humoral contra virus homólogos y heterólogos de 7 vacunas experimentales inactivadas del VDVB en bovinos y en dos modelos experimentales (ovinos y cobayos). Las vacunas conteniendo VDVB Singer, Oregon, NADL y polivalentes indujeron seroconversión en los tres modelos y se alcanzaron títulos de anticuerpos mayores de 2. La vacuna con VDVB genotipo 2 VS-115, NCP, no resultó inmunogénica. La vacuna genotipo 2 125C sólo indujo baja respuesta humoral en ovinos, mientras que la VS-115, NCP, no resultó inmunogénica. En bovinos se determinó la respuesta a virus homólogos y heterólogos a 60 dpv, lo que indica un alto grado de inmunidad cruzada entre la mayoría de las cepas estudiadas. Cuando los sueros bovinos fueron ensayados con la cepa de campo de Argentina 00-693, los niveles de reacción cruzada fueron más bajos; esto sugiere la necesidad de una vigilancia epidemiológica sostenida a fin de identificar y caracterizar las cepas emergentes del VDVB. La óptima correlación en el modelo bovino-ovino y bovino-cobayo indica su utilidad para evaluar la inmunogenicidad de vacunas inactivadas de VDVB.

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD- 420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.

Se estudió la interacción de aislamientos de campo de Argentina del VDVB (Pestivirus, Flaviridae) en las líneas celulares continuas MDBK, BoTur y BHK-21. Se utilizaron los virus de campo genotipo 1b, 99/134, 00/693 (casos compatibles con enfermedad de las mucosas) y 04P7016 (cuadro respiratorio) y las cepas de referencia genotipo 1a Singer y NADL. Además se evaluó la interacción de VDVB-NADL con las líneas celulares experimentales de bovino RD-420 y NCL-1 y de riñón porcino (PKZ). Se usaron 2 protocolos de infección. Los títulos virales observados dependieron del virus y del tiempo de infección y no así del modo de infección. Mientras que MDBK y BoTur resultaron susceptibles a la infección, BHK-21 y PKZ no lo fueron. Los virus NADL, 00/693 y 04/89 incrementaron su título entre las 24 y las 48 h p.i. en BoTur para mantenerlo posteriormente; los virus 99/134 y 04P7016 no presentaron variaciones y la cepa Singer presentó título máximo a las 24 h p.i para luego descender. La cinética del virus NADL en las células MDBK, RD-420 y NCL-1 tuvo un incremento de título para MDBK y NCL-1 entre las 24 y 48 h p.i que descendió a las 72 h p.i. La interacción virus-línea celular no estaría relacionada con el sub-genotipo del virus (1a o 1b), ni con el cuadro clínico; las células MDBK y NCL-1 serían más susceptibles a la replicación del VDVB.

A retrospective study was performed on 169 beef and dairy calves aged from 1 to 7 days old submitted to the Diagnostic Laboratories at INTA Balcarce, Argentina. Bacterial culture was performed for aerobic and microaerophilic organisms. Samples from spleen and lymph nodes, and peripheral blood mononuclear cells were also cultured for viral isolation on cell culture. Bovine rotavirus was detected by direct-ELISA. Multiple tissue samples were fixed in 10% formalin, routinely processed and stained with hematoxylin and eosin for microscopic examination. Etiological diagnosis was made in 70 of the 169 calves. Infectious agents were identified in 49 cases, the most common being Escherichia coli. When the histopathological examination was performed in cases with undetermined diagnosis, it was noted that 44 specimens had histological lesions, which suggested the presence of an infectious agent. In order to characterize the causes of bovine neonatal mortality, the protocols and methodology should be improved in further works.

Se realizó un estudio restrospectivo en 169 terneros muertos 1 a 7 días después del nacimiento pertenecientes a rodeos para carne y leche, remitidos a los Laboratorios de Diagnóstico del INTA Balcarce, Argentina. Para detectar organismos aeróbicos y microaerófilos se realizó el cultivo bacteriano. Para el aislamiento viral sobre cultivo celular, se recolectaron muestras de bazo, ganglios linfáticos y sangre periférica. El rotavirus bovino fue identificado por ELISA directo. Se efectuó el examen microscópico de diferentes tejidos, los cuales fueron fijados en formol al 10%, procesados y teñidos con hematoxilina y eosina. Se obtuvo un diagnóstico etiológico en 70 de los 169 terneros. Se identificaron agentes infecciosos en 49 casos, siendo el más común Escherichia coli. En los casos con diagnóstico indeterminado, el examen histopatológico realizado determinó que 44 especímenes poseían lesiones compatibles con la presencia de agentes infecciosos. Es necesario mejorar los protocolos y las metodologías de trabajo a los fines de caracterizar las causas de mortalidad neonatal en bovinos.

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

The aim of this study was to determine the consequences of the infection of BVDV genotype II (BVDV-2) in calves with preexisting immunity to BVDV-1 (induced by infection with wild type BVDV (Group A, 4 calves), or induced by vaccination with a killed BVDV-1 product (Group B, 2 calves). Group A and B were intranasally exposed to a field isolate of BVDV-2 (25ml x 10-7.8 TCID50/ml). Calves of Group A had erosions on the oral mucosa from 3 to 4 day post-challenge (DPC); moreover, intense salivation, mucous nasal secretion and sporadic diarrhea were observed from 4-14 DPC. Only one calf of Group B had diarrhea at 5 DPC. The average value of peripheral blood leukocytes (PBL) previous to BVDV-2 challenge was 8,775 PBL/ml (±603) in calves of Group A and 9,975 PBL/ml (±1308) in calves of Group B. After BVDV-2 challenge there was leukopenia in Groups A and B. The minimal number of PBL of 5,875 PBL/ml (±311) was observed in Group A at 6 PDC (P <0.05). There were differences (P <0,05) in the average of PBL during the 17 DPC among challenged animals and uninfected controls. Moreover, a difference in the number of blood lymphocytes was observed at 6-7 DPC between virus exposed and control calves. BVDV was isolated from ocular swabs of 3 calves of Group A at 13 and 17 DPC and from PBL at 9 and 11 DPC. The neutralizing antibody titer (NT) to both genotypes of BVDV was higher (P <0.05) in calves of Group A. Previous to virus challenge calves in this group had NT of 1:125 (geometric mean) and 1:128 to BVDV-1 and -II, respectively. The NT rose to 1:2048 at 17 DPC to both Genotypes of BVDV. Calves in Group B had a NT of 1:16 to both BVDV-1 and -II previous to BVDV-2 challenge. At 17 DPC the NT rose to 1:64 and to 1:181 to BVDV-1 and -II, respectively. These results allow us to conclude that antibodies generated by natural infection or vaccination with BVDV-1 increased for BVDV-1 and -2 after the experimental challenge with BVDV-2.