RESUMO
Neurological disorders in ruminants have an important impact on veterinary health, but very few host-specific in vitro models have been established to study diseases affecting the nervous system. Here we describe a primary neuronal dorsal root ganglia (DRG) culture derived from calves after being conventionally slaughtered for food consumption. The study focuses on the in vitro characterization of bovine DRG cell populations by immunofluorescence analysis. The effects of various growth factors on neuron viability, neurite outgrowth and arborisation were evaluated by morphological analysis. Bovine DRG neurons are able to survive for more than 4 weeks in culture. GF supplementation is not required for neuronal survival and neurite outgrowth. However, exogenously added growth factors promote neurite outgrowth. DRG cultures from regularly slaughtered calves represent a promising and sustainable host specific model for the investigation of pain and neurological diseases in bovines.
Assuntos
Gânglios Espinais/patologia , Animais , Bovinos , Células Cultivadas , Técnicas de Cocultura , Microscopia Eletrônica de TransmissãoRESUMO
AIMS: Isoprene (2-methyl-1,3-butadiene; C(5) H(8) ) is naturally produced by photosynthesis and emitted in the atmosphere by the leaves of many herbaceous, deciduous and woody plants. Fermentative yeast and fungi (Ascomycota) are not genetically endowed with the isoprene production process. The work investigated whether Ascomycota can be genetically modified and endowed with the property of constitutive isoprene production. METHODS AND RESULTS: Two different strategies for expression of the IspS gene in Saccharomyces cerevisiae were employed: (i) optimization of codon usage of the IspS gene for specific expression in S. cerevisiae and (ii) multiple independent integrations of the IspS gene in the rDNA loci of the yeast genome. Copy number analysis showed that IspS transgenes were on the average incorporated within about 25% of the endogenous rDNA. Codon use optimization of the Pueraria montana (kudzu vine) IspS gene (SckIspS) for S. cerevisiae showed fivefold greater expression of the IspS protein compared with that of nonoptimized IspS (kIspS). With the strategies mentioned earlier, heterologous expression of the kudzu isoprene synthase gene (kIspS) in S. cerevisiae was tested for stability and as a potential platform of fermentative isoprene production. The multi-copy IspS transgenes were stably integrated and expressed for over 100 generations of yeast cell growth and constitutively produced volatile isoprene hydrocarbons. Secondary chemical modification of isoprene to a number of hydroxylated isoprene derivatives in the sealed reactor was also observed. CONCLUSION: Transformation of S. cerevisiae with the Pueraria montana var. lobata (kudzu vine) isoprene synthase gene (IspS) conferred to the yeast cells constitutive isoprene hydrocarbons production in the absence of adverse or toxic effects. SIGNIFICANCE AND IMPACT OF THE STUDY: First-time demonstration of constitutive isoprene hydrocarbons production in a fermentative eukaryote operated through the mevalonic acid pathway. The work provides concept validation for the utilization of S. cerevisiae, as a platform for the production of volatile hydrocarbon biofuels and chemicals.
Assuntos
Alquil e Aril Transferases/genética , Engenharia Genética , Hemiterpenos/biossíntese , Saccharomyces cerevisiae/metabolismo , Butadienos , Códon , DNA Ribossômico/genética , Fermentação , Pentanos , Plasmídeos , Pueraria/enzimologia , Pueraria/genética , Saccharomyces cerevisiae/genética , Transformação Genética , TransgenesRESUMO
AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.
Assuntos
Tronco Encefálico , Encéfalo/metabolismo , Caderinas/metabolismo , Plexo Corióideo/metabolismo , Encefalite/veterinária , Listeria monocytogenes/metabolismo , Listeriose/veterinária , Animais , Encéfalo/microbiologia , Tronco Encefálico/metabolismo , Bovinos , Plexo Corióideo/microbiologia , Encefalite/metabolismo , Encefalite/microbiologia , Cabras , Listeriose/metabolismo , Listeriose/microbiologia , Dados de Sequência Molecular , OvinosRESUMO
Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.
Assuntos
Bovinos/genética , Encefalopatia Espongiforme Bovina/genética , Predisposição Genética para Doença , Sulfotransferases/genética , Animais , Encefalopatia Espongiforme Bovina/metabolismo , Proteínas PrPC/metabolismo , Sulfotransferases/metabolismo , Carboidrato SulfotransferasesRESUMO
Abnormal patterns of cell death, including increased apoptosis, can influence homeostasis of ligaments and could be involved in the pathogenesis of cranial cruciate ligament (CCL) rupture. Increased nitric oxide (NO) production has been implicated as a stimulus to increased apoptosis in articular cartilage. This study investigated apoptotic cell death in ruptured canine CCL (CCL group, n = 15), in ruptured CCL of dogs treated with oral L-N6-(1-iminoethyl)-lysine (L-NIL), a selective NO-synthetase(NOS)-inhibitor, (L-NIL group, n = 15) and compared the results with normal canine CCL (control group, n = 10). Orally administered L-NIL at a dosage of 25mg/m2 of body surface area was effective in inhibiting NO production in the articular cartilage of dogs in the L-NIL group, but it did not significantly influence the increased quantity of apoptotic cells found in ruptured CCL specimens. The results of this study suggest that apoptosis of ligamentocytes in the canine CCL is not primarily influenced by increased NO production within the stifle joint.
Assuntos
Ligamento Cruzado Anterior/patologia , Doenças do Cão/patologia , Coxeadura Animal/patologia , Lisina/análogos & derivados , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Ruptura Espontânea/veterinária , Animais , Ligamento Cruzado Anterior/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Doenças do Cão/tratamento farmacológico , Doenças do Cão/enzimologia , Cães , Inibidores Enzimáticos/uso terapêutico , Lisina/uso terapêutico , Óxido Nítrico Sintase/antagonistas & inibidores , Ruptura Espontânea/tratamento farmacológicoRESUMO
Cell-based vaccination strategies to induce functional tumor-specific T cells in cancer patients have focused on using autologous dendritic cells. An alternative approach is to use RNA-loaded CD40 activated B cells (CD40-B) that are highly efficient antigen-presenting cells capable of priming naive T cells, boosting memory T-cell responses and breaking tolerance to tumor antigens. The use of tumor RNA as the antigenic payload allows for gene transfer without viruses or vectors and permits major histocompatibility complex (MHC)-independent, multiple-antigen targeting. Here, we use CD40L transfected K562 cells to generate functional CD40-B cells from the peripheral blood of humans and dogs. Testing of RNA-loaded CD40-B cells in dogs allows not only for its development in veterinary medicine but also for determination of its safety and efficacy in a large animal model of spontaneous cancer prior to initiation of human clinical trials. We found that CD40-B cells from healthy humans, healthy dogs and tumor-bearing dogs express increased levels of immune molecules such as MHC and CCR7. Moreover, RNA-loaded CD40-B cells induce functional, antigen-specific T cells from healthy dogs and dogs with lymphoma. These findings pave the way for immunotherapy trials using tumor RNA-loaded CD40-B cells to stimulate antitumor immunity in a large animal model of spontaneous neoplasia.
Assuntos
Doenças do Cão/terapia , Terapia Genética/métodos , Imunoterapia Adotiva/métodos , Linfoma/terapia , Linfoma/veterinária , RNA Neoplásico/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Sequência de Bases , Antígenos CD40/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Doenças do Cão/imunologia , Cães , Humanos , Imunofenotipagem , Ativação Linfocitária , Linfoma/imunologia , Dados de Sequência Molecular , Receptores CCR7/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , TransfecçãoRESUMO
Signaling lymphocyte activation molecule (SLAM) or CD150 can function as a receptor for the canine distemper virus (CDV) in vitro. The expression of SLAM was studied using immunohistochemistry in order to evaluate the presence and distribution of the receptor in dogs in vivo. Additionally, receptor expression was assessed after experimental infection of dogs with CDV. In 7 control dogs without distemper virus, the receptor was found in various tissues, mostly on cells morphologically identified as lymphocytes and macrophages. In 7 dogs with early distemper lesions characterized by presence of the virus, higher numbers of SLAM-expressing cells were found in multiple tissues recognized as targets of CDV compared with those in control dogs. These findings suggest that SLAM, a putative distemper receptor, is expressed in dogs in vivo. Additionally, virus infection is associated with up-regulation of SLAM, potentially causing an amplification of virus in the host.
Assuntos
Antígenos CD/metabolismo , Vírus da Cinomose Canina/metabolismo , Cinomose/virologia , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Cães , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Regulação para Cima , Células VeroRESUMO
CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.
Assuntos
Doenças do Sistema Nervoso Central/veterinária , Doenças do Cão/imunologia , Antígenos Comuns de Leucócito/metabolismo , Microglia/imunologia , Transdução de Sinais , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/imunologia , Doenças do Sistema Nervoso Central/patologia , Doenças do Cão/patologia , Cães , Feminino , Citometria de Fluxo/métodos , Citometria de Fluxo/veterinária , Antígenos Comuns de Leucócito/biossíntese , Leucócitos Mononucleares/imunologia , Masculino , Microglia/citologiaRESUMO
Switzerland is controlling Transmissible Spongiform Encephalopathies (TSE) in cattle (BSE) and small ruminants (scrapie). Since BSE is potentially transmissible to sheep, goats or pigs through feeding of contaminated meat and bone meal, implementation of an active surveillance programme for TSE in these species is discussed. The aim of this pilot study was to obtain preliminary data on the prevalence ofTSE and other neurological disorders in these populations. For that purpose, a total of 398 perished and 825 slaughtered adult small ruminants and pigs was examined for the presence of neuropathological changes. None of these animals revealed positive for TSE. However, the investigations demonstrated that perished sheep and goats exhibited a higher prevalence of relevant neuropathological changes when compared with slaughtered animals. From these results, it is concluded that perished small ruminants are probably a risk population for TSE and should be considered as target populations for an active surveillance programme.
Assuntos
Encefalopatia Espongiforme Bovina/epidemiologia , Doenças Priônicas/veterinária , Scrapie/epidemiologia , Animais , Bovinos , Contaminação de Alimentos/prevenção & controle , Cabras , Projetos Piloto , Doenças Priônicas/epidemiologia , Fatores de Risco , Vigilância de Evento Sentinela/veterinária , Ovinos , Especificidade da Espécie , Suínos , Suíça/epidemiologiaRESUMO
Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.
Assuntos
Ceratopogonidae/imunologia , Doenças dos Cavalos/imunologia , Hipersensibilidade/veterinária , Mordeduras e Picadas de Insetos/veterinária , Animais , Western Blotting/veterinária , Estudos de Coortes , Feminino , Cavalos , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/imunologia , Mordeduras e Picadas de Insetos/imunologia , Proteínas e Peptídeos Salivares/imunologia , Estações do AnoRESUMO
REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.
Assuntos
Doenças dos Cavalos/diagnóstico , Hipersensibilidade/veterinária , Testes Imunológicos/veterinária , Mordeduras e Picadas de Insetos/veterinária , Leucotrienos/biossíntese , Dermatopatias/veterinária , Animais , Feminino , Seguimentos , Liberação de Histamina , Doenças dos Cavalos/imunologia , Cavalos , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Testes Imunológicos/métodos , Testes Imunológicos/normas , Mordeduras e Picadas de Insetos/diagnóstico , Mordeduras e Picadas de Insetos/imunologia , Leucócitos/metabolismo , Masculino , Recidiva , Reprodutibilidade dos Testes , Estações do Ano , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Fatores Sexuais , Dermatopatias/diagnóstico , Dermatopatias/imunologiaRESUMO
Small ruminants infected with scrapie show a large range of often unspecific clinical symptoms. The most-often described signs, locomotion, sensibility and behavioural disorders and emaciation, rarely occur together, and cases have been described in which only one of those signs was detectable.Thus, formulating a well-circumscribed definition of a clinical suspect case is difficult. Most animals with CNS-effecting diseases such as listeriosis, polioencephalomacia, cerebrospinal nematidiasis and enterotoxemia will, in a thorough neurological examination, show at least some scrapie-like symptoms. Among the 22 neurological field cases examined in this study, a goat with cerebral gliomatosis and hair lice showed the closest similarity to clinical scrapie. The unilateral deficiency of the cerebral nerves has potential as an clinical exclusion criterion for scrapie. However, the laboratory confirmation--or exclusion--of scrapie remains important. It thus needs to be realized that a consistent and thorough examination of neurologically diseased small ruminants (including fallen stock) is the backbone of a good surveillance system for these diseases. This should be a motivation for submitting adult sheep and goats for neuropathological examination.
Assuntos
Doenças das Cabras/diagnóstico , Doenças das Cabras/epidemiologia , Scrapie/diagnóstico , Scrapie/epidemiologia , Animais , Diagnóstico Diferencial , Feminino , Doenças das Cabras/patologia , Cabras , Incidência , Masculino , Exame Neurológico/veterinária , Scrapie/patologia , Vigilância de Evento Sentinela/veterinária , Ovinos , Suíça/epidemiologiaRESUMO
Monitoring of transmissible spongiform encephalopathy (TSE) in Swiss sheep and goats is based on the examination of animals from different sources. In this study, frequencies and proportions of the different diagnoses were compared between routinely submitted sheep and goats, notified scrapie suspects as well as fallen stock. Meningitis/ encephalitis cases were significantly more frequent (OR = 2.2) in the scrapie suspect group when compared to the routine submissions. Metabolic-toxic encephalopathy was seen more frequently within the fallen stock. Rare neurological diagnoses were more frequent among scrapie suspects and routine submissions when compared to fallen stock. Listeriosis was diagnosed equally frequent among the scrapie suspects and routine submissions but less frequent in fallen stock. Scrapie prevalence among the fallen stock and the routine submissions was 0 (zero), with 95% certainty that prevalence is < 1%. The examined animals are representative for most of the Swiss regions with considerable sheep and goat production. Continuation of the detailed neuropathological examination of small ruminants from these three groups, substituted by actively testing a sufficiently large sample of fallen stock and possibly also healthy-slaughtered adult sheep and goats for transmissible spongiform encephalopathies would ensure a good surveillance within the small ruminant population.
Assuntos
Doenças das Cabras/diagnóstico , Doenças do Sistema Nervoso/veterinária , Doenças dos Ovinos/diagnóstico , Animais , Diagnóstico Diferencial , Feminino , Doenças das Cabras/epidemiologia , Cabras , Masculino , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/epidemiologia , Exame Neurológico/veterinária , Razão de Chances , Prevalência , Doenças Priônicas/diagnóstico , Doenças Priônicas/epidemiologia , Doenças Priônicas/veterinária , Fatores de Risco , Scrapie/diagnóstico , Scrapie/epidemiologia , Vigilância de Evento Sentinela/veterinária , Ovinos , Doenças dos Ovinos/epidemiologia , Suíça/epidemiologiaRESUMO
Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.
Assuntos
Vírus da Cinomose Canina/patogenicidade , Cinomose/virologia , Doenças do Cão/virologia , Dermatoses do Pé/veterinária , Queratinócitos/virologia , Animais , Antígenos Virais/metabolismo , Proliferação de Células , Células Cultivadas , Cinomose/patologia , Doenças do Cão/patologia , Cães , Dermatoses do Pé/patologia , Dermatoses do Pé/virologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Queratinócitos/citologia , Queratinócitos/fisiologia , Antígeno Ki-67/metabolismo , Nucleoproteínas/metabolismo , RNA ViralRESUMO
Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.
Assuntos
Obstrução das Vias Respiratórias/imunologia , Obstrução das Vias Respiratórias/veterinária , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Doenças dos Cavalos/imunologia , Obstrução das Vias Respiratórias/sangue , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/genética , Feminino , Cavalos , Separação Imunomagnética/veterinária , Masculino , Poaceae/imunologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Estatísticas não ParamétricasRESUMO
Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.
Assuntos
Vírus da Cinomose Canina/fisiologia , Cinomose/metabolismo , Doenças do Cão/metabolismo , Epiderme/metabolismo , Dermatoses do Pé/veterinária , NF-kappa B/metabolismo , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Cinomose/patologia , Cinomose/virologia , Doenças do Cão/patologia , Doenças do Cão/virologia , Cães , Epiderme/patologia , Epiderme/virologia , Dermatoses do Pé/patologia , Dermatoses do Pé/virologia , Imuno-Histoquímica/veterinária , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/virologia , Organismos Livres de Patógenos Específicos , Fator de Transcrição RelARESUMO
The susceptibility of red foxes (Vulpes vulpes) to European bat lyssavirus type 1 (EBLV-1) infection was examined. Eight foxes were inoculated intramuscularly (i.m.) with 10(4.9) foci-forming units (FFU) (n = 4) and 10(5.1) FFU (n = 4) and observed for up to 90 days. All foxes showed manifestations of a neurologic disorder (e.g. seizures, myoclonus, agitation), starting as early as 5 days post-infection (p.i.). Subsequently, all animals showed improvement followed by one or more relapses. One fox was killed 3 days after it recovered, 26 days post-infection. Two other foxes were also killed 38 and 54 days post-infection after severe neurologic signs returned. All foxes developed a humoral immune response against EBLV-1 as determined in serum and brain tissues. However, no rabies virus antigen was detected in the brain, other tissues and secretions examined (e.g. salivary gland, saliva, tonsils, lungs) by using different standard diagnostic techniques [fluorescent antibody test, reverse transcription polymerase chain reaction (RT-PCR), rabies tissue culture inoculation test], with the exception of one fox in which EBLV-1 RNA was detected by RT-PCR in only the spinal cord. Brain tissues showed moderate to severe multifocal, mononuclear encephalomyelitis in the three foxes that were killed during the observation period, although no EBLV-1 virus was detectable in these tissues.
Assuntos
Raposas , Lyssavirus/patogenicidade , Raiva/veterinária , Animais , Anticorpos Antivirais/análise , Suscetibilidade a Doenças/veterinária , Europa (Continente) , Imunofluorescência/veterinária , Lyssavirus/classificação , Lyssavirus/genética , Lyssavirus/imunologia , Lyssavirus/isolamento & purificação , RNA Viral/análise , Raiva/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.
Assuntos
Códon de Iniciação , Vírus da Cinomose Canina/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Vírus da Cinomose Canina/fisiologia , Genes Virais , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Plasmídeos , Biossíntese de Proteínas , RNA Mensageiro/fisiologia , RNA Viral/genética , RNA Viral/fisiologiaRESUMO
The proliferation of footpad keratinocytes of canine distemper virus (CDV)-infected dogs was investigated. Footpads of 19 dogs inoculated experimentally with a virulent distemper strain (A75/17) and of two noninoculated control dogs were collected at necropsy. Dogs were divided into four groups according to results of the postmortem examination: dogs with severe distemper (group 1), dogs with mild distemper (group 2), inoculated dogs without distemper (group 3) and noninoculated dogs (group 4). There was no distinct difference of epidermal thickness among the four groups. Infection of the footpad epidermis with CDV was demonstrated using immunohistochemistry for viral nucleoprotein and in situ hybridization for nucleoprotein messenger ribonucleic acid (mRNA). Only group 1 dogs had viral antigen and mRNA in the footpad epidermis with the same distribution. Footpad epidermis of group 1 dogs had more mitotic figures in the basal layer, and significantly more basal keratinocytes were positive for the proliferation markers Ki-67 and proliferating cell nuclear antigen. Double-staining for Ki-67 and viral nucleoprotein identified rare double-labeled basal keratinocytes. These findings suggest that the presence of CDV particles in the footpad epidermis is associated with keratinocyte proliferation.