RESUMO
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.
Assuntos
Doenças dos Bovinos/virologia , Regulação da Expressão Gênica/imunologia , Herpesvirus Bovino 1/metabolismo , Herpesvirus Bovino 5/metabolismo , Rinotraqueíte Infecciosa Bovina/virologia , Receptores Toll-Like/metabolismo , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Rinotraqueíte Infecciosa Bovina/imunologia , Sistema Respiratório/metabolismo , Receptores Toll-Like/genética , Regulação para CimaRESUMO
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.
Assuntos
Herpesvirus Bovino 4/isolamento & purificação , Herpesvirus Bovino 4/fisiologia , Replicação Viral/genética , Animais , Argentina , Bovinos , Doenças dos Bovinos/virologia , Linhagem Celular , DNA Viral/genética , Feminino , Variação Genética , Genótipo , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/genética , Filogenia , Timidina Quinase/genética , Vagina/virologia , Esfregaço Vaginal/veterinária , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/fisiologia , Rinotraqueíte Infecciosa Bovina/virologia , Ativação Viral , Doença Aguda , Animais , Encéfalo/virologia , Bovinos , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/isolamento & purificação , Sistema Nervoso/virologia , Gânglio Trigeminal/virologiaRESUMO
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.
Assuntos
Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/isolamento & purificação , Herpesvirus Bovino 5/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Virologia/métodos , Animais , Bovinos , Cães , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Células Madin Darby de Rim Canino , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
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.
Assuntos
Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Neospora/isolamento & purificação , Animais , Animais Recém-Nascidos , Anticorpos Antiprotozoários/sangue , Argentina/epidemiologia , Bioensaio/veterinária , Encéfalo/parasitologia , Bovinos , Doenças dos Bovinos/epidemiologia , Coccidiose/epidemiologia , Coccidiose/parasitologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imuno-Histoquímica/veterinária , Masculino , Camundongos , Camundongos Knockout , Repetições de Microssatélites , Dados de Sequência Molecular , Tipagem de Sequências Multilocus/veterinária , Neospora/genética , Neospora/imunologia , Reação em Cadeia da Polimerase , Estudos SoroepidemiológicosRESUMO
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.
Assuntos
Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/virologia , Encefalite Viral/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 5/patogenicidade , Meningoencefalite/veterinária , Sistema Nervoso/metabolismo , Receptores Toll-Like/metabolismo , Administração Intranasal , Animais , Bovinos , Doenças dos Bovinos/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/virologia , DNA Viral/metabolismo , Encefalite Viral/metabolismo , Encefalite Viral/patologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Herpesvirus Bovino 5/genética , Herpesvirus Bovino 5/isolamento & purificação , Meningoencefalite/metabolismo , Meningoencefalite/patologia , Sistema Nervoso/patologia , Sistema Nervoso/virologia , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologiaRESUMO
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.
Assuntos
Aminoquinolinas/farmacologia , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Herpesvirus Bovino 5/imunologia , Receptores Toll-Like/imunologia , Aminoquinolinas/uso terapêutico , Animais , Bovinos , Doenças dos Bovinos/imunologia , Linhagem Celular , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Imiquimode , Análise dos Mínimos Quadrados , Leucócitos Mononucleares , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transdução de Sinais/imunologia , Receptores Toll-Like/agonistas , Replicação Viral/imunologiaRESUMO
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.
Assuntos
Antígenos de Protozoários/imunologia , Doenças dos Bovinos/prevenção & controle , Coccidiose/veterinária , Neospora/imunologia , Vacinas Protozoárias/imunologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/prevenção & controle , Feminino , GravidezRESUMO
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.
Assuntos
Coccidiose/patologia , Coccidiose/parasitologia , Doenças Fetais/parasitologia , Neospora/patogenicidade , Complicações Parasitárias na Gravidez/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Coccidiose/imunologia , DNA de Protozoário/genética , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Repetições de Microssatélites , Neospora/classificação , Neospora/genética , Neospora/isolamento & purificação , GravidezRESUMO
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.