RESUMEN
This study provides an initial analysis of the toll-like receptors (TLRs) that might be implicated in alpha-herpesvirus infection of the bovine respiratory system. A significant variation in the expression of TLR3 and TLRs 7-9 during bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) acute infections and particularly an up-regulation during viral reactivation in respiratory tissues has been demonstrated. Furthermore, viral distribution in the respiratory tract of BoHV-1- and BoHV-5-infected calves at different stages of the infectious cycle was analysed. The wide distribution of BoHV DNA in the respiratory tract during acute infection was restricted during latent infection and the subsequent reactivation of BoHV-1 and BoHV-5. Overall, the findings presented here contribute to the knowledge on the replication and dissemination of bovine alpha-herpesviruses. Furthermore, some of the immune factors triggered in the host that determine the different outcomes of infection by two closely related pathogens of cattle have been elucidated.
Asunto(s)
Enfermedades de los Bovinos/virología , Regulación de la Expresión Génica/inmunología , Herpesvirus Bovino 1/metabolismo , Herpesvirus Bovino 5/metabolismo , Rinotraqueítis Infecciosa Bovina/virología , Receptores Toll-Like/metabolismo , Animales , Bovinos , Enfermedades de los Bovinos/metabolismo , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Rinotraqueítis Infecciosa Bovina/inmunología , Sistema Respiratorio/metabolismo , Receptores Toll-Like/genética , Regulación hacia ArribaRESUMEN
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to the Rhadinovirus genus, which is increasingly associated with various problems of the reproductive tract of cattle. In Argentina, analysis of BoHV-4 strains isolated from cervico-vaginal mucus of aborted cows revealed a high genetic divergence among strains, which could be classified in three different groups: Genotype 1 comprises Movar-like strains (European prototype), Genotype 2 includes DN599-like strains (American prototype) and Genotype 3 corresponds to a novel genotype group. Understanding the replication behavior in cell cultures and the molecular characteristics of this pathogen of cattle is critical for the rational design of in vitro experiments. The aim of this work was to quantitatively evaluate the replication properties of different Argentinean BoHV-4 strains and to characterize their phylogenetic relationships. Significant differences were evident among the virus titers of the different BoHV-4 isolates in vitro. The most conserved gene was the major capsid protein (ORF25). The glycoprotein B (gB), glycoprotein H (gH), and thymidine kinsase (TK) genes displayed both synonymous and non-synonymous substitutions, with the highest diversity observed for gB, which displayed amino acid substitutions in 24 out of the 178 positions examined. Strains 09/759, 12/512, and 07/568 presented a deletion encompassing amino acid position 27 to 35, whereas strains 07/435 and 09/227 had a deletion from position 28 to 35. Two strains, 07/435 and 09/227, also displayed the highest divergence compared to the other strains analyzed. This study provides information about the in vitro replication and behavior of nine field isolates of BoHV-4. These findings are relevant since available information on the in vitro growth characteristics of BoHV-4 strains is scarce. The results from this study may also be useful for establishing comparisons with other related viruses.
Asunto(s)
Herpesvirus Bovino 4/aislamiento & purificación , Herpesvirus Bovino 4/fisiología , Replicación Viral/genética , Animales , Argentina , Bovinos , Enfermedades de los Bovinos/virología , Línea Celular , ADN Viral/genética , Femenino , Variación Genética , Genotipo , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 4/genética , Filogenia , Timidina Quinasa/genética , Vagina/virología , Frotis Vaginal/veterinaria , Proteínas del Envoltorio Viral/genéticaRESUMEN
Bovine herpesvirus type 1 (BoHV-1) is responsible for respiratory and genital disease in cattle. BoHV-1 encephalitis is only occasionally reported. However, several cases of neurological disease have been recently attributed to BoHV-1. In this study, the distribution and pathological alterations caused by two BoHV-1 strains in the nervous system of experimentally infected calves during acute infection and reactivation are described. Calves were inoculated intranasally with BoHV-1 Los Angeles (BoHV-1.LA) or Cooper (BoHV-1.Cooper) strains. Acutely infected calves were euthanased at 6 days (BoHV-1.Cooper, n = 2) and 7 days post-inoculation (BoHV-1.LA, n = 2). Latently infected calves that were given dexamethasone to induce reactivation were euthanased at 2 days (BoHV-1.Cooper, n = 2) or 5 days (BoHV-1.LA, n = 2) after dexamethasone administration. Both BoHV-1 strains were isolated from the brains of acutely infected calves. Distribution of viral DNA in the neural tissues was similar for both strains. During reactivation, neither BoHV-1.LA nor BoHV-1.Cooper was isolated from any brain section or trigeminal ganglia in infected calves. Macroscopic lesions were not evident in any group. In BoHV-1.LA infected calves, microscopic lesions were found in the brain but not in the trigeminal ganglia. Microscopic lesions in the brain of BoHV-1.Cooper infected calves were not as evident as in BoHV-1.LA infected animals. However, mononuclear infiltrates and neuronophagia were present in trigeminal ganglia. The results of this study demonstrated that respiratory BoHV-1 strains are able to replicate and disseminate within the bovine nervous tissue and provide evidence of the neuroinvasiveness of BoHV-1 strains.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/fisiología , Rinotraqueítis Infecciosa Bovina/virología , Activación Viral , Enfermedad Aguda , Animales , Encéfalo/virología , Bovinos , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/aislamiento & purificación , Sistema Nervioso/virología , Ganglio del Trigémino/virologíaRESUMEN
Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.
Asunto(s)
Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/aislamiento & purificación , Herpesvirus Bovino 5/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Virología/métodos , Animales , Bovinos , Perros , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Células de Riñón Canino Madin Darby , Desnaturalización de Ácido Nucleico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
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
In this study, the expression levels of viral Toll-like receptors (TLRs) in the nervous system of bovine herpesvirus type 5 (BoHV-5)-infected calves were investigated. A significant increase in the expression of TLRs 3 and 7-9 was found in the anterior cerebral cortex during acute infection and viral reactivation. In the trigeminal ganglia, only TLR9 expression was significantly affected. The magnitude of the increase was lower in BoHV-1-infected calves, suggesting that a restricted immune response might protect against exacerbated inflammatory responses in the brain. This work describes, for the first time, the involvement of TLRs 3 and 7-9 in the recognition of BoHV in the bovine nervous system, indicating that the expression of these receptors might be associated with the development of neurological disease. Modulation of the signalling pathways mediated by TLRs might provide an effective approach to control the neuro-immune response to BoHV-5, which may be responsible for neurological lesions.
Asunto(s)
Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/virología , Encefalitis Viral/veterinaria , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 5/patogenicidad , Meningoencefalitis/veterinaria , Sistema Nervioso/metabolismo , Receptores Toll-Like/metabolismo , Administración Intranasal , Animales , Bovinos , Enfermedades de los Bovinos/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/virología , ADN Viral/metabolismo , Encefalitis Viral/metabolismo , Encefalitis Viral/patología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Herpesvirus Bovino 5/genética , Herpesvirus Bovino 5/aislamiento & purificación , Meningoencefalitis/metabolismo , Meningoencefalitis/patología , Sistema Nervioso/patología , Sistema Nervioso/virología , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 9/metabolismo , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/patología , Ganglio del Trigémino/virologíaRESUMEN
The involvement of Toll-like receptors (TLRs) in bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) infections has not been analyzed. In this study, the role of TLR signaling on virus replication was investigated. Blood leukocytes consistently express TLRs. Thus, our approach was to study in vitro the effects of agonist stimulation of TLRs expressed by peripheral blood leukocytes on BoHV-1 and BoHV-5 replication. Furthermore, the patterns of TLRs 3, 7-9 expression on virus-infected-bovine leukocytes were analyzed. Only Imiquimod (TLR7/8 agonist) showed anti-viral activity on infected MDBK cells. This is the first evidence that the timely activation of TLR7/8 signaling is effective in impairing BoHV-1 and 5 replication, thereby providing an experimental indication that Imiquimod may be a promising immune modulator. This work describes, for the first time, the expression patterns of TLRs in BoHV-1- or BoHV-5-infected-bovine leukocytes, suggesting the involvement of TLR7 and TLR9 in the recognition of these viruses.
Asunto(s)
Aminoquinolinas/farmacología , Enfermedades de los Bovinos/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/inmunología , Herpesvirus Bovino 5/inmunología , Receptores Toll-Like/inmunología , Aminoquinolinas/uso terapéutico , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Línea Celular , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Imiquimod , Análisis de los Mínimos Cuadrados , Leucocitos Mononucleares , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Transducción de Señal/inmunología , Receptores Toll-Like/agonistas , Replicación Viral/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
The current report was prompted by an atypical outbreak of mucosal disease that occurred in a beef herd in the southwestern part of Buenos Aires Province, Argentina, where a total of 9/41 (21.9%) yearling bulls died. Blood samples from 73 bulls and 189 heifers were tested for evidence of persistent BVDV infection with Bovine Viral Diarrhea Virus (BVDV). Non-cytopathic BVDV was isolated from 7 (9.6%) 24- to 36-month-old bulls, and 3 (1.6%) 36-month-old heifers. Non-cytopathic BVDV was also detected in the seminal plasma of three of six persistently infected (PI) bulls. Furthermore, a 171 bp genomic fragment of BVDV was consistently detected by nested RT-PCR in one of the two samples of the commercial semen used for artificial insemination, indicating that this semen could be a possible source of infection for the whole herd. To evaluate the possible reproductive consequences of PI heifers and bulls, ovaries and semen were obtained from PI cattle for in vitro assays. The in vitro fertilization of oocytes with semen from PI bulls was associated with decreased cleavage and embryo development rates. Additionally, non-cytopathic BVDV was isolated from the follicular fluid of PI heifers. Genetic typing revealed that all isolates BVDV from the present study had a high percentage of homology and that all of the fragments from the RT-PCR clearly fit with the BVDV 1b cluster. These findings confirm the negative impact that BVDV can have on the reproductive performance of cattle and the importance of applying the proper sanitary controls to minimize the risk of BVDV infection.
Asunto(s)
Aborto Veterinario/virología , Diarrea Mucosa Bovina Viral/transmisión , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Inseminación Artificial/veterinaria , Semen/virología , Animales , Argentina/epidemiología , Bovinos , Femenino , Masculino , Reacción en Cadena de la Polimerasa/veterinaria , Embarazo , ARN Viral/aislamiento & purificación , Esparcimiento de VirusRESUMEN
Bovine herpesvirus 4 (BoHV-4) is a γ-herpesvirus that has been isolated both from apparently healthy animals and from cattle with a variety of clinical signs, including post-partum endometritis and abortion. BoHV-4 causes either a persistent or a latent infection in cells of the monocyte/macrophage lineage. Two groups of BoVH-4 strains have been defined based on their restriction patterns: the Movar-like strains (European prototype) and the DN 599-like strains (American prototype). The purpose of the present study was to genetically characterize wild type BoHV-4 strains isolated from vaginal discharges of aborted cows in Argentina. The virus was identified by isolation and nested PCR in all vaginal discharge samples from aborted cows, either as a sole agent or in association with other pathogens. Restriction enzyme profiling and phylogenetic analysis demonstrated that there is a high genetic variability among the studied field isolates. The existence of three groups of strains, which were designated as genotypes 1, 2 and 3, is described. Genotypes 1 and 2 possibly correspond to the Movar-like and DN 599-like groups, respectively, whereas Genotype 3 corresponds to a novel group. Two viral strains did not cluster into any of these three groups, indicating that other genotypes could be circulating in Argentina. These results suggest a complex epidemiological background for the Argentinean BoHV-4 strains, probably influenced by independent events of genetic drift. This hypothesis cannot be rejected based on the available data. However, there is no direct evidence supporting this possibility. Thus, it seems speculative to suggest that interspecific jumps are responsible for the observed phylogenetic grouping. On the other hand, our analyses suggest a geographical structure for the observed viral genotypes, since genotypes 1 and 2 included the European (Movar-like) and American (DN599-like) reference strains, respectively. Geographic dispersion is known to be a driver of herpes viruses diversification, and independent evolution in geographical isolated places ensures the emergence of particular mutations in each location due to genetic drift (Compans, 2007; Zong et al., 1999). Therefore, at this point, the genetic drift hypothesis is the one that requires less ad-hoc considerations and thus, to our understanding, is the one that fits to the findings from this study. The involvement of this genetic variability in the detection and pathogenesis of BoHV-4 remains to be investigated.
Asunto(s)
Enfermedades de los Bovinos/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 4/genética , Aborto Veterinario/virología , Animales , Argentina , Bovinos , Enfermedades de los Bovinos/genética , Línea Celular , Femenino , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 4/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Embarazo , Vagina/virología , Frotis Vaginal/veterinariaRESUMEN
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 work was to study the in vitro replication of bovine herpesvirus types 1 and 5 (BoHV-1 and 5) at the beginning and end of the logarithmic growth phase of Madin-Darby Bovine Kidney (MDBK) cells. The replication kinetics and size of lysis and infection plaques of the field isolates 09/210 (BoHV-1) and 97/613 (BoHV-5) and the reference strains BoHV-1.1 Los Angeles 38 (LA38), BoHV-1.1 Cooper, BoHV-5a N569 and BoHV-5b A663 were evaluated. The highest mean virus titre was recorded for N569, followed by LA38 and 97/613. For most of the viruses, the virus titre values increased from 24 h post-infection (hpi) up to 48 hpi and then, they remained unchanged up to 72 hpi. However, the virus titre for 09/210 was significantly lower and a slight, steady increase was observed from 24 to 72 hpi. Furthermore, the largest lysis and infection plaques were recorded for 97/613 and LA38, respectively. According to this work, it is evident that there is a relationship between the replication of BoHV and the multiplication stage of MDBK cells. The results of this study contribute to the understanding of the replication behaviour in cell cultures of several strains of BoHV, which is critical for the rational design of in vitro experiments and vaccine production.
Asunto(s)
Herpesvirus Bovino 1/fisiología , Herpesvirus Bovino 5/fisiología , Replicación Viral , Animales , Bovinos , Línea Celular , Proliferación Celular , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 5/crecimiento & desarrollo , Factores de Tiempo , Carga Viral , Ensayo de Placa Viral , Cultivo de Virus/métodosRESUMEN
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 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.
Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Bovinos/inmunología , Coccidiosis/veterinaria , Neospora/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/farmacología , Western Blotting/veterinaria , Bovinos , Enfermedades de los Bovinos/parasitología , Coccidiosis/inmunología , Coccidiosis/parasitología , ADN Protozoario/genética , Ensayo de Inmunoadsorción Enzimática/veterinaria , ISCOMs/inmunología , Isotipos de Inmunoglobulinas/inmunología , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interferón gamma/inmunología , Neospora/genética , Neospora/patogenicidad , Parasitemia/genética , Reacción en Cadena de la Polimerasa/veterinaria , Distribución Aleatoria , Factores de Tiempo , Vacunación/veterinariaRESUMEN
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.
Asunto(s)
Diarrea Mucosa Bovina Viral/virología , Virus de la Diarrea Viral Bovina/crecimiento & desarrollo , Síndrome Hemorrágico de los Bovinos/virología , Cultivo de Virus/métodos , Replicación Viral , Animales , Argentina/epidemiología , Diarrea Mucosa Bovina Viral/epidemiología , Bovinos , Técnicas de Cultivo de Célula/métodos , Línea Celular/virología , Cricetinae , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Perros , Femenino , Síndrome Hemorrágico de los Bovinos/epidemiología , Riñón/citología , Masculino , Mesocricetus , Especificidad de Órganos , Porcinos , Testículo/citología , Útero/citologíaRESUMEN
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.
Asunto(s)
Virus de la Diarrea Viral Bovina/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Bovinos , Virus de la Diarrea Viral Bovina/genética , Relación Dosis-Respuesta Inmunológica , Genotipo , Cobayas , Modelos Animales , Pruebas de Neutralización , Ovinos , Especificidad de la Especie , Vacunas de Productos Inactivados/inmunologíaRESUMEN
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.
Asunto(s)
Animales , Bovinos , Cricetinae , Perros , Femenino , Masculino , Diarrea Mucosa Bovina Viral/virología , Virus de la Diarrea Viral Bovina/crecimiento & desarrollo , Síndrome Hemorrágico de los Bovinos/virología , Técnicas In Vitro , Replicación Viral , Cultivo de Virus/métodos , Argentina/epidemiología , Diarrea Mucosa Bovina Viral/epidemiología , Técnicas de Cultivo de Célula/métodos , Línea Celular/virología , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Síndrome Hemorrágico de los Bovinos/epidemiología , Riñón/citología , Mesocricetus , Especificidad de Órganos , Porcinos , Testículo/citología , Útero/citologíaRESUMEN
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.
Asunto(s)
Animales , Bovinos , Cobayas , Virus de la Diarrea Viral Bovina/inmunología , Vacunas Virales/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Relación Dosis-Respuesta Inmunológica , Virus de la Diarrea Viral Bovina/genética , Genotipo , Modelos Animales , Pruebas de Neutralización , Ovinos , Especificidad de la Especie , Vacunas de Productos Inactivados/inmunologíaRESUMEN
From 2003 through 2007, serum samples from 5594 dairy and beef heifers and cows in Argentina were assessed to quantify the association between presence of Neospora caninum antibodies and history of abortion, type of exploitation, and age category of animals. Animals with a history of abortion were 85% more likely (P<0.01) to be positive to N. caninum than animals without a record of abortion. For a given category (age) of animals, being in a dairy operation increased the odds of being N. caninum-positive. Replacement dairy heifers were 76% more likely (P<0.01) to be N. caninum-positive than beef cows. These results suggest that postnatal exposure may be more frequent in dairy operations than in beef herds and provide insight into the epidemiology of the disease in one of the most important livestock production regions of the world.