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1.
Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.  相似文献   

2.
Lymphoproliferative response (LPR) was studied in 19 lambs orally infected (Group I) with Mycobacterium avium subsp. paratuberculosis (MAP) with in vitro lymphocyte stimulation test using MTT dye reduction assay. The non-specific LPR against Con A and specific LPR against sonicated antigen and johnin PPD (purified protein derivatives) were estimated on preinfection (0 day) and various days postinfection period (15 to 330 dpi) in the animals, which were classified according to histological and bacteriological evidence of paratuberculosis infection. Of the two antigens used, johnin PPD was found to be superior in terms of consistency and uniformity of response over an observation period of about a year. Significantly (P < 0.05) higher LPR were observed in the infected sheep during postinfection period, as compared with preinfection values and values from uninfected control sheep. It was evident from the present study that the LPR in histologically infected animals fluctuated during the long course of infection and had a definite relationship with the gut pathology and the mycobacterial load. The LPR were stronger but variable in sheep with grades 1, 2 and 3 lesions (paucibacillary) and increased progressively from 30 dpi onwards. The sheep with the advanced lesions (grade 4, multibacillary) showed progressive decline in LPR till 120 dpi after initial stronger response at 30 dpi. Most of the animals were detected by LPR before initiation of faecal shedding of MAP. The results suggested that repeated testing was required while screening an infected flock for detecting most of the positive animals.  相似文献   

3.
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   

4.
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. This study examined the movement of peripheral blood mononuclear cells (PBMCs) infected with MAP and the dissemination of MAP following mycobacteraemia induced by IV inoculation over a time frame of 3 days.  相似文献   

5.
Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.  相似文献   

6.
《Veterinary microbiology》2015,175(2-4):275-285
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n = 9) and naturally infected cows in the subclinical (n = 10) and clinical (n = 13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-γ) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.  相似文献   

7.
Early detection of Johne’s disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is essential to reduce transmission; consequently, new diagnostic techniques and approaches to detect MAP or markers of early MAP infection are being explored. The objective was to identify biomarkers associated with MAP infection at 6 and 9 months after oral inoculation. Therefore, gene expression analysis was done using whole blood cells obtained from MAP-infected calves. All MAP-inoculated calves had a cell-mediated immune response (IFN-γ) to Johnin PPD specific antigens, and 60% had an antibody response to MAP antigens. Gene expression analysis at 6 months after inoculation revealed downregulation of chemoattractants, namely neutrophil beta-defensin-9 like peptide (BNBD9-Like), S100 calcium binding protein A9 (s100A9) and G protein coupled receptor 77 (GPR77) or C5a anaphylatoxin chemotactic receptor (C5a2). Furthermore, BOLA/MHC-1 intracellular antigen presentation gene was downregulated 9 months after inoculation. In parallel, qPCR experiments to evaluate the robustness of some differentially expressed genes revealed consistent downregulation of BOLA/MHC-I, BNBD9-Like and upregulation of CD46 at 3, 6, 9, 12, and 15 months after inoculation. In conclusion, measuring the expression of these genes has potential for implementation in a diagnostic tool for the early detection of MAP infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0096-5) contains supplementary material, which is available to authorized users.  相似文献   

8.
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   

9.
Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSPA 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.  相似文献   

10.
Mycobacterium avium subspecies paratuberculosis (MAP), the causal agent of paratuberculosis, was detected by quantitative real‐time IS900 PCR in the follicular fluid from the reproductive tracts of cows originating from one infected herd. As well as being detected in follicular fluid of cows shedding bacteria in their faeces, MAP was also detected in the follicular fluid of one apparently healthy, non‐shedding individual cow. The finding of MAP in follicular fluid is unexpected and could contribute to the lower viability of embryos and resultant lower pregnancy rate. In addition to finding contaminated follicular fluid, vaginal and uterine flush fluids were determined to be positive for the presence of MAP in 75% and 56.3% of the time of the cattle currently shedding MAP in their faeces, respectively. The presence of MAP in different parts of the reproductive tract was seen in clinically as well as subclinically infected cows. These findings extend our currently scant and contradictory knowledge about the dissemination of MAP in the reproductive tract of female cattle.  相似文献   

11.
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   

12.
Johne's disease (JD), or paratuberculosis is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). There may be a long subclinical phase with no signs of clinical disease.  相似文献   

13.

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.  相似文献   

14.

Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence of PTB in Indian cattle. A total of 213 cattle, were subjected to multiple diagnostic tests viz. Johnin PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P?<?0.01) different in case-control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.

  相似文献   

15.
There are inconsistent results for the association of Mycobacterium avium subspecies paratuberculosis (MAP) infection with production and reproduction in dairy cows. Determination of these associations in each region is essential to encourage participation of dairy cattle producers in disease control programs. This study was conducted in Shiraz, southern Iran, to quantify the association of subclinical MAP infection with 305-day milk production and calving interval in Iranian Holsteins. A total of 21 dairy herds were selected for the study and in each herd, quarter milk samples were collected from ten to 12 dairy cows for PCR analysis. Data about parity, calving interval, length of lactation period, total milk production and 305-day milk production were also provided for each animal. Overall, 252 individual milk samples were collected. Herd- and individual-level prevalence of MAP infection were 23.8% (95% CI, 6.2–41.4%) and 3.2% (95% CI, 1.3–5.1%), respectively based on IS900 nested PCR. The results for 305-day milk production revealed a 248 kg reduction in positive cows compared with negative ones (P = 0.009). When cows from positive herds were compared with cows from negative herds, a 335-kg reduction in 305-day milk production (P = 0.005) and a 30-day increase in calving interval (P = 0.057) were observed in the former group. These findings support the previous results that paratuberculosis infection is negatively associated with the performance of the animals.  相似文献   

16.

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log105 to log107Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 104–107 mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.  相似文献   

17.
In the present study efficacy of single intradermal Johnin test, acid fast staining of faecal smear and IS 900 faecal polymerase chain reaction tests was evaluated in 200 goats for detection of Mycobacterium avium subsp paratuberculosis. Two hundred goats comprising 150 goats from an organised farm in Trichur district and 50 goats reared under field condition at farmers premise from Malappuram district of Kerala state formed the study population. Faecal smear from all the 200 goats was stained by Ziehl–Neelsen acid fast stain and faecal polymerase chain reaction (PCR) specific for M. avium subsp paratuberculosis (MAP); IS 900 was performed on all samples. All the animals were subjected to single intradermal Johnin test. Out of 200 goats screened for paratuberculosis, six goats (3%), 11 goats (5.5%) and 42 goats (21%) were found positive by Ziehl–Neelsen acid fast staining of faecal smear, single intradermal Johnin test and IS 900 PCR respectively. Results of the present study indicate that amplification of IS 900 insertion element was the most specific and sensitive diagnostic detection method. Single intradermal Johnin test and Ziehl–Neelsen acid fast staining did not show any significant difference.  相似文献   

18.
Bovine paratuberculosis (Johne's disease), a chronic and debilitating disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a major cause of chronic ruminant enteritis. A national eradication program has been in place in South Korea since the first report of the disease in 1967; however, only limited data on bovine paratuberculosis in South Korea are available. Some research, such as investigations of the reactivity of animal sera against MAP antigens, has been done in localized areas and in limited animal species. Compared with the worldwide situation, the development of diagnostic methods in South Korea has shown similar results even though some data were obtained from international collaborative studies. MAP is considered by some to be zoonotic, noting an association with Crohn's disease, although this issue is still controversial; however, research into this association is limited. Decisions based on disease priorities have hampered active progress in research on the disease. In this paper, we reviewed the available results generated from South Korea compared with global research. Finally, we propose a theme for future research.  相似文献   

19.
Maximum inactivation (83–98%) of proliferating Mycobacterium bovis and/or Mycobacterium avium sensitized bovine lymphocytes was obtained when the lymphocytes were treated with 10?4 M of 5-bromodeoxyuridine (BUdR) and light. Inactivation was specific though not total whether the cultures were first stimulated with M. bovis PPD or M. avium PPD, though slightly higher inactivation resulted when cultures were first stimulated with M. bovis PPD. There was, however, a statistically significant suppression (P < 0.05) of the counts/min of cultures initially stimulated with either M. bovis PPD or M. avium PPD and later restimulated with either of the antigens after BUdR-light treatment. Thus experiments showed that inactivation occurs only to those cells responding by proliferation to a particular stimulant. There is a potential for the use of this assay for differentiating infection due to either M. bovis or M. avium if total inactivation of proliferating lymphocytes could be achieved.  相似文献   

20.
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   

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