A recombinant 64 kilodalton protein of Mycobacterium bovis bacillus Calmette-Guerin specifically stimulates human T4 clones reactive to mycobacterial antigens.

A recombinant 64 kD protein of Mycobacterium bovis bacillus Calmette-Guerin (BCG) (antigen A), which amounted to approximately 2% of an E. coli lysate, was tested for its capacity to stimulate human T4 clones reactive to mycobacterial proteins. Two out of four crossreactive clones, established from a patient with tuberculoid leprosy, which could be stimulated by protein preparations of M. leprae and M. tuberculosis, and by particulate M. bovis BCG were also reactive to antigen A without further enrichment from E. coli lysate. In addition, BCG-reactive T cell clones from two of three healthy PPD+ donors reacted with antigen A. This finding shows that human T cell clones may be useful for probing gene-cloned proteins of potential value for vaccination against diseases where protection is mediated exclusively by T cells.

The recombinant 65-kD heat shock protein of Mycobacterium bovis Bacillus Calmette-Guerin/M. tuberculosis is a target molecule for CD4+ cytotoxic T lymphocytes that lyse human monocytes.

Since little is known about Tc cells in the human immune response to intracellular parasites, we have studied the role of Tc cells in response to M. bovis Bacillus Calmette-Guerin (BCG). Donors whose PBMC responded to BCG, purified protein derivative (PPD), and the recombinant 65-kD heat shock protein (HSP) of BCG generated BCG/PPD-specific CD4+ effector T lymphocytes that lysed PPD as well as recombinant 65-kD-pulsed monocytes. Nonpulsed or irrelevant antigen-pulsed target cells were lysed to a much lower but still significant extent. PPD-stimulated effector lymphocytes of a recombinant 65-kD nonresponder lysed PPD but not recombinant 65-kD-pulsed monocytes. Recombinant 65-kD-educated effector lymphocytes lysed both recombinant 65-kD- and PPD-pulsed monocytes. In addition, these effector cells efficiently lysed nonpulsed target cells. These results demonstrate that in recombinant 65-kD responders, the recombinant 65-kD HSP of BCG is an immunodominant target as well as a triggering molecule for BCG/PPD-specific CD4+ cytotoxic T cells that lyse autologous monocytes. The implications of these findings with respect to the role of the 65-kD HSP in autoimmunity are discussed.

Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis.

OBJECTIVE: To investigate outbreaks of multidrug-resistant tuberculosis (TB) by using DNA fingerprint databases. DESIGN: Investigation of two outbreaks of multidrug-resistant TB in separate hospitals in Spain by restriction fragment length polymorphism (RFLP) and spoligotyping. Outbreak strains were compared with more than 1500 RFLPs of Mycobacterium tuberculosis complex strains isolated in Spain and 6000 RFLPs from 30 different countries. METHODS: Standardized IS6110 DNA fingerprinting and 'spoligotyping' was used to type multidrug-resistant isolates belonging to the M. tuberculosis complex amongst the outbreak cases. The DNA types were matched against DNA fingerprint databases in Spain and The Netherlands. RESULTS: The DNA typing analysis indicated that a single multidrug-resistant Mycobacterium bovis strain was responsible for a nosocomial outbreak in a hospital in Spain involving at least 16 HIV-infected patients with non-treatable to multidrug-resistant TB. Introduction of the fingerprint type of this strain to the international database revealed a single matching strain. This strain was also isolated from an HIV-infected patient in The Netherlands who had died from multidrug-resistant TB. This patient had previously been hospitalized in Spain, where a multidrug-resistant TB nosocomial outbreak involving 20 HIV-infected patients was ongoing. The strains causing this outbreak were also identified as M. bovis with an identical DNA pattern to those strains isolated in the Spanish hospital and the patient in The Netherlands. CONCLUSIONS: The use of centralized DNA databases can help to identify rapidly the origin and transmission routes of multidrug-resistant TB across international boundaries and the potential use of such an early warning surveillance system for investigation of nosocomial multidrug-resistant TB outbreaks between HIV-infected patients. To our knowledge this is the first report of transmission of multidrug-resistant M. bovis between hospitals.

Predominace of a novel Mycobacterium tuberculosis genotype in the Delhi region of India.

Using IS 6110 -restriction fragment length polymorphism (RFLP) and spoligotyping, genetic variations of 83 Mycobacterium tuberculosis strains isolated from tuberculosis patients from two wards in a hospital in Delhi and a rural chest clinic near Delhi were analysed. The vast majority of the isolates (75%) were closely related and this novel genogroup was designated the 'Delhi type'. Both drug-sensitive and drug-resistant strains were found among strains of this genogroup. A minority of the strains harboured a single IS 6110 copy and only one strain belonged to the Beijing genotype, a genotype that is predominant in other parts of Asia. A comparison of the RFLP and spoligotype with existing data suggests that the predominance of Delhi genogroup is geographically limited to the Indian subcontinent and perhaps to specific regions in India. Despite the high prevalence of the M. tuberculosis strains of the Delhi type, the strains could easily be discriminated due to polymorphisms in the IS 6110 patterns. Future studies may disclose the genetic characteristics of strains belonging to the Delhi genotype, analogous to the recently observed virulence among the Beijing genogroup.

Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats.

We studied the presence of bacterial antigens in rat tissues. We produced a monoclonal antibody (MAb 2E9) directed against intestinal flora-derived peptidoglycan-polysaccharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed by immunohistology with a previously described MAb 2-4 directed to muramyl dipeptide. Other lymphoid organs did not stain positively with 2E9 and 2-4. Neonatal and young rats showed no staining of the spleen, but positivity could be induced by injecting peptidoglycan-polysaccharide complexes systemically. We conclude that bacterial fragments are present in splenic macrophages of conventional rats.

Natural antibodies to 65 kD mycobacterial heat shock protein in rats do not correlate with susceptibility for Mycobacterium tuberculosis induced adjuvant arthritis.

Natural antibodies to 65 kD heat shock protein (hsp65) of Mycobacterium bovis were found in the sera of Lewis rats. The levels of these natural hsp65 antibodies differed substantially between the individual rats. Each rat was subsequently tested for its susceptibility to develop arthritis following injection of M. tuberculosis in incomplete Freund adjuvant. It was found that the incidence and severity of the induced arthritis did not differ between groups of Lewis rats with relatively high and relatively low natural antibody levels to hsp65. Inoculation of rats without natural antibodies to hsp65 with intestinal contents did not induce hsp65 antibodies, although the rats were able to respond to the antigen.

Are intestinal bacteria involved in the etiology of rheumatoid arthritis? Review article.

Observations in bowel-related joint diseases give support to this hypothesis. In Crohn's disease and ulcerative colitis, the bowel wall inflammation is complicated in about 20% of the patients by joint inflammation. Bowel infection by Salmonella, Shigella and Yersinia can provoke joint inflammation and supports an etiological link between bowel bacteria and arthritis. The arthropathic properties of the most abundant group of intestinal bacteria, i.e. the obligate anaerobic bacteria, were studied in an animal model. Cell wall fragments (CWF), with peptidoglycan as the major component, from some Eubacterium and Bifidobacterium species induced a severe chronic polyarthritis in Lewis rats after a single intraperitoneal injection. Eubacterium was found in numbers of 10(8)-10(9) per gram in stools of healthy subjects and rheumatoid arthritis (RA) patients. CWF of isolated strains of E. aerofaciens were arthropathic. Soluble peptidoglycan polysaccharide complexes (PG-PS) originating from the obligate anaerobic flora were purified from human intestinal contents. PG-PS from ileostomy fluid that proved to be less processed by intestinal enzymes induced chronic arthritis in rats after a single administration in oil in the base of the tail. It was concluded that the human intestinal bowel contains soluble bacterial cell wall products that are arthropathic in an animal model. Peptidoglycan (PG) or its subunits was reported to be present in mammalian tissues. Immunohistochemical studies from our group showed the presence of intestinal PG-PS in sections of normal rat spleen. Bacterial cell wall or PG-induced joint inflammation in rats is proven to be absolutely dependent on functional T cells. T-cell lines were isolated from the lymph nodes of rats with an E. aerofaciens CWF arthritis. A helper T-cell line B13 was in vivo arthritogenic in knee or ankle joints upon intravenous injection in rats and proliferated in vitro on syngeneic spleen cells alone, but was additionally stimulated by intestinal PG-PS and E. aerofaciens CWF. It was postulated that the arthritogenic T cells that seem to be autoreactive are, in fact, recognizing bacterial PG-PS on antigen-presenting cells (APC). It is generally accepted that RA is a T-cell-dependent process and that therefore the reaction is directed at small peptides bound by the major histocompatibility complex of APC. The only peptides present in arthritis inducing intestinal PG-PS and in CWF are PG peptides interlinking the sugar chains. We feel that the immunoreaction against PG peptides plays a pivotal role in experimental and human arthritis of an unknown etiology.

Degradation of intestinal glycoproteins by Bacteroides vulgatus.

Bacteroides vulgatus, isolated from a patient with Crohn's disease, produced in gnotobiotic rats 7 constitutive enzymes that might be concerned with the degradation of intestinal glycoproteins. Furthermore Bacteroides vulgatus caused an almost complete loss of blood group antigenicity of the intestinal glycoproteins. Enzymes with the potency to release toxic compounds from hepatic conjugates and plant glycosides, beta-glucuronidase and beta-glucosidase, respectively, were only detectable in small amounts. These findings indicate that Bacteroides vulgatus, which accounts for 40% of the total flora of patients with Crohn's disease, may play a role in the pathogenesis of Crohn's disease, by increasing the break-down of the mucus layer and therefore damaging its protective function.

Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. International Union Against Tuberculosis and Lung Disease, Tuberculosis in Animals Subsection.

The Tuberculosis in Animals Subsection of the International Union Against Tuberculosis and Lung Disease (IUATLD) recently identified a need to standardize the deoxyribonucleic acid (DNA) strain typing of Mycobacterium bovis. The standard method for strain typing of M. tuberculosis isolates cannot be directly extrapolated to M. bovis due to the low copy number of IS6110 identified in the majority of M. bovis strains, particularly from cattle. To improve the resolution of M. bovis strains, alternative methods and additional DNA probes have been investigated. In combination with studies of published literature, laboratories performing M. bovis DNA fingerprinting were surveyed. Results of these surveys allowed us to reach consensus and to make recommendations for DNA typing of M. bovis isolates, which hopefully will lead towards a standardized approach to the DNA fingerprinting of this organism. This approach, in conjunction with conventional epidemiological traceback approaches, should facilitate more accurate and effective investigations into the epidemiology, maintenance and transmission of M. bovis within and between man and domesticated, feral and wild animals, both at a local and a global level.

Estimation of serial interval and incubation period of tuberculosis using DNA fingerprinting.

OBJECTIVE: To determine the frequency distributions of serial interval and incubation period of tuberculosis within 4 years of transmission, and to identify correlates of serial intervals and incubation periods. METHODS: DNA fingerprints were obtained for all isolates from all culture-positive patients notified in The Netherlands from 1993 to 1996. Patient information was obtained from the National Tuberculosis Register. Results from contact investigations were provided by public health services. Source cases and secondary cases of tuberculosis were identified, based on 1) identical DNA fingerprints, and 2) epidemiological confirmation of contact. Under-representation of long intervals were corrected for by weighting cases. RESULTS: A total of 69 source-secondary case couples were identified. The geometric mean serial interval was 29.5 weeks (95% confidence interval [CI] 22.8-38.2 weeks) and the geometric mean incubation period 20.8 weeks (95% CI 15.5-27.8 weeks). Serial intervals and incubation periods tended to increase with age (P > 0.05). Three secondary cases with human immunodeficiency virus infection showed very short incubation periods (P > 0.05). CONCLUSION: Using a new methodology, the distribution of incubation periods of tuberculosis gave results consistent with earlier studies.

Molecular epidemiology of tuberculosis in Cuba outside of Havana, July 1994-June 1995: utility of spoligotyping versus IS6110 restriction fragment length polymorphism.

SETTING: Molecular typing has become an important tool for examining the extent of active transmission of tuberculosis. OBJECTIVES: To examine transmission of tuberculosis in Cuba using IS6110 restriction fragment length polymorphism (RFLP) typing and to evaluate the utility of spoligotyping. DESIGN: One hundred and sixty Mycobacterium tuberculosis strains isolated over a one year period in Cuba were subjected to RFLP and spoligotyping. RESULTS: Forty-eight percent of the isolates were found in 19 clusters of strains with identical RFLP patterns. In general, cluster sizes were limited, except for two large institutional outbreaks. Age was strongly inversely correlated to clustering. Most streptomycin-resistant isolates were found in clusters. Fifteen spoligotype clusters comprised 78% of the isolates. Significantly different IS6110 RFLP types subdivided 11 spoligotype clusters, whereas none of the IS6110 clusters were subdivided by spoligotyping. CONCLUSIONS: Considering the short study period, 48% clustering is high, indicating that recent transmission plays an important role in Cuba. Although resistance is still a minor problem, transmission of streptomycin-resistant strains occurs. The high polymorphism observed with IS6110 RFLP indicates that this marker is useful for future molecular epidemiological studies in Cuba. Spoligotyping appeared less suitable for population-based studies.

Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes.

Ninety-two Mycobacterium bovis isolates from cattle, deer and badgers in Northern Ireland and the Republic of Ireland were genotyped by spacer-oligotyping (spoligotyping) and 67 of these were analysed by restriction fragment length polymorphism (RFLP). RFLP analysis was performed using three DNA probes, PGRS, DR and IS6110. Forty-seven of the M. bovis isolates were from 45 different sources; these were typed using both RFLP and spoligotyping. These 47 isolates could be differentiated into 24 different RFLP types and 15 distinct spoligotypes. Although RFLP was found to be more discriminatory compared to the present spoligotyping technique, spoligotyping was able to differentiate 21 RFLP type 'ACA' isolates into three different patterns. The remaining 45 M. bovis isolates were from a small case study, involving infected cattle, deer and badgers from the same geographic region. All these isolates were analysed by spoligotyping and a selection of 20 isolates were RFLP typed. All the isolates in the case study had the same spoligotype pattern with the exception of one cervine isolate. Similarly all the isolates typed by RFLP had the same pattern. Consequently, the predominant strain in the case study was not host restricted. The consistency between the results obtained using the two techniques indicates the potential value of both techniques for epidemiological studies. Spoligotyping was found to be a much more rapid technique and easier to perform, requiring less sophisticated computer software for strain typing. Spoligotyping results were more readily documented and analysed and the technique was also more suitable than RFLP analysis for large-scale screening studies.

[Transmission of sensitive and resistant strains of Mycobacterium tuberculosis in The Netherlands, 1993-1995, studied by means of DNA fingerprinting]. / Transmissie van gevoelige en resistente Mycobacterium tuberculosis-stammen in Nederland, 1993-1995, onderzocht met DNA-'fingerprinting'.

OBJECTIVE: Determination of the contribution of recent transmission of both sensitive and drug-resistant Mycobacterium tuberculosis strains to the number of tuberculosis cases in the Netherlands. DESIGN: Descriptive study. SETTING: National Institute for Public Health and Environment, Bilthoven, the Netherlands. METHODS: Since 1993 all isolates of M. tuberculosis in the Netherlands are sent to the national reference laboratory for surveillance purposes. The strains with IS6110 DNA fingerprint, isolated from January 1993 to July 1995, were analysed for clustering of DNA patterns (clustering of identical DNA patterns was assumed to represent recent transmission of tuberculosis). A transmission index was calculated from the ratio of the number recently infected tuberculosis patients and the number of source patients. RESULTS: Among 2,217 M. tuberculosis isolates, 1,313 unique DNA fingerprints were observed, while 264 DNA patterns occurred more than once. 904 (41%) DNA fingerprints were part of a cluster of identical fingerprints. The mean cluster size was 3.42. The 232 resistant strains showed significantly less clustering (33% versus 42%, p < 0.02) and a smaller transmission index (0.27 versus 0.42, p < 0.02) compared with sensitive strains. CONCLUSION: Recent transmission contributes to the magnitude of the tuberculosis problem in the Netherlands. The epidemiological situation would, however, lead to gradual elimination of the disease were it not for introduction of tuberculosis from other countries. Transmission of resistant strains is relatively limited. Micro-epidemics caused by resistant M. tuberculosis strains were not observed.

[Recombinant DNA studies, a controversial subject which is also promising from the point of view of veterinary medicine (author's transl)]. / Recombinant DNA onderzoek; een controversieel onderwerp dat (ook diergeneeskundige) beloften inhoudt.

The hereditary characters of organisms are bound up in the DNA of their genomes. Genetic information is expressed by a complicated mechanism. The process by which this occurs and the control of the degree of expression in bacteria and a number of the other lower organisms are known only in broad outline. There usually is no exchange of DNA between organisms of unrelated species in nature. "Foreign" DNA can be linked to bacterial plasmid DNA and multiplied in the laboratory as a recombinant DNA molecule in bacteria using the recently discovered so-called restriction enzymes. Expression of bacterial DNA can thus be accomplished in a different, unrelated bacterium. In principle, this means that the natural barriers between unrealated species of organisms may be passed by. Human and animal DNA can also be multiplied in bacteria by this method. Previous recombinant DNA studies showed that the mode of expression of genetic information in the higher organisms differs markedly from that in bacteria. The initially sanguine expectations regarding the practical use of recombinant DNA research, for instance in the production of biologically important substances by bacteria, will therefore possibly not be realized at short notice. On the other hand this technique has added considerably to the knowledge of expression of genes within a short space of time. This is particularly true of the mechanisms of pathogenicity of E. coli, the bacterium which plays such an important role in veterinary medicine. The recombinant DNA technique is briefly reviewed in the present paper. The uses are broadly outlined, and one of these, which could be of importance in veterinary medicine, is described in greater detail. International discussions of potential hazards, and therefore also of the admissibility of this type of experiment, are griefly summarized.

[Reduction of the number of tetracycline-resistant strains of Salmonella in the Netherlands (author's transl)]. / Afname van het aantal tetracycline-resistente Salmonella stammen in Nederland.

Since 1974, tetracycline resistance in salmonellae of human and porcine origin has decreased nation-wide in the Netherlands. This decrease had coincided with the ban on incorporation of tetracycline in animal feeds for growth-promotion.