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.

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.

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.

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.

Translocation of an ampicillin resistance determinant within an R-factor aggregate in Salmonella panama.

The molecular properties of the plasmids of a natural isolate of Salmonella panama have been studied. This strain, Sp477, harbours 5 different plasmids: the conjugative plasmid pRI477TF (molecular weight 20 megadaltons), the two non-conjugative plasmids, pRI477A and pRI477S, coding for ampicillin and streptomycin plus sulfonamide resistance respectively (molecular weights of both 5.6 megadaltons) and two cryptic plasmids with molecular weights of 1.0 and 2.7, megadaltons respectively. After conjugal transfer to Escherichia coli the ampicillin resistance determinant was frequently found to be integrated into pRI477TF or pRI477S. The translocatable sequence on pRI477A, designated as Tn901, resembles the TnA sublcass transposon TnA(1).

Penicillinase-producing Neisseria gonorrhoeae in the Netherlands: epidemiology and genetic and molecular characterization of their plasmids.

Penicillinase-producing Neisseria gonorrhoeae strains were isolated in the Netherlands with increasing frequency during the period of 1976 to 1979. About 3% of the gonococci isolated in the first half of 1979 produced penicillinase. In contrast to the period of 1976 to 1977, most penicillinase-producing N. gonorrhoeae infections during the period of 1978 to 1979 were contracted in the Netherlands. The results of genetic and molecular studies on 80 penicillinase-producing N. gonorrhoeae strains were similar to earlier observations of others: resistance plasmids of only two sizes, 4.5 and 3.3 megadaltons (Md), occurred in penicillinase-producing N. gonorrhoeae strains, and these encoded for the TEM-1 enzyme. The 4.5-Md plasmid could be transferred to Escherichia coli when it coexisted with a plasmid of 24 Md. The latter plasmid was present in the vast majority of the strains carrying the 4.5-Md plasmid. One strain carried a cryptic 7.5-Md plasmid in addition to the commonly found 2.5-Md plasmid. Two penicillinase-producing strains of Haemophilus parainfluenzae isolated were found to carry a 3.3-Md plasmid species which was indistinguishable from the 3.3-Md gonococcal resistance plasmids. No plasmid deoxyribonucleic acid was found in two strains of penicillinase-producing Branhamella catarrhalis, and these strains produced a penicillinase different from the TEM-1 enzyme.

Cloning and expression of a deoxyribonucleic acid fragment that encodes for the adhesive antigen K99.

Deoxyribonucleic acid fragments of the naturally occurring conjugative K99 plasmid were cloned into vectors pBR322 and pBR325. The smallest deoxyribonucleic acid segment obtained that still expressed K99 was 4.5 megadaltons in size. With regard to the serological, adhesive, and morphological properties, no differences in the nature of the K99 antigen was observed between Escherichia coli strains carrying recombinant plasmids and those carrying pRI9901. Furthermore, the regulation of K99 expression from the recombinant plasmid deoxyribonucleic acid was similar to that from pRI9901. the low level of K99 expression in E. coli K-12 compared with natural K99-producing isolates seems to be host specific.

Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment.

A polymerase chain reaction with nested primer pairs based on the DNA sequence of the 39-kDa bmp gene of Treponema pallidum subsp. pallidum is described. The method allowed the detection of purified T. pallidum DNA equivalent to the amount of DNA in a single bacterium and was specific for T. pallidum subspecies. After concentration of DNA, using diatomaceous earth, it was possible to detect about 100 treponemes in 1 ml of cerebrospinal fluid. Cerebrospinal fluid samples from a total of 29 symptomatic and asymptomatic patients with neurosyphilis were tested for the presence of treponemal DNA before and at various intervals after intravenous treatment with penicillin. Prior to the penicillin treatment, we detected T. pallidum DNA in 5 of 7 patients with acute symptomatic neurosyphilis, in none of the 4 patients with chronic symptomatic neurosyphilis tested before treatment, and in 2 of 16 patients with asymptomatic neurosyphilis. Unexpectedly, T. pallidum DNA was also often detected in cerebrospinal fluid long after intervenous treatment with penicillin, sometimes up to 3 years after therapy.

Polymerase chain reaction and synthetic DNA probes: a means of distinguishing the causative agents of syphilis and yaws?

Synthetic DNA probes specific for either the tpf-1 gene of Treponema pallidum subsp. pallidum Nichols or the tyf-1 gene of Treponema pallidum subsp. pertenue CDC 2575 were used for hybridization with in vitro-amplified chromosomal DNAs of 10 different Treponema isolates. tpf-1 and tyf-1 differ only in one nucleotide at residue 123, and three of four syphilis strains were of the Nichols type, whereas five of six yaws strains were of the CDC 2575 type, indicating that the nucleotide at position 123 of the tpf-1 or tyf-1 gene is not a definitive trait for either T. pallidum subspecies.