RESUMO
Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.
Assuntos
Células Endoteliais , Macrófagos , Bovinos , Animais , Macrófagos/fisiologia , Linhagem Celular , Fagocitose , Células AlimentadorasRESUMO
Paratuberculosis is a chronic enteritis of ruminants caused by the facultative intracellular pathogen Mycobacterium avium subsp. paratuberculosis. The Th1 response inhibits the proliferation of M. avium subsp. paratuberculosis during the early subclinical stage. However, we have previously shown that immune inhibitory molecules, such as prostaglandin E2 (PGE2), suppress M. avium subsp. paratuberculosis-specific Th1 responses as the disease progresses. To date, the mechanism underlying immunosuppression during M. avium subsp. paratuberculosis infection has not been elucidated. Therefore, in the present study, we investigated the function of cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed by peripheral blood mononuclear cells (PBMCs) from cattle with paratuberculosis because CTLA-4 expression is known to be elevated in T cells under an M. avium subsp. paratuberculosis experimental infection. M. avium subsp. paratuberculosis antigen induced CTLA-4 expression in T cells from cattle experimentally infected with M. avium subsp. paratuberculosis. Interestingly, both PGE2 and an E prostanoid 4 agonist also induced CTLA-4 expression in T cells. In addition, a functional assay with a bovine CTLA-4-immunogobulin fusion protein (CTLA-4-Ig) indicated that CTLA-4 inhibited gamma interferon (IFN-γ) production in M. avium subsp. paratuberculosis-stimulated PBMCs, while blockade by anti-bovine CTLA-4 monoclonal antibody increased the secretion of IFN-γ and tumor necrosis factor alpha production in these PBMCs. These preliminary findings show that PGE2 has immunosuppressive effects via CTLA-4 to M. avium subsp. paratuberculosis. Therefore, it is necessary to clarify in the future whether CTLA-4-mediated immunosuppression facilitates disease progression of paratuberculosis in cattle.
Assuntos
Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Antígeno CTLA-4/metabolismo , Interferon gama , Leucócitos Mononucleares , Fator de Necrose Tumoral alfa/metabolismo , Abatacepte/metabolismo , Terapia de Imunossupressão , Prostaglandinas E/metabolismo , Prostaglandinas/metabolismo , Anticorpos Monoclonais/metabolismoRESUMO
Johne's disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.
Assuntos
Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática , Fezes , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.
Assuntos
Paratuberculose/epidemiologia , Paratuberculose/prevenção & controle , Criação de Animais Domésticos , Animais , Animais Selvagens/microbiologia , Notificação de Doenças/normas , Incidência , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/economia , Ruminantes/microbiologiaRESUMO
Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.
Assuntos
Imunidade Adaptativa/imunologia , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/patologia , Dinoprostona/imunologia , Dinoprostona/metabolismo , Paratuberculose/imunologia , Paratuberculose/patologia , Animais , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Bovinos , Doenças dos Bovinos/microbiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismoRESUMO
Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-γ) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-γ responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doenças dos Bovinos/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígeno B7-H1/biossíntese , Antígeno B7-H1/imunologia , Bovinos , Doenças dos Bovinos/microbiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/imunologia , Depleção Linfocítica , Macrófagos/imunologia , Masculino , Paratuberculose/microbiologia , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Here, we report the complete genome sequence of Mycobacterium avium subsp. paratuberculosis strain 42-13-1, isolated from cattle presenting with chronic diarrhea caused by Johne's disease in Japan, which was assembled via long- and short-read hybrid assembly.
RESUMO
Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.
Assuntos
Antígeno B7-H1/imunologia , Doenças dos Bovinos/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Animais , Derrame de Bactérias/efeitos dos fármacos , Derrame de Bactérias/imunologia , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Doenças dos Bovinos/parasitologia , Masculino , Paratuberculose/tratamento farmacológicoRESUMO
Paratuberculosis or Johne's disease (JD), is a chronic infectious disease causing intractable diarrhea in cattle, which leads to less productivity, such as decreased milk yield, and lower daily weight gain. As a control measure against JD in cattle, national serological surveillance has been conducted in Japan since 1998. To conduct modeling studies that are useful to evaluate the effectiveness of control measures against JD, reliable parameter values, such as length of time from infection to the start of fecal shedding or antibody expression, are especially important. These parameters in the Japanese cattle population are assumed to be different from those in other countries with a higher prevalence of JD or in experimental infection settings; therefore, they must be estimated for the cattle population in Japan. Data from national surveillance conducted in Tokachi District, Hokkaido Prefecture, were used for this study. Using data from JD diagnostic tests for all cattle in Tokachi District between 1998 and 2014, all testing histories for infected animals were estimated as the number of tested cattle and positive cattle at each age of month for both fecal and antibody tests. A deterministic mathematical model for JD development, from infection to fecal shedding and antibody expression in infected cattle, was constructed to obtain the probability of testing positive when applied to both fecal and antibody tests at a given age. Likelihood was obtained from these estimated test results and best values for parameters were obtained using the Markov Chain Monte-Carlo method. Fifty-five percent of infected cattle were projected to have a transient shedding period, which was estimated to start 12 months after infection and last for 4 months. Persistent shedding was projected to occur in all infected cattle, and estimated to begin 7-84 months from infection. Following persistent shedding, antibody expression was estimated to start 7 months later. These values are useful for developing models to evaluate the status of JD infection and the effectiveness of control measures in the Japanese cattle population.
Assuntos
Anticorpos Antibacterianos/sangue , Derrame de Bactérias , Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/microbiologia , Animais , Bovinos , Indústria de Laticínios , Fezes/microbiologia , Japão , Modelos Teóricos , Mycobacterium avium subsp. paratuberculosis/imunologiaRESUMO
The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.
Assuntos
Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Doenças dos Ovinos/microbiologia , Animais , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Fezes/microbiologia , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Radiometria/métodos , Radiometria/veterinária , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/diagnóstico , Estatísticas não ParamétricasRESUMO
A 14-month-old Japanese black beef steer presented with severe chronic diarrhea and emaciation and was euthanized. Postmortem examination showed thickened and corrugated intestinal mucosa and enlarged granulomatous mesenteric lymph nodes with caseating necrosis. Numerous epithelioid cells and multinucleated giant cells infiltrated in the lamina propria and the submucosal tissue of the intestines. These cells were also observed in the systemic organs. Many acid-fast bacilli were detected in the cytoplasm of these cells and were identified as 'Mycobacterium avium subsp. hominissuis' (Mah) on the basis of the results of molecular examinations and immunohistochemistry. These findings indicate that Mah can cause systemic mycobacteriosis, and this unique infection needs to be distinguished from Johne's disease and tuberculosis in cattle.
Assuntos
Intestinos/microbiologia , Intestinos/patologia , Mycobacterium avium/fisiologia , Tuberculose/veterinária , Animais , Bovinos , Diarreia/veterinária , Granuloma/microbiologia , Imuno-Histoquímica , Masculino , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
An efficient protocol for the manual fluorescent MGIT culture system combined with rapid confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) growth in the broth culture was established and evaluated for the detection of viable MAP in direct quantitative PCR (QPCR) positive bovine feces. Manually detected fluorescence emissions from MGIT tubes were analyzed objectively using an open source software, ImageJ. For molecular confirmation of MAP growth, DNA samples harvested by simply boiling the broth, an inexpensive and time- and labor-saving DNA preparation method, yielded adequate results. The sheep strain of MAP required longer incubation time relative to the cattle strain, suggesting that the MGIT system may not support well the growth of ovine isolates as described previously. Of 61 direct QPCR positive bovine feces, the recovery rate of MAP in the MGIT system (62.3%) was significantly higher (P<0.05) than that using 7H10 agar-based slants (44.3%). The time to obtain a final result for fecal culture by the MGIT system was several weeks earlier compared to solid media. In MGIT culture positive samples, the time to detect fluorescence was correlated with the DNA quantity detected in fecal QPCR. As a positive result in the direct fecal QPCR test does not mean fecal excretion of viable MAP, bacterial isolation by fecal culture could be conducted to verify the QPCR result. For this purpose, the manual MGIT system is a sensitive and rapid culture method at least for bovine samples.
Assuntos
Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/microbiologia , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Meios de Cultura , DNA Bacteriano/química , DNA Bacteriano/genética , Fezes/microbiologia , Microscopia de Fluorescência/veterinária , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterináriaRESUMO
Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.
Assuntos
Doenças dos Bovinos/microbiologia , Enoil-CoA Hidratase/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/microbiologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Enoil-CoA Hidratase/genética , Ensaio de Imunoadsorção Enzimática/normas , Mycobacterium avium subsp. paratuberculosis/enzimologia , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/sangue , Paratuberculose/imunologia , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Sensibilidade e EspecificidadeRESUMO
Diagnostic tests used for Johne's disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Doenças dos Ovinos/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Fezes/microbiologia , Modelos Logísticos , Estudos Longitudinais , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/imunologia , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Fatores de TempoRESUMO
The aim of this study was to assess the humoral immune responses in MAP-infected and uninfected sheep against 27 MAP stress-associated recombinant proteins that were regulated in in vitro models of physiological stress. These include evaluation of 5 proteins, which were previously reported by Gumber et al. (2009b), using serum samples from sheep with a wide range of disease stages. For purification of recombinant his-tagged proteins expressed as an insoluble protein, on-column refolding purification was applied as well as one-step denaturing purification. All purifications together resulted in a total of 48 recombinant antigen preparations. In antibody ELISA tests, 23 of these, representing 18 MAP proteins, showed significant differences in responses between infected and uninfected sheep. Recombinant antigen preparations MAP2281c, MAP3555 (refolded form), and MAP0711c (refolded form) when incorporated in an ELISA, had similar sensitivity to a commercial antibody ELISA test at the cutpoint of 90% specificity, and showed relatively high values in receiver operating characteristic (ROC) curve analysis. However, as some of the sera from uninfected sheep also reacted to recombinant antigens, further development of the assays is necessary prior to practical application. Compared to the commercial antibody ELISA, MAP0593c, MAP2281c, MAP2411, MAP3555, and MAP3200 detected more infected sheep with a lower grade of lesion, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected sheep at an early stage of infection. This is consistent with the hypothesis of latency or dormancy in subclinical mycobacterial infection.
Assuntos
Proteínas de Bactérias/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Paratuberculose/microbiologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Recombinantes de Fusão/imunologia , Ovinos , Estresse Fisiológico/imunologiaRESUMO
In this study, humoral immune responses in cattle against Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins were assessed longitudinally by ELISA during the first 30 weeks after MAP infection. A total of 11 MAP genes previously identified by proteomic analysis were selected for cloning and expression. These included possible general stress-associated proteins of MAP and proteins expressed in vivo in MAP-infected sheep at an early stage of infection. An increase in the antibody levels against 5 recombinant antigen preparations (MAP1027c, MAP1339, MAP1588c, MAP1589c and MAP2411) was seen in MAP-infected calves (n=16) but not in control calves (n=3) over the time examined. Antibody responses were recorded as early as two weeks post-inoculation, and 87.5% of the inoculated cattle responded to at least one of the five immunogenic antigen preparations within the first 30 weeks of infection, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected cattle at a relatively early stage after infection and therefore stimulate the host's immune system. It has been assumed that the sensitivity of antibody ELISA tests is dependent on the stage of infection and the age of the animals. However, we have provided some evidence that humoral immunity occurs at an early stage of paratuberculosis and can be detected using appropriate antigens such as MAP stress-associated proteins.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/imunologia , Doenças dos Bovinos/microbiologia , Proteínas de Choque Térmico/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/imunologia , Distribuição de Qui-Quadrado , Ensaio de Imunoadsorção Enzimática/veterinária , Proteínas de Choque Térmico/genética , Imunidade Humoral/imunologia , Estudos Longitudinais , Masculino , Paratuberculose/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.
Assuntos
Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Doenças dos Ovinos/diagnóstico , Carneiro Doméstico/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Células Cultivadas , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Fezes/microbiologia , Estudos Longitudinais , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Ovinos/microbiologia , Doenças dos Ovinos/microbiologiaRESUMO
The growth pattern and protein expression profiles of sheep (S) and cattle (C) strains of Mycobacterium avium subsp. paratuberculosis (MAP) under oxidative and nitrosative stress were characterised. Oxidative stress was induced using 0.05% (v/v) H(2)O(2) in BACTEC medium, and was lethal for an inoculum of 10(4) cells. However, an inoculum of 10(7) cells survived and proteomic changes were observed at 7 days. Nitrosative stress was induced using 1mM NaNO(2); it slowed the growth of an inoculum of 10(4) cells, but both strains recovered quickly when resuscitated in fresh media. Silver staining showed higher sensitivity for detection of 2D spots compared to SYPRO Ruby staining. A total of 18 proteins were regulated under oxidative and/or nitrosative stress. The expression of four antioxidant enzymes (AhpC, AhpD, OxcA and SodA) and four proteins involved in fatty acid metabolism (DesA2, FadA6_3, FabG and FadE19) was altered, together with a range of other proteins. Only one protein, AhpC was differentially regulated in both strains of MAP. Seven proteins (DesA2, AhpC, AhpD, Ppa, FabG, and hypothetical proteins MAP2411 and MAP 1885c) were identified in previous in vitro studies with temperature, hypoxia and/or nutrient starvation stressors and may be general stress response proteins of MAP. Prior studies have identified immune responses directed against AhpC and Ppa in animals with Johne's disease, expression of sodA and ppa within macrophages, and reduced virulence of impA mutants in mice, highlighting the relevance of proteomic studies using these in vitro stress models for pathogenesis studies.
Assuntos
Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Proteoma/imunologia , Espécies Reativas de Nitrogênio/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Sobrevivência Celular/imunologia , Eletroforese em Gel Bidimensional , Compostos Organometálicos/química , Paratuberculose/microbiologia , Coloração pela Prata/veterinária , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Interleukin-10 (IL-10) is not only an essential immunoregulator in host immunity, but also it accounts for the intracellular survival of mycobacteria because of its inhibitory activity against anti-mycobacterial functions of macrophage. It has been also indicated that blood cells from calves infected with Mycobacterium avium subsp. paratuberculosis (Map) produce a large amount of IL-10 after stimulation with Map antigen, and it leads to suppression of Interferon-gamma (IFN-gamma) production in T-cells. This characteristic expression of IL-10 in Map-infected cattle seems to be playing important roles in the pathogenesis of Johne's disease caused by Map, and could be an important diagnostic indicator. The aim of this study was to investigate the diagnostic significance of IL-10 production from blood cells stimulated by a PPE (Proline-Proline-Glutamic acid) protein family of Map. The recombinant PPE protein, Map41, which has been reported as one of the IFN-gamma inducing antigens of Map, also strongly induced IL-10 from macrophages obtained from infected calves. The elicited IL-10 production in response to Map41 from experimentally infected calves was as early as 2 weeks after the inoculation of Map, and the IL-10 production was detected earlier than that of IFN-gamma. The blood cells from calves immunized with Map produced higher amounts of IL-10 against Map41 stimulation than those of calves immunized with various Mycobacterium species. Furthermore, this IL-10 induction also showed high specificity to Map in guinea pigs experimentally infected with various Mycobacterium species. These observations suggest that IL-10 assay is a useful diagnostic method in the early stage of Johne's disease.