Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.     

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.     

Interaction of antigen presenting cells with mycobacteria

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNF and IL-10 whereas macrophages produced TNF, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

Objectives To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis .
Design Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates.
Procedure DNAs from 35 Australian isolates of M para-tuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared.
Results The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales.
Conclusion Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis , and could be used to study the transmission of strains in Australia.     

The ranging behaviour and habitat use of rabbits on farmland and their potential role in the epidemiology of paratuberculosis

Grazing herbivores avoid grass swards contaminated with faeces as the ingestion of faeces is a common route of micro- and macro-parasite transmission. The recent novel finding that herbivores do not avoid grass swards contaminated with rabbit faeces suggests that disease risk posed to herbivores by rabbits is determined by rabbit ranging and excretory behaviour. Using as a case study rabbits and the risk Mycobacterium avium sub-species paratuberculosis (M. a. paratuberculosis) poses to cattle, the interaction between rabbits and grazing pasture was studied on an infected farm in the east of Scotland in spring and autumn 2000. Radio telemetry, burrow surveys and faecal pellet count data were collected on two areas (Areas 1 and 2) of the farm with different habitat mosaics, to study the potential effects of season and habitat on the spatial distribution of rabbits faeces and thus disease in the environment. Twenty one rabbits were radio tracked and a total of 902 fixes collected. Mean home range sizes (100% minimum convex polygons) were between 2.0 and 7.1 ha per rabbit per season. Home ranges were significantly larger in spring, and in Area 1 which had more moor and woodland and less rough pasture. Rabbits used rough pasture most in Area 1 and gorse scrub in Area 2. In both areas, significantly more burrows were located in gorse scrub than in any other habitat. Most faecal pellets were deposited on the moorland habitat of Area 2 in autumn. In habitats to which grazing livestock had access, the mean rate of faecal deposition was 8571 pellets per ha per day. The greatest risk of disease transmission occurred in habitats of poor grazing quality (e.g., gorse scrub) which were used by rabbits for burrowing and thus contained high concentrations of faeces. The findings of the study are discussed in relation to management practices aimed at reducing disease risk to livestock, including the fencing of scrub and the reduction of rabbit population size to prevent expansion of rabbit burrows from scrub into grazing pastures.     

Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

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.     

牛分枝杆菌Ag85A基因在大肠埃希氏菌中的表达

以BamHⅠ和EcoRⅠ双酶切已构建的pGEM-T-85A和pET28a（＋），并将纯化的Ag85A基因亚克隆至pET28a（＋）中，构建出原核表达质粒pET28a-85A。将pET28a-85A转化至感受态E．coli BL21（DE3）中，经IPTG诱导和SDS-PAGE分析，可见约32ku的外源蛋白带。Western-blotting分析表明，该蛋白具有牛分枝杆菌的抗原性。     

牛分枝杆菌mpb64和Ag85B融合基因在大肠埃希氏菌中的原核表达

以牛分枝杆菌Vallee株基因组DNA为模板,应用PCR扩增获得mpb64和Ag85B两个目的基因片段,采用重叠延伸剪接技术（SOE）剪接mpb64和Ag85B,得到融合基因mpb64-Ag85B;将融合基因片段先克隆于pMD 18-T载体,再亚克隆到表达载体pET32a（＋）中,得到重组质粒pET64-85.该重组质粒经核苷酸序列测定,显示其中的外源片段与期望的序列一致;其BL21（DE3）转化菌经IPTG诱导表达带有6个组氨酸标签的融合蛋白;用Ni2＋螯合层析方法纯化融合蛋白,Western-blotting分析结果显示,该融合蛋白能与抗牛分枝杆菌阳性血清发生反应.     

An Enzyme-linked Immunosorbent Assay using Immonoaffinity-purified Antigen in the Diagnosis of Caprine Paratuberculosis and Its Comparison with Conventional ELISAs

An enzyme-linked immunosorbent assay (APA-ELISA) using an immunoaffinity-purified antigen was developed and compared with the unabsorbed and absorbed ELISA procedures, using a crude antigenic preparation, for its efficacy in detecting antibodies in goat sera against Mycobacterium avium paratuberculosis. Serum samples from 89 goats belonging to three different flocks, two with a history and evidence of paratuberculosis and one without it, were subjected to each ELISA, which had been standardized on known positive sera from goats experimentally infected with paratuberculosis. Faecal culture, faecal examination and histopathology were used as indicators of infection. The diagnostic sensitivities of the unabsorbed, absorbed and APA-ELISA were 81.8%, 77.3% and 77.3% and the specificities were 90.6%, 93.7% and 96.8%, respectively. The positive predictive values of APA-ELISA (94.4%) was the highest, followed by absorbed ELISA (80.9%) and unabsorbed ELISA (72.0%). The negative predictive values for APA-ELISA, absorbed ELISA and unabsorbed ELISA were 93.0%, 92.7% and 93.8%, respectively. The results indicated the value of APA-ELISA in avoiding the need to absorb individual test sera with Mycobacterium phlei and giving more consistent results than the absorbed ELISA. The APA-ELISA was also better than the other two procedures in terms of specificity and positive predictive values.     

Granulomatous lesions in lymph nodes of slaughter pigs bacteriologically negative for Mycobacterium avium subsp. avium and positive for Rhodococcus equi

The prevalence of granulomatous lesions in lymph nodes of pigs was studied. From January till August 2004 in two slaughterhouses in The Netherlands 2,116,536 pigs were examined for the presence of granulomatous lesions in the sub-maxillary lymph nodes. In 15,900 (0.75%) of these pigs, lesions could be detected. Nine farms with the highest incidence of lesions were selected for a more detailed pathological and bacteriological examination. On these farms, the prevalence of lesions in sub-maxillary lymph nodes ranged from 2.3 to 5.7% with a mean of 3.0%. From 1276 pigs that were sampled, 98 (7.7%) displayed granulomatous lesions in the sub-maxillary lymph nodes and one (0.1%) pig showed lesions in its mesenteric lymph node. Mycobacterium avium subsp. avium (MAA) could not be isolated from the lymph nodes of the 99 pigs with lesions and from a selection of lymph nodes (n = 61) of pigs without lesions. Rhodococcus equi was isolated from 44 out of 98 (44.9%) of the sub-maxillary lymph nodes with granulomatous lesions and from two mesenteric lymph nodes without lesions. A comparison of former studies and the current results indicate that the prevalence of MAA infections in slaughter pigs has strongly decreased over the last decade, whereas R. equi is highly prevalent. The high incidence of granulomatous lesions associated with the bacteriological presence of R. equi could be considered as a serious cause of misdiagnosis of MAA infections in cases where meat inspection is carried out by inspection for granulomatous changes of lymph nodes only.