RESUMO
Apart from prion protein genotype, the factors determining the host range and susceptiblity for specific transmissible spongiform encephalopathy agents remain unclear. It is known that bovine atypical L-BSE can transmit to a range of species including primates and humanised transgenic mice. It is important, therefore, that there is as broad an understanding as possible of how such isolates might present in food animal species and how robust they are on inter- and intra-species transmission to inform surveillance sytems and risk assessments. This paper demonstrates that L-BSE can be intracerebrally transmitted to sheep of several genotypes, with the exception of ARR/ARR animals. Positive animals mostly present with a cataplectic form of disease characterized by collapsing episodes and reduced muscle tone. PrP accumulation is confined to the nervous system, with the exception of one animal with lymphoreticular involvement. In Western blot there was maintenance of the low molecular mass and glycoform profile associated with L-BSE, irrespective of ovine host genotype, but there was a substantially higher N-terminal antibody signal relative to the core-specific antibody, which is similar to the ratio associated with classical scrapie. The disease phenotype was maintained on experimental subpassage, but with a shortened survival time indicative of an original species barrier and subsequent adaptation. Passive surveillance approaches would be unlikely to identify such cases as TSE suspects, but current statutory active screening methods would be capable of detecting such cases and classifying them as unusual and requiring further investigation if they were to occur in the field.
Assuntos
Encefalopatia Espongiforme Bovina/transmissão , Doenças dos Ovinos/transmissão , Animais , Western Blotting/veterinária , Encéfalo/patologia , Bovinos , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/patologia , Ensaio de Imunoadsorção Enzimática/veterinária , Fenótipo , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/mortalidade , Doenças dos Ovinos/patologiaRESUMO
BACKGROUND: Atypical bovine spongiform encephalopathies (BSEs), classified as H-type and L-type BSE based on the Western immunoblot profiles, are naturally occurring diseases in cattle, which are phenotypically different to classical BSE. Transmission studies in cattle using the intracerebral route resulted in disease where the phenotypes were maintained irrespective of BSE type but clinically affected cattle with a shorter survival time displayed a nervous form whereas cattle with a longer survival time displayed a dull form. A second transmission study is reported here where four cattle were intracerebrally inoculated with brain tissue from experimentally infected cattle presenting with either the nervous or dull form of H- or L-type BSE to determine whether the phenotype is maintained. RESULTS: The four inoculated cattle were culled at 16.5-19.5 months post inoculation after presenting with difficulty getting up, a positive scratch response (all) and dullness (three cattle), which was not observed in two non-inoculated control cattle, each housed with either group of inoculated cattle. Only the inoculated cattle had detectable prion protein in the brain based on immunohistochemical examination, and the Western immunoblot profile was consistent with the H-type or L-type BSE of the respective donor cattle. CONCLUSIONS: Second passage of H-type and L-type BSE in cattle produced a TSE where the majority of cattle displayed the dull form regardless of clinical disease form of the donor cattle. The pathological and molecular phenotypes of H- and L-type BSE were maintained.
Assuntos
Encefalopatia Espongiforme Bovina/patologia , Animais , Western Blotting/veterinária , Encéfalo/patologia , Bovinos , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/mortalidade , Encefalopatia Espongiforme Bovina/transmissão , Feminino , Masculino , Fenótipo , Análise de SobrevidaRESUMO
BACKGROUND: The majority of atypical bovine spongiform encephalopathy (BSE) cases so far identified worldwide have been detected by active surveillance. Consequently the volume and quality of material available for detailed characterisation is very limiting. Here we report on a small transmission study of both atypical forms, H- and L-type BSE, in cattle to provide tissue for test evaluation and research, and to generate clinical, molecular and pathological data in a standardised way to enable more robust comparison of the two variants with particular reference to those aspects most relevant to case ascertainment and confirmatory diagnosis within existing regulated surveillance programmes. RESULTS: Two groups of four cattle, intracerebrally inoculated with L-type or H-type BSE, all presented with a nervous disease form with some similarities to classical BSE, which progressed to a more dull form in one animal from each group. Difficulty rising was a consistent feature of both disease forms and not seen in two BSE-free, non-inoculated cattle that served as controls. The pathology and molecular characteristics were distinct from classical BSE, and broadly consistent with published data, but with some variation in the pathological characteristics. Both atypical BSE types were readily detectable as BSE by current confirmatory methods using the medulla brain region at the obex, but making a clear diagnostic distinction between the forms was not consistently straightforward in this brain region. Cerebellum proved a more reliable sample for discrimination when using immunohistochemistry. CONCLUSIONS: The prominent feature of difficulty rising in atypical BSE cases may explain the detection of naturally occurring cases in emergency slaughter cattle and fallen stock. Current confirmatory diagnostic methods are effective for the detection of such atypical cases, but consistently and correctly identifying the variant forms may require modifications to the sampling regimes and methods that are currently in use.
Assuntos
Encéfalo/patologia , Encefalopatia Espongiforme Bovina/patologia , Ácido 3-Hidroxibutírico/sangue , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Comportamento Animal/fisiologia , Bilirrubina/sangue , Western Blotting/veterinária , Bovinos , Eletrólitos/sangue , Encefalopatia Espongiforme Bovina/sangue , Encefalopatia Espongiforme Bovina/genética , Fibrinogênio/análise , Globulinas/análise , Glutationa Peroxidase/sangue , Haptoglobinas/análise , Imuno-Histoquímica/veterinária , Masculino , Albumina Sérica/análise , Ureia/sangue , Vitamina E/sangueRESUMO
The tuberculin skin test has been used for the diagnosis of bovine and human tuberculosis (TB) for over a hundred years. However, the specificity of the test is compromised by vaccination with the Mycobacterium bovis-derived vaccine strain bacille Calmette-Guérin (BCG). Since current promising vaccines against bovine TB are based on heterologous prime-boost combinations that include BCG, there is a need for diagnostic tests for differentiating infected from vaccinated animals (DIVA). The application of antigens such as ESAT-6 and CFP-10 for DIVA has so far been realized largely through their application in the blood-based gamma interferon release assay. In the current study, we have reassessed the potential of such antigens as skin test reagents for DIVA in cattle. A cocktail of the Mycobacterium tuberculosis complex recombinant protein antigens ESAT-6, CFP-10, MPB70, and MPB83 elicited delayed-type hypersensitivity (DTH) skin test responses in 78% of naturally infected tuberculin-positive cattle. Importantly, this cocktail induced no skin responses in BCG-vaccinated cattle despite them being sensitized for strong tuberculin responses. Further optimization of skin test antigen combinations identified that the inclusion of Rv3615c (Mb3645c) enhanced skin test sensitivity in naturally infected cattle without compromising specificity. In addition, we demonstrate for the first time the utility of synthetic peptides as promising skin test antigens for bovine TB for DIVA. Our data provide a promising basis for the future development of skin tests for DIVA with practical relevance for TB diagnosis in both veterinary and clinical settings.
Assuntos
Vacina BCG/imunologia , Técnicas Bacteriológicas/métodos , Doenças dos Bovinos/diagnóstico , Mycobacterium bovis/imunologia , Tuberculose Bovina/diagnóstico , Animais , Antígenos de Bactérias , Proteínas de Bactérias , Bovinos , Sensibilidade e Especificidade , Testes CutâneosRESUMO
BACKGROUND: Active surveillance for transmissible spongiform encephalopathies in small ruminants has been an EU regulatory requirement since 2002. A number of European countries have subsequently reported cases of atypical scrapie, similar to previously published cases from Norway, which have pathological and molecular features distinct from classical scrapie. Most cases have occurred singly in flocks, associated with genotypes considered to be more resistant to classical disease. Experimental transmissibility of such isolates has been reported in certain ovinised transgenic mice, but has not previously been reported in the natural host. Information on the transmissibility of this agent is vital to ensuring that disease control measures are effective and proportionate. RESULTS: This report presents the successful experimental transmission, in 378 days, of atypical scrapie to a recipient sheep of homologous genotype with preservation of the pathological and molecular characteristics of the donor. This isolate also transmitted to ovinised transgenic mice (Tg338) with a murine phenotype indistinguishable from that of Nor 98. CONCLUSION: This result strengthens the opinion that these cases result from a distinct strain of scrapie agent, which is potentially transmissible in the natural host under field conditions.
Assuntos
Encéfalo/metabolismo , Surtos de Doenças/veterinária , Proteínas PrPSc/metabolismo , Scrapie/transmissão , Animais , Western Blotting/veterinária , Encéfalo/patologia , Europa (Continente)/epidemiologia , União Europeia , Predisposição Genética para Doença , Genótipo , Imuno-Histoquímica/veterinária , Proteínas PrPSc/administração & dosagem , Scrapie/epidemiologia , Scrapie/genética , Scrapie/metabolismo , OvinosRESUMO
Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10)CFU/10ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.
Assuntos
Adesinas Bacterianas/fisiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/fisiologia , Flagelos/fisiologia , Doenças dos Suínos/microbiologia , Animais , Aderência Bacteriana/fisiologia , Células Cultivadas/microbiologia , Células Cultivadas/ultraestrutura , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Microscopia Eletrônica de Transmissão/métodos , Mutação/fisiologia , Técnicas de Cultura de Órgãos/veterinária , SuínosRESUMO
The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.
Assuntos
Adenoviridae/genética , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacina BCG/genética , Vacina BCG/imunologia , Mycobacterium bovis/imunologia , Vacinação/métodos , Animais , Antígenos de Bactérias/administração & dosagem , Vacina BCG/administração & dosagem , Bovinos , Vias de Administração de Medicamentos , Expressão Gênica , Masculino , Tuberculose Bovina/prevenção & controleRESUMO
Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1 log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13 °C, 14 °C, 12 °C and 10 °C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals.
Assuntos
Vírus da Febre Suína Clássica/isolamento & purificação , Peste Suína Clássica/virologia , Carne/virologia , Animais , Peste Suína Clássica/transmissão , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/patogenicidade , Genótipo , Masculino , Músculos/virologia , Suínos , Temperatura , Carga Viral/veterinária , Inativação de VírusRESUMO
Mycobacterium bovis (M. bovis) causes major economy and public health problem in numerous countries. In Great Britain, despite the use of a test-and-slaughter strategy, the incidence of bovine tuberculosis (bTB) in cattle has steadily risen in recent years. One strategy being considered to reduce the burden of bTB in cattle is the development of an efficient vaccine. The only current potentially available vaccine against tuberculosis, live attenuated M. bovis bacille Calmette-Guérin (BCG), has demonstrated variable efficacy in both humans and cattle and the development of improved vaccination strategies for cattle is a research priority. In this study we assessed the immunogenicity in cattle of two recombinant BCG strains, namely BCG Pasteur Δzmp1::aph and BCG Danish Δzmp1. By applying a recently defined predictive immune-correlate of protection (T cell memory responses measured by cultured ELISPOT), we have compared these two recombinant BCG with wild-type BCG Danish SSI. Our results demonstrated that both strains induced superior T cell memory responses compared to wild-type BCG. These data provide support for the prioritisation of testing BCG Danish Δzmp1 in vaccination/M. bovis challenge studies to determine its protective efficacy.
Assuntos
Vacina BCG/imunologia , Memória Imunológica , Linfócitos T/imunologia , Tuberculose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Bovinos , Interferon gama/imunologia , Masculino , Mycobacterium bovis , Perforina/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Vaccination is being considered as part of a sustainable strategy for the control of bovine tuberculosis (BTB) in the UK. The live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used experimentally to vaccinate cattle against BTB. However, BCG confers partial protection against BTB and therefore, there is a need to develop improved vaccines. BTB vaccine efficacy experiments require the use of biosafety level 3 facilities which are expensive to maintain, generally oversubscribed and represent a bottle neck for the testing of vaccine candidates. One indicator of the induction of protective responses would be the ability of the host's immune response to control/kill mycobacteria. In this work we have evaluated an intranodal BCG challenge for the selection of vaccine candidates at biosafety level 2 which are capable of inducing mycobactericidal responses. To our knowledge, this is the first such report. Whilst BCG only confers partial protection, it is still the standard against which other vaccines are judged. Therefore we tested the BCG intranodal challenge in BCG (Danish strain) vaccinated cattle and showed that vaccinated cattle had lower BCG cfu counts than naïve cattle at 14 and 21 days after intranodal challenge with BCG (Tokyo strain). This model could help prioritize competing TB vaccine candidates and exploration of primary and secondary immune responses to mycobacteria.
Assuntos
Linfonodos/microbiologia , Mycobacterium bovis/isolamento & purificação , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Carga Bacteriana , Bovinos , Interferon gama/sangue , Interleucina-17/sangue , Tuberculose Bovina/imunologiaRESUMO
We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.
Assuntos
Vacina BCG/imunologia , Técnicas de Laboratório Clínico/métodos , Interleucina-2/análise , Mycobacterium bovis/imunologia , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Medicina Veterinária/métodos , Animais , Vacina BCG/administração & dosagem , Bovinos , Células Cultivadas , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucócitos Mononucleares/imunologiaRESUMO
In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1ß and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.
Assuntos
Antígenos de Bactérias/imunologia , Imunidade Celular , Imunidade Humoral , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Bovinos , Proliferação de Células , Imidazóis/administração & dosagem , Imunoglobulina G/sangue , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A peptide cocktail derived from the mycobacterial antigens ESAT-6, CFP-10, and Rv3615c allowed differentiation between Mycobacterium bovis-infected and M. bovis bacillus Calmette-Guérin (BCG)-vaccinated cattle when used as a skin test reagent for a "DIVA" test (i.e., a test capable of differentiating infected and uninfected vaccinated animals). Addition of the antigen Rv3020c improves the diagnostic sensitivity without compromising specificity in the face of BCG or Johne's disease vaccination.
Assuntos
Antígenos de Bactérias , Mycobacterium bovis/imunologia , Tuberculose Bovina/diagnóstico , Medicina Veterinária/métodos , Animais , Vacina BCG/imunologia , Bovinos , Diagnóstico Diferencial , Peptídeos , Sensibilidade e Especificidade , Testes Cutâneos/métodos , Tuberculose Bovina/imunologiaRESUMO
Experiments in small animal models have indicated that intranasal vaccination confers a greater degree of protection against TB than other routes such as intradermal (i.d.) or intramuscular. In this work, using a prime-boost vaccination strategy, we have compared in cattle vaccinated with BCG as a priming vaccine the boosting capabilities of Ad5-85A delivered either via the endobronchial (e.b.) or i.d. route. We show that Ad5-85A delivered through either route induced comparable peripheral blood antigen specific responses, and that both i.d. and e.b. routes induced bronchioalveolar lavage cells (BALC) that produced antigen-specific IFNgamma. We also show that, regardless of the route of boosting, the kinetics of peripheral blood and BALC responses, as assessed by antigen specific IFNgamma production, are different with systemic responses being detectable earlier than mucosal responses. These results contribute to our understanding on how different vaccination strategies may affect different compartments of the immune response and in turn to the development of safer and more effective vaccines.
Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Imunização Secundária/métodos , Adenoviridae/genética , Administração por Inalação , Animais , Antígenos de Bactérias/genética , Vacina BCG/genética , Bovinos , Injeções Intradérmicas , Interferon gama/metabolismo , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Suínos/imunologia , Suínos/virologia , Vacinação/métodos , Animais , Anticorpos Neutralizantes/imunologia , Peste Suína Clássica/prevenção & controle , Peste Suína Clássica/transmissão , Vírus da Febre Suína Clássica/patogenicidade , Interferon gama/metabolismo , Masculino , Linfócitos T/imunologia , Linfócitos T/virologia , Fatores de TempoRESUMO
We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a "test package," although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.
Assuntos
Anticorpos Antibacterianos/sangue , Camelídeos Americanos , Interferon gama/sangue , Teste Tuberculínico/veterinária , Tuberculose/veterinária , Animais , Antígenos de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Mycobacterium bovis/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , Tuberculina/sangue , Tuberculina/imunologia , Teste Tuberculínico/métodos , Tuberculose/diagnóstico , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-gamma (IFN-gamma) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1beta (IL-1beta) whilst confirming stable IFN-gamma and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1beta and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-gamma responses.
Assuntos
Doenças dos Bovinos/metabolismo , Dessensibilização Imunológica/veterinária , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Teste Tuberculínico/veterinária , Tuberculose/veterinária , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-10/sangue , Interleucina-1beta/sangue , Fatores de Tempo , Tuberculose/diagnóstico , Tuberculose/metabolismoRESUMO
The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].