RESUMO
Abortions cause heavy economic losses for the bovine sector. The use of a standardized panel of analyses covering a large spectrum of pathogens responsible of abortion in cattle allowed demonstrating the direct involvement of at least one pathogen in 57% of analysed abortions in the southern part of Belgium. This result suggests a margin of improvement in the diagnostic efficacy. In order to evaluate the interest to broaden the list of pathogens included in the panel of analyses, the implication of bovine herpesvirus 4 (BoHV-4) in abortion was assessed by two different studies. In the first study, coupled serology was performed after abortion on 714 dams to identify specific seroconversion against BoHV-4. The overall seroconversion in cows was 19.5%, with a higher frequency in primiparous compared to multiparous females. In addition, the type of breed (beef cattle) and the time period from the fourth quarter 2008 until the last quarter 2009 were significantly related to the seroconversion of cows. The second study investigated the virus ability to infect the foetus. In this study, 368 cases of bovine abortions were specifically tested for BoHV-4, using PCR on foetus tissues and ELISA on dam and foetus sera. The results showed a maternal seroprevalence of 64.7%, a foetal seroprevalence of 0.8% and a PCR prevalence in foetuses of 1.1%, demonstrating the ability of BoHV-4 to infect the foetus.
Assuntos
Feto Abortado/virologia , Aborto Animal/epidemiologia , Doenças dos Bovinos/epidemiologia , Infecções por Herpesviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Aborto Animal/virologia , Animais , Bélgica , Bovinos/genética , Bovinos/fisiologia , Doenças dos Bovinos/virologia , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/fisiologia , Paridade , Prevalência , Estações do Ano , Soroconversão , Estudos Soroepidemiológicos , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologiaRESUMO
This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.
Assuntos
Aborto Animal/patologia , Morte Celular , Proliferação de Células , Citocinas/genética , Infecções por Herpesviridae/veterinária , Aborto Animal/virologia , Animais , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/fisiologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Placenta/patologia , Placenta/virologia , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Útero/patologia , Útero/virologiaRESUMO
Bovine herpesvirus type 4 (BHV-4) is related to many different conditions: infertility, postpartal metritis, vulvovaginitis, mastitis, encephalitis, calf pneumonia, keratoconjunctivitis, cutaneous lesions, digital dermatitis and abortion. In this study a retrospective PCR examination of 100 extracted DNA samples from aborting cows was performed in order to determine: prevalence of BHV-4 in abortive cattle, whether coinfections BHV-4 with other abortifacient pathogens are present in the same sample and to determine the month of gestation when BHV-4 associated abortions were detected. Out of 100 examined samples, the BHV-4 genome was detected in 21 samples (21%). In two samples we detected coinfection of BHV-4 with bovine viral diarrhea virus (BVDV) and in one with Neospora caninum. Most of the BHV-4-associated abortions were detected during the seventh month of gestation. It was concluded that an active BHV-4 infection was present among cows that aborted on the farms examined. The high prevalence of the BHV-4 genome in abortion material suggests that this virus may have cause the abortions. Further studies and examinations are needed to establish causative connection between presence of BHV-4 and abortion.
Assuntos
Aborto Animal/virologia , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/isolamento & purificação , Infecções Tumorais por Vírus/veterinária , Feto Abortado/virologia , Animais , Bovinos , DNA Viral/isolamento & purificação , Infecções por Herpesviridae/virologia , Prevalência , Infecções Tumorais por Vírus/virologiaRESUMO
Fetal tissues and placenta from a third trimester Mediterranean miniature donkey (Equus asinus) abortion were submitted to the Washington State University, Washington Animal Disease Diagnostic Laboratory for abortion diagnosis. Microscopic examination of formalin-fixed tissues revealed multifocal necrotizing placentitis. Several cells within the necrotic foci contained large, eosinophilic, intranuclear inclusions. Virus isolation from fresh, frozen placenta identified a cytopathic, syncytia-forming virus. Polymerase chain reaction (PCR) from the cultured virus using degenerate universal herpesvirus primers amplified a 699-base pair portion of the DNA polymerase gene. The PCR amplicon had 96.7% nucleotide identity with the DNA polymerase gene of Equid herpesvirus 7 (EHV-7; asinine herpesvirus 2), a gammaherpesvirus. An identical sequence was obtained when the same degenerate herpesvirus primers were used for PCR on the formalin-fixed placenta. Additionally, the amplicon had complete identity with short sequences of asinine herpesviruses that have been published in association with interstitial pneumonia in donkeys. EHV-7 has previously been isolated from nasal secretions of normal donkeys and mules. Our report describes a case of abortion associated with EHV-7 or a similar virus.
Assuntos
Aborto Animal/virologia , Equidae , Gammaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/veterinária , Animais , DNA Polimerase Dirigida por DNA/genética , Gammaherpesvirinae/genética , Infecções por Herpesviridae/virologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA/veterinária , Proteínas Virais/genéticaRESUMO
Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle with respiratory infections, vulvovaginitis, mastitis, abortions, endometritis and from apparently healthy animals throughout the world. Although it has not yet been established as causal agent of a specific disease entity, it is primarily associated with reproductive disorders of cattle. This virus can infect a wide range of species, either in vivo or in vitro. Two groups of prototype strains were originated from the first isolates: the DN599-type strains (American group) and the Movar-type strains (European group). In Argentina, BoHV-4 was isolated and characterized in 2007 from vaginal discharge samples taken from cows that had aborted. So far, more than 40 isolates, mainly associated with aborting bovine females have been registered in our country.
Assuntos
Aborto Animal/virologia , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/isolamento & purificação , Infecções Tumorais por Vírus/veterinária , Aborto Animal/epidemiologia , Animais , Anticorpos Antivirais/sangue , Apoptose , Argentina/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Causalidade , Efeito Citopatogênico Viral , Endométrio/virologia , Feminino , Genoma Viral , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/classificação , Herpesvirus Bovino 4/patogenicidade , Herpesvirus Bovino 4/fisiologia , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Transtornos Puerperais/veterinária , Transtornos Puerperais/virologia , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologia , Tropismo Viral , Virulência , Ativação ViralRESUMO
In spite of extensive research, the mechanisms of reproductive disease associated with Porcine Reproductive and Respiratory Syndrome virus (PRRSv) are still poorly understood. The objectives of this large scale study were to evaluate associations between viral load and fetal preservation, determine the impact of type 2 PRRSv on fetal weights, and investigate changes in ORF5 PRRSv genome in dams and fetuses during a 21-day period following challenge. At gestation day 85 (±1), 114 gilts were experimentally infected with type 2 PRRSv, while 19 gilts served as reference controls. At necropsy, fetuses were categorized according to their preservation status and tissue samples were collected. PRRSv RNA concentrations were measured in gilt serum collected on days 0, 2, 6, and 21 post-infection, as well as in gilt and fetal tissues collected at termination. Fetal mortality was 41±22.8% in PRRS infected litters. Dead fetuses appeared to cluster in some litters but appeared solitary or random in others. Nine percent of surviving piglets were meconium-stained. PRRSv RNA concentration in fetal thymus, fetal serum and endometrium differed significantly across preservation category and was greatest in tissues of meconium-stained fetuses. This, together with the virtual absence of meconium staining in non-infected litters indicates it is an early pathological condition of reproductive PRRS. Viral load in fetal thymus and in fetal serum was positively associated with viral load in endometrium, suggesting the virus exploits dynamic linkages between individual maternal-fetal compartments. Point mutations in ORF5 sequences from gilts and fetuses were randomly located in 20 positions in ORF5, but neither nucleotide nor amino acid substitutions were associated with fetal preservation. PRRSv infection decreased the weights of viable fetuses by approximately 17%. The considerable variation in gilt and fetal outcomes provides tremendous opportunity for more detailed investigations of potential mechanisms and single nucleotide polymorphisms associated with fetal death.
Assuntos
Aborto Animal/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Proteínas do Envelope Viral/genética , Animais , Endométrio/virologia , Feminino , Peso Fetal , Estudos de Associação Genética , Genótipo , Idade Gestacional , Fenótipo , Polimorfismo de Nucleotídeo Único , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Gravidez , RNA Viral/genética , Análise de Sequência de DNA , Sus scrofa , Suínos , Timo/virologia , Carga ViralRESUMO
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.
Assuntos
Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/genética , Aborto Animal/virologia , Animais , Argentina , Bovinos , Doenças dos Bovinos/genética , Linhagem Celular , Feminino , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Gravidez , Vagina/virologia , Esfregaço Vaginal/veterináriaRESUMO
Equine herpesvirus-1 (EHV-1) can be classified into distinct groups by single nucleotide polymorphisms (SNPs) in their genomes. Only a few of these can be associated with a special attribute of the virus. Differences in the ORF30 region can determine the neuropathogenic potential, while by substitutions in the ORF68 region several strain groups can be made. In previous studies no connection was found between the neuropathogenic potential and the SNPs in ORF68, but the occurrence of members of distinct groups in different outbreaks can facilitate epidemiological investigations because the geographical distribution of a particular group is very often specific. The present study aimed at the molecular examination and grouping of 35 EHV-1 strains isolated from aborted equine fetuses in Hungary between 1977 and 2008. Genotyping was based on the comparison of nucleotide sequences of a polymorphic segment located in the ORF68 region, which had previously been found to be a useful tool for classification. After sequencing this region, the Hungarian EHV-1 isolates could be classified into seven groups. Only 23 of the 35 isolates belonged to the formerly described groups, while the SNPs of 12 isolates diverged, and four new groups could be set up. In addition, phylogenetic analysis was performed to compare the ORF68 sequences of the Hungarian strains with the sequences of isolates from Europe, America and Australia. The number of newly formed groups suggests that the further analysis of unknown EHV-1 isolates would involve the emergence of extended numbers of new groups, which can impair the usability of this grouping method.
Assuntos
Aborto Animal/virologia , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Polimorfismo de Nucleotídeo Único , Proteínas do Envelope Viral/genética , Feto Abortado/virologia , Animais , Sequência de Bases , Genótipo , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/epidemiologia , Cavalos , Hungria/epidemiologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , Estudos RetrospectivosRESUMO
Equid herpesvirus 1 (EHV-1) infection has a signifcant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identifcation of specifc EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-fve EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplifed and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.
La infección por Herpesvirus equino 1 (EHV-1) tiene un signifcativo impacto económico en la producción equina mundial al causar abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos. La identifcación de genes específcos relacionados con la virulencia y patogenicidad de este virus ha sido el propósito de varios grupos de investigación. En este trabajo se analizaron diferentes regiones genómicas de cepas argentinas de EHV-1 para determinar la posible relación entre la estructura genómica y la virulencia o los signos clínicos producidos. Veinticinco cepas aisladas de diferentes casos clínicos observados entre los años 1979 y 2007 y dos cepas de referencia fueron amplifcadas y secuenciadas. El alineamiento de las secuencias se realizó con el programa Clustal X versión 1.92; el programa Bio-Edit versión 7.05 permitió deducir la secuencia de aminoácidos. Solo se observaron cambios menores, no se encontraron variaciones que pudieran estar relacionadas con la diferencia de virulencia observada previamente en el modelo ratón. No se hallaron variantes genómicas. Las regiones genómicas analizadas no permitieron diferenciar cepas abortigénicas de aquellas aisladas de muertes neonatales.
Assuntos
Animais , Camundongos , Genoma Viral , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Sequência de Aminoácidos , Aborto Animal/epidemiologia , Aborto Animal/virologia , Argentina/epidemiologia , Sequência de Bases , DNA Viral/genética , Genes Virais , Cavalos , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/epidemiologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Virulência/genéticaAssuntos
Encefalomielite/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/virologia , Reação em Cadeia da Polimerase/veterinária , Aborto Animal/virologia , Animais , Encéfalo/virologia , Primers do DNA , Sondas de DNA/normas , DNA Polimerase Dirigida por DNA/genética , Encefalomielite/sangue , Encefalomielite/diagnóstico , Encefalomielite/virologia , Feminino , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/sangue , Cavalos , Placenta/virologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Gravidez , Sensibilidade e Especificidade , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.
Assuntos
Aborto Animal/veterinária , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/isolamento & purificação , Infecções Tumorais por Vírus/veterinária , Feto Abortado/virologia , Aborto Animal/virologia , Animais , Bovinos , Sondas de DNA , DNA Viral/análise , Feminino , Infecções por Herpesviridae/virologia , Imuno-Histoquímica , Rim/patologia , Rim/virologia , Fígado/patologia , Fígado/virologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Gravidez , Baço/patologia , Baço/virologia , Infecções Tumorais por Vírus/virologiaRESUMO
Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5-99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3-84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.
Assuntos
Vírus da Encefalomiocardite/genética , Sus scrofa/virologia , Aborto Animal/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Cardiovirus/veterinária , Infecções por Cardiovirus/virologia , China , Primers do DNA/genética , DNA Viral/genética , Encefalomielite Enzoótica Suína , Vírus da Encefalomiocardite/classificação , Vírus da Encefalomiocardite/isolamento & purificação , Feminino , Dados de Sequência Molecular , Gravidez , Complicações Infecciosas na Gravidez/veterinária , Complicações Infecciosas na Gravidez/virologia , Doenças dos Suínos/virologia , Proteínas Estruturais Virais/genéticaRESUMO
Diagnostic studies on aborted placentas of cattle usually do not determine any reason for abortions. In this paper, five bovine herpesviruses (BoHVs) and some bacteriological agents were investigated by several methods in 33 aborted bovine placentas. Inclusion bodies, PCR and in situ DNA hybridization proved the presence of BoHV-4 DNA in six (18.18%) tested placentas. Positive DNA hybridization signal localized BoHV-4 DNA to placental epithelial cells macrophages and lymphocytes. By destroying epithelial cells and provoking local immune response BoHV-4 infection may inhibit physiological functions of the placenta during gestation. This finding is a further sign that BoHV-4 plays an active role in reproductive disorders of cattle.
Assuntos
Aborto Animal/virologia , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/isolamento & purificação , Doenças Placentárias/veterinária , Infecções Tumorais por Vírus/veterinária , Feto Abortado , Animais , Bovinos , DNA Viral/química , DNA Viral/genética , Feminino , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/genética , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Linfócitos/patologia , Macrófagos/patologia , Doenças Placentárias/virologia , Reação em Cadeia da Polimerase/veterinária , Gravidez , Infecções Tumorais por Vírus/virologiaRESUMO
Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.
Assuntos
Herpesvirus Equídeo 1/isolamento & purificação , Cavalos/virologia , Aborto Animal/epidemiologia , Aborto Animal/virologia , Animais , Sequência de Bases , Clonagem Molecular , República Tcheca/epidemiologia , DNA Viral/genética , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Dados de Sequência Molecular , Gravidez , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas do Envelope Viral/genéticaRESUMO
Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR-ELISA system. A competitive PCR-ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR-ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms.
Assuntos
Aborto Animal/diagnóstico , Infecções por Herpesviridae/diagnóstico , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase/métodos , Aborto Animal/virologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Cavalos , Masculino , Sondas de Oligonucleotídeos , Plasmídeos , Gravidez , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas do Envelope Viral/genéticaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is a common cause of reproductive failure and abortion in swine. The mechanism of abortion is not fully defined, and the effect of the virus on luteal function has not been explored. In this study, we exposed late-term pregnant swine to varied doses of PRRSV strain NADC-8 and evaluated effects on ovarian function by serial determination of plasma progesterone levels and by microscopic evaluation of ovarian pathologic alterations combined with immunohistochemistry and in situ hybridization to detect PRRSV antigen. We identified no specific trend in plasma progesterone level associated with PRRSV infection status and no microscopic ovarian lesions. PRRSV antigen was not demonstrated in ovarian tissues by immunohistochemistry or in situ hybridization at necropsy 21 days postexposure. Based on these findings, it does not appear that either a direct or an indirect effect on luteal function contributes to PRRSV-induced abortion.
Assuntos
Idade Gestacional , Ovário/fisiopatologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Progesterona/sangue , Doenças dos Suínos/virologia , Aborto Animal/virologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Antígenos Virais/imunologia , Feminino , Imuno-Histoquímica , Hibridização In Situ , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Gravidez , Suínos , Doenças dos Suínos/fisiopatologiaRESUMO
The polymerase chain reaction and DNA in-situ hybridization were used to study sections of uterine tissue collected from mares near the time of abortion due to equid herpesvirus-1 (EHV-1) infection. These techniques revealed viral nucleic acids in endothelial cells of endometrial arterioles, in accordance with previously published immunohistological data. In addition, however, they revealed nucleic acids in cellular debris within endometrial glands and diffusing across the placenta at sites of microcotyledonary infarction. Perivascular leucocytes were generally negative for viral DNA, despite marked perivascular cuffing. These data provided further support for the central role of the vascular endothelial cell in the pathogenesis of EHV-1 abortion and demonstrated direct transplacental spread of nucleic acids at sites of microcotyledonary infarction and across the endometrial glands in the vicinity of vascular lesions.
Assuntos
Aborto Animal/virologia , DNA Viral/análise , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/virologia , Aborto Séptico/veterinária , Animais , Arteríolas/patologia , Arteríolas/virologia , Endométrio/irrigação sanguínea , Endométrio/patologia , Endométrio/virologia , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Feminino , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/patologia , Cavalos , Hibridização In Situ , Reação em Cadeia da Polimerase , GravidezRESUMO
Virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization methods were compared for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Seven aborted fetuses and 6 stillborn piglets naturally infected with PRRSV were used in the study. Viral antigen and viral nucleic acid were detected in macrophages and dendritic cells in the spleen, tonsil, lymph nodes, and thymus; in macrophages of liver, heart, and lung; and in endothelial cells and myocytes of the heart. Viral antigen and viral nucleic acid were most consistently detected in the spleen. Of the 13 samples, 6 were positive for PRRSV by all 4 techniques. Four (31%) samples were positive for PRRSV by RT-PCR, in situ hybridization, and virus isolation. Two (15%) samples were positive for PRRSV by virus isolation, RT-PCR, and in situ hybridization. One (8%) was positive for PRRSV by virus isolation and RT-PCR. The RT-PCR identified the presence of PRRSV more frequently than the other methods. However, when only formalin-fixed tissues are submitted, immunohistochemistry and in situ hybridization would be useful methods for the detection of PRRSV antigen and nucleic acid.
Assuntos
Aborto Animal/virologia , Morte Fetal/veterinária , Síndrome Respiratória e Reprodutiva Suína/patologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Aborto Animal/patologia , Animais , Feminino , Morte Fetal/patologia , Morte Fetal/virologia , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Macrófagos/patologia , Macrófagos/virologia , Reação em Cadeia da Polimerase/métodos , Síndrome Respiratória e Reprodutiva Suína/embriologia , Gravidez , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Bovine viral diarrhea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. This study was designed to establish the effects of BVDV infection on estradiol, progesterone and PGF2alpha secretion in the cow. Seven BVDV-free cows were challenged with non-cytopathogenic BVDV (strain Pe 515: 5x10(6) tissue culture infected dose50) so that peak viremia occurred during the initial phase of luteal development in a synchronized estrous cycle. Ovulation was also synchronized in 7 sham-infected animals. Within 2 wk of inoculation, viremia, leukopenia and serum neutralizing antibodies were recorded in all of the BVDV-infected cows but not the sham-infected animals. Between Day 4 and Day 9 post estrus the BVDV-infected cows had significantly (P<0.01) lower plasma estradiol levels than the sham-infected animals. However, the BVDV infection did not alter rectal temperatures, plasma progesterone concentrations or PGF2alpha secretion 17, 18 and 19 d post estrus. These data highlight a potential causal link between BVDV viremia, endocrine dysfunction and poor fertility in the cow.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/fisiopatologia , Vírus da Diarreia Viral Bovina , Dinoprosta/metabolismo , Estradiol/metabolismo , Progesterona/metabolismo , Aborto Animal/virologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Dinoprosta/sangue , Estradiol/sangue , Feminino , Infertilidade Feminina/veterinária , Infertilidade Feminina/virologia , Contagem de Leucócitos , Gravidez , Progesterona/sangueRESUMO
In an attempt to determine if pregnant mice could be protected from abortion subsequent to challenge with equine herpesvirus-1 (EHV-1) in the mouse model of EHV-1 disease, female BALB/c mice were inoculated with baculovirus-expressed EHV-1 glycoprotein B (bac-gB), wild-type baculovirus (bac-wt), rabbit kidney (RK-13) or baby hamster kidney (BHK-21) cells. Using an ELISA, antibodies against EHV-1 were detected in the serum of mice following two injections of bac-gB and were enhanced by a third injection, after which low levels of neutralising antibody were also detected. After mating, mice in the bac-gB, bac-wt and RK-immunised groups were infected intranasally with 10(7) pfu of EHV-1 on day 16 of pregnancy. All challenged mice experienced body weight loss post-infection (pi). However, postnatally, the gB-immunised group demonstrated body weight gain which was not seen in the other groups. There were no maternal deaths in the gB-immunised group but 1/6 bac-wt-immunised and 3/6 RK-immunised mice died post-challenge. Litter survival rate was significantly higher (p < 0.001) for the gB-immunised dams (54%) than that of either the bac-wt-(9%) or RK-immunised (0%) dams and the mean body weight of young from the surviving bac-wt-immunised litter was significantly (p = 0.021) lower than either the gB-immunised group or the BHK-immunised unchallenged group at 10 days of age. The virus was not isolated from any foetus from a gB-immunised dam. However, the virus was detected in 9% of foetuses from bac-wt-immunised and 21% of foetuses from RK-immunised dams.