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1.
In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.  相似文献   

2.
Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.  相似文献   

3.
The gamma interferon assay was evaluated for diagnosis of paratuberculosis in goats with special emphasis on false positive reactions. Four categories of herds were tested: (A) herds that had a history of paratuberculosis, had given positive Mycobacterium avium subsp. paratuberculosis fecal samples and were vaccinated against paratuberculosis; (B) herds that had been vaccinated but had never shown clinical signs of paratuberculosis nor given positive M. a. paratuberculosis fecal samples; and (C) non-vaccinated herds without paratuberculosis. To extend the analysis of samples from young goats free of paratuberculosis, animals less than 18 months of age from non-vaccinated herds without paratuberculosis, category D, were included. Heparinized blood was stimulated with purified protein derivate (PPD) from M. a. paratuberculosis for 24 h and plasma was assayed for the presence of gamma interferon. Results were recorded as the difference between OD values of PPD stimulated and control samples. Vaccinated animals from herds with paratuberculosis, category A, showed significant higher gamma interferon responses than animals from vaccinated herds without paratuberculosis, category B. In both these groups the responses were correlated to age with higher responses in younger animals. Some of the vaccinated animals in herds without paratuberculosis had a gamma interferon response lasting for several years, which demonstrate a long lasting interference with diagnostic testing in vaccinated goats. Only three of the 121 non-vaccinated animals free of paratuberculosis in category C had responses against PPD (corrected OD values at 0.2, 0.24 and 0.5), and none of the 255 young animals in category D had corrected OD values exceeding 0.2. This indicates that false positive reactions do not appear to the same extent in young goats as in young cattle. We conclude that the low responses of non-infected goats could make the gamma interferon assay useful in monitoring the paratuberculosis status of non-vaccinated herds. However, more information about the early gamma interferon responses of naturally infected goats and the presence of false negative samples are needed.  相似文献   

4.
In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.  相似文献   

5.
The purpose of the present work was to demonstrate cell-mediated immune response to paratuberculosis in experimentally infected animals, using quantification of interleukin-2 receptor (IL-2R) expression on activated lymphocytes by means of in vitro stimulation with Mycobacterium avium ssp. paratuberculosis-derived purified protein derivative (PPDp). A whole-blood technique was developed, and optimal conditions for quantification of IL-2R expression on caprine lymphocytes, using monoclonal antibodies (anti-bovine IL-2R-alpha) and low cytometrical analysis, were determined. Different PPDp-antigen concentrations and incubation times were compared. The whole-blood method was also compared to the more traditional IL-2R assay using peripheral blood mononuclear cultures (Hesketh et al., 1993). Cross-reactivity to Mycobacterium avium was studied at different mycobacteria-PPD concentrations. An immune response could be demonstrated in animals infected with Mycobacterium avium ssp. paratuberculosis. We found that a PPDp concentration of 10microgml(-1) together with an incubation time of 72h, gave the best results using the whole-blood method. The whole-blood method eliminates many laborious steps involved in lymphocyte separation, and the effects of all the constituents of blood are expressed in a way which corresponds more to in vivo conditions. The risk of selecting subpopulations of lymphocytes during cell separation is avoided.  相似文献   

6.
OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.  相似文献   

7.
Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for these mycobacterial infections are examined with regards to the importance of their natural reservoirs.  相似文献   

8.
The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.  相似文献   

9.
During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M. scrofulaceum strains. By PCR, only one animal was positive for M. bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population.  相似文献   

10.
Paratuberculosis or Johne's disease of livestock, which is caused by Mycobacterium avium subsp. paratuberculosis (MAP), has increased in prevalence and expanded in geographic and host ranges over about 100 years. The slow and progressive spread of MAP reflects its substantial adaptation to its hosts, the technical limitations of diagnosis, the lack of practical therapeutic approaches, the lack of a vaccine that prevents transmission and the complexity and difficulty of the on-farm control strategies needed to prevent infection. More recently evidence has accumulated for an association of MAP with Crohn's disease in humans, adding to the pressure on animal health authorities to take precautions by controlling paratuberculosis. Mycobacterial infections invoke complex immune responses but the essential determinants of virulence and pathogenesis are far from clear. In this review we compare the features of major diseases in humans and animals that are caused by the pathogenic mycobacteria M. ulcerans, M. avium subsp. avium, M. leprae, M. tuberculosis and MAP. We seek to answer key questions: are the common mycobacterial infections of humans and animals useful "models" for each other, or are the differences between them too great to enable meaningful extrapolation? To simplify this, the immunopathogenesis of mycobacterial infections will be defined at cellular, tissue, animal and population levels and the key events at each level will be discussed. Many pathogenic processes are similar between divergent mycobacterial diseases, and at variance between virulent and avirulent isolates of mycobacteria, suggesting that the research on the pathogenesis of one mycobacterial disease will be informative for the others.  相似文献   

11.
More accurate tests are required to test cattle which have reacted positively in the tuberculin skin test. For this purpose, a range of mycobacterial antigens, MPB59, MPB64, MPB70, MPB83, ESAT-6 and CFP10, were used either as recombinant proteins or as synthetic peptides in the whole blood interferon-gamma (IFN-gamma) test. Groups of uninfected cattle with typical 'non-specificity' problems were targeted, in particular animals with skin tuberculosis, animals vaccinated against Johne's disease and animals that were positive in the standard purified protein derivative (PPD)-based IFN-gamma test. The two study groups consisted of 74 Mycobacterium bovis-culture positive animals and 72 uninfected animals, all of which tested positive in the caudal fold tuberculin skin test eight to 28 days before the blood test. The use of combinations of ESAT-6 and CFP10 antigens, either as recombinant proteins or peptides, detected similar percentages of M bovis-infected animals as the PPD-based IFN-gamma test, but produced significantly fewer false positive reactions. The PPD-based IFN-gamma test was very effective in differentiating animals vaccinated against Johne's disease that were skin-test positive from those with bovine tuberculosis, and the use of PPD or specific mycobacterial antigens minimised the number of false positive reactions in animals with skin tuberculosis.  相似文献   

12.
Methods to improve the ELISA test to detect Mycobacterium avium subsp. paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne's disease. In the present study, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.  相似文献   

13.
Mycobacterium avium paratuberculosis is the causative agent of Johne disease, a chronic ulcerative intestinal condition in ruminant animals. Owing to the predominance of cellular response in subclinical forms of the infection, identification of M. a. paratuberculosis antigens eliciting host cell-mediated immune (CMI) reaction is crucial for early control of the disease. A 35 kDa protein of M. a. paratuberculosis was studied for its ability to elicit CMI responses using a mouse model. Lymphoproliferation and IFN-γ response were used to measure the CMI response. Recombinant 35 kDa protein (P35) stimulated proliferation of mouse mononuclear splenocytes sensitized with M. a. paratuberculosis. The P35 elicited increased nitrite production from mononuclear splenocytes from M. a. paratuberculosis-sensitized mice. In addition, RT-PCR-based semiquantitative IFN-γ measurement showed that stimulation with P35 is associated with significant expression of IFN-γ mRNA in M. a. paratuberculosis-sensitized mouse splenocytes. The results indicate that the 35 kDa protein of M. a. paratuberculosis is associated with CMI response in the host.  相似文献   

14.
Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good.Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations.Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity.The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.  相似文献   

15.
In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.  相似文献   

16.
The host immune response to infection with Mycobacterium paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T cell populations serves to activate macrophages to kill ingested M. paratuberculosis as well as activate other T cell subsets to contain the infection. This paper reviews the current knowledge of T cell immune responses in M. paratuberculosis infection based upon clinical studies and research using mouse models.  相似文献   

17.
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18.
Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.  相似文献   

19.
Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n=5) were vaccinated with M. bovis CFP formulated with either Emulsigen or Polygen adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-gamma (IFN-gamma) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-gamma responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-gamma responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen+CpG or CFP/Polygen+CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen+CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.  相似文献   

20.
The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3)CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.  相似文献   

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