共查询到20条相似文献，搜索用时 31 毫秒
1.
Mikkelsen H Aagaard C Nielsen SS Jungersen G 《Veterinary immunology and immunopathology》2011,143(1-2):46-54
Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assays use purified protein derivative of MAP (PPDj) which are complex antigen mixtures with low specificity. The objectives of this study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The study included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was an animal with ≥ 2 positive tests for ≥ 4 antigens, resulting in 13 cases and 13 non-cases. IFN-γ levels of cases were higher compared to IFN-γ levels of non-cases (P<0.05). The results of the IFN-γ assay using PPDj did not correlate well with the results using the novel antigens. PPDj produced elevated IFN-γ responses of samples from both the non-infected and the MAP infected herd, indicating unspecific IFN-γ responses and showed low consistency. Three latency proteins, LATP-1, LATP-2 and LATP-3 gave positive IFN-γ tests that correlated very well with the case definition suggesting high immunogenicity. Three tested antigens, LATP-2, MAP-1 and MAP-2 have no homologue in the M. avium subsp. avium or M. bovis genome and could be promising diagnostic antigens, especially LATP-2 correlated highly with the case definition. 相似文献
2.
Mikkelsen H Aagaard C Nielsen SS Jungersen G 《Veterinary immunology and immunopathology》2012,147(1-2):69-76
Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies. 相似文献
3.
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 2–7 months 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. 相似文献
4.
Wiebren Santema Marije Overdijk Judith Barends Jeroen Krijgsveld Victor Rutten Ad Koets 《Veterinary microbiology》2009,138(1-2):191-196
Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens.One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N = 20) were significantly higher (p < 0.01) than in control animals (N = 20), despite the conserved nature of this protein.In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases. 相似文献
5.
Suelee Robbe-Austerman Judith R Stabel Daniel G Morrical 《Journal of veterinary diagnostic investigation》2007,19(1):88-90
Cell-mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the Johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While these CMI tests tend to be relatively specific in noninfected flocks, in MAP-infected flocks, these tests often identify animals that cannot be confirmed infected by any other reference test, including necropsy and culture. The aim of this study was to determine if antigen exposure by inhalation or oral ingestion of killed MAP organisms would cause a detectable CMI response in sheep. Forty-eight lambs 4 months of age were randomly divided into a control group, an orally exposed group (dosed with 1 x 10(10) autoclaved MAP organisms 3 times), and an inhalation-exposed group (dosed once with 1 x 10(5) dead organisms). Lambs were skin tested and/or bled pre-exposure and 1, 2, 3, 4, and 12 months postexposure. No significant difference was seen with either the oral- or inhalation-exposed groups of lambs versus controls with either the IFN-gamma ELISA or the skin test at any time pre- or postexposure. These results suggest that infection/invasion of MAP organisms must occur in order to have a positive skin test or IFN-gamma ELISA beyond the false-positive rate. Simple exposure is not enough to elicit a detectable CMI response. 相似文献
6.
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. 相似文献
7.
8.
Rauw F Anbari S van den Berg T Lambrecht B 《Veterinary immunology and immunopathology》2011,143(1-2):27-37
The detection of ChIFN production after ex vivo antigenic-stimulation of T-lymphocytes has been evaluated for the first time, as a tool to assess cell-mediated immunity (CMI) after avian influenza (AI) infection in 10-day-old SPF chickens. Preliminarily, recall antigens have been produced either by concentrating and inactivating the whole virus or by dissociating the viral proteins. Biologically and structurally intact forms of the viral proteins were isolated by non-ionic detergents while heating, chemical agents and ionic detergent used for virus inactivation altered the antigenic viral components. The n-octyl-B-D-gluco-pyranoside treatment at low temperature was very efficient to produce AI antigenic proteins used for evaluation of ChIFN production after ex vivo antigenic-stimulation of splenic and peripheral lymphocytes. In addition, protocols to isolate lymphocytes from the respiratory tract - the trachea and the lung - have been adapted for local CMI evaluation after similar ex vivo recall assay. Specific AI CMI in the spleen, the blood and the lung was detected for 5 weeks after low pathogenic AI (LPAI) infection in chickens, while further development is needed for tracheal CMI measurement. 相似文献
9.
Akoolo L Pellé R Saya R Awino E Nyanjui J Taracha EL Kanyari P Mwangi DM Graham SP 《Veterinary immunology and immunopathology》2008,121(3-4):216-221
East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated. 相似文献
10.
An immunoblotting protocol for the detection of antibodies to Mycoplasma crocodyli was developed using sonicated antigen of the reference strain 266/93. Immunoblotting detected nine reacting antigens, of which the 33 and 40kDa antigens were immunodominant. There was no difference in reactivity of the antigens against sera obtained from vaccinated and infected crocodiles. Both antigens are candidates for other serological and molecular studies. This is the first report to develop and apply an immunoblotting test for detection of antibody to M. crocodyli infection in crocodiles. 相似文献
11.
Silvestre R Santarém N Cunha J Cardoso L Nieto J Carrillo E Moreno J Cordeiro-da-Silva A 《Veterinary parasitology》2008,158(1-2):23-30
Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections. 相似文献
12.
Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB). Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay. Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a 'relative gold standard' for each antigen and were used to perform a receiver operating curve analysis. The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi. 相似文献
13.
An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys. 相似文献
14.
de Waal T 《Veterinary parasitology》2012,189(1):65-74
Microscopy still remains the gold standard procedure for the diagnosis of many protozoan infections in animals, but the specific identification requires skilled and experienced personnel. Immunoassays, detecting antibodies or specific protozoan antigens, have been developed but often lack sensitivity and specificity due to close relationship between many protozoa. Recent research has focussed almost exclusively on molecular based techniques for the identification and quantification of parasite DNA in samples. Opinion differ on most appropriate targets to use and there are very few diagnostic kits available making comparison between laboratories difficult. Future research needs to focus on robust, cheap field diagnostic assays. 相似文献
15.
Ostertagia ostertagi soluble antigens were prepared by gel electrophoresis and electrophoretic transfer onto nitrocellulose for enzyme-linked immunosorbent assays with serum probes. Serologic responses to L3-derived antigen of approximately 32 kDa may be unique and diagnostic for cattle harboring inhibited larvae, or pre-Type II ostertagiasis. Specificity was evaluated by comparing sera from pre-Type II cattle to sera from Type I, uninfected, Fascioloides magna infected, Fasciola hepatica infected or Cooperia oncophora infected cattle. 相似文献
16.
Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection. 相似文献
17.
Adams AA Sturgill TL Breathnach CC Chambers TM Siger L Minke JM Horohov DW 《Veterinary immunology and immunopathology》2011,139(2-4):128-140
Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ+ CD5+T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease. 相似文献
18.
The nature of cell-mediated immune (CMI) responses was studied in cross-bred bovine calves, immunised by attenuated and allogeneic macroschizonts of Theileria annulata. The CMI responses were also investigated in calves, destined to survive or die of tropical theileriosis (Theileria annulata) induced by a virulent dose of sporozoites or macroschizont-infected lymphoblasts. Calves suffering fatal theileriosis showed poor CMI response. Microcytotoxicity assay revealed an enhanced population of specific cytotoxic cells amongst the peripheral blood lymphocytes (PBL) of calves resolving the infection successfully. The E rosette assay showed proliferation of T cells and the assay for macrophage migration inhibition factor (MIF) demonstrated antigen sensitised cells in the PBL. Calves, immunised by allogeneic and attenuated macroschizont-infected lymphoblasts or those recovering from virulent macroschizont-induced infection, showed protective CMI responses with patterns similar to those appearing after non-fatal sporozoite infection. 相似文献
19.
20.
Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-gamma assay and faecal culture techniques 总被引：2，自引：0，他引：2
Infections with Mycobacterium avium subsp. paratuberculosis (MAP) can be latent for years without affecting the animal, but the animal may become infectious or clinical at some point. Diagnosis of paratuberculosis can be a challenge primarily in latent stages of the infection, and different diagnosis interpretations are usually required by the variety of decision makers. The objective of this paper was to provide a critical review of reported accuracies of ELISA tests, interferon-gamma assays (IFN-gamma) and faecal culture (FC) techniques used for diagnosis of three defined target conditions: MAP infected, MAP infectious and MAP affected animals. For each animal species, target condition and diagnostic test-type, sensitivities (Se) and specificities (Sp) were summarised based on a systematic, critical review of information in literature databases. The diagnostic test information often varied substantially for tests of the same type and make, particularly ELISA, which was the most frequently reported test-type. Comparison of the various tests accuracies was generally not possible, but stratification of test-evaluations by target condition improved the interpretation of the test accuracies. Infectious and affected animals can often be detected, but Se for infected animals is generally low. A main conclusion of the review was that the quality of design, implementation and reporting of evaluations of tests for paratuberculosis is generally poor. Particularly, there is a need for better correspondence between the study population and target population, i.e. the subjects chosen for test evaluation should reflect the distribution of animals in the population where the test is intended to be used. 相似文献