Glycolipid antigen for use in diagnostic assays for bovine tuberculosis.

A glycolipid antigen, was isolated, purified and characterized from Mycobacterium bovis An5. Chemical analysis (thin-layer chromatography, nuclear magnetic resonance and infrared spectra) showed that this glycolipid was a 2,3-di-O-acyl trehalose (DAT), similar to the DAT of M. tuberculosis. This antigen was used to establish ELISA-based serodiagnostic tests for M. bovis-infected cattle. The sensitivity and specificity of the assay were investigated using sera of cattle from tuberculosis-free herds and from tuberculosis-infected herds. No correlation was found between DAT-ELISA and the skin test, nor between DAT-ELISA and interferon-gamma with bovine purified protein derivative. The antibody titres were not related to cell-mediated immunity. Although the antigen was highly specific (95.9%), the sensitivity of DAT-ELISA, as judged from assays in bacteriologically confirmed tuberculosis, was low (29 to 36.8%). The low sensitivity of ELISA might also be attributed to a reciprocal relationship between B-cell proliferation and T-cell protective immunity.

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

Molecular characterization of environmental mycobacterium strains by PCR-restriction fragment length polymorphism of hsp65 and by sequencing of hsp65, and of 16S and ITS1 rDNA.

Fifteen mycobacterial strains from the environment, not clearly identifiable by biochemical properties, were analyzed with molecular markers: PCR-restriction enzyme analysis of hsp65 and sequencing of hsp65, and of the internal transcribed spacer 1 (ITS1) and 16S rDNA. The 16S rDNA sequencing closely related the strains to a slow-growing mycobacterial group including Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium genavense, Mycobacterium triplex and Mycobacterium heidelbergense. A stretch of T bases at the level of 16S rDNA enabled the separation of M. simiae and M. lentifiavum from M. genavense, M. triplex and M. heidelbergense; hence the attribution of some environmental strains to the first or second group. But the distances between the two clades were very short and the relative positions of environmental strains and of reference strains were not resolved in terms of node robustness (low bootstrap values) in the distance tree. However, the hsp65 restriction profiles suggested assigning six strains to the M. lentiflavum species, although these strains had been found closely related to M. genavense and M. triplex from 16S rDNA nucleotide signatures. The clustering of environmental strains into the same three clusters was deduced from analysis of three sequence data (hsp65, and ITS1 and 16S rDNA), but the taxonomic affiliation of environmental strains to reference strains remained tentative. Among environmental strains and reference strains, the distances found from hsp65 sequences had the same amplitude as those found between different strains of Mycobacterium gordonae. From ITS1 rDNA sequences, the distances found between the strains of the Mycobacterium avium complex also had the same amplitude as those found between environmental strains and reference strains. From our results, it appears that the environmental strains and the reference strains could constitute a complex of subspecies or closely related species. Their taxonomic status must be confirmed by DNA/DNA hybridization experiments.

Isolation and characterization of serologically reactive lipooligosaccharides from Mycobacterium tuberculosis.

Two major highly polar antigenic glycolipids were isolated from recent isolates of Mycobacterium tuberculosis from a wide range of geographical origins. The occurrence of these polar glycolipids was demonstrated by isolation, purification and chromatographic characterization and/or serological procedures in 12 strains. Based on their chromatographic properties, these polar glycolipids belong to the lipooligosaccharide family. Preliminary data on the use of these newly described antigens in the serodiagnosis of tuberculosis is presented.

High frequency ultrasound in the preoperative staging of primary melanoma: a statistical analysis.

High frequency sonography has been shown to be a useful tool in the preoperative staging of malignant melanoma. In the present study sonometric and histometric data concerning tumour thickness were compared, using appropriate statistical methods, in order to assess the accuracy of ultrasonography. From December 1997 all pigmented lesions suspected of being melanoma were preoperatively assessed by a 20 MHz ultrasound B scan. The results of these ultrasound examinations were compared with histometric data. Pearson's correlation coefficient and absolute and relative differences were used for statistical analysis. Of the 261 examined lesions, 193 were malignant melanoma. A high correlation between sonometry and histometry was computed (r = 0.95), with an absolute difference of 0.32 +/- 0.03 mm (mean +/- SEM) and a mean relative difference of 27.2% (95% confidence interval 23-31.4%). The highest correlation was found in melanoma > or = 1.51 mm thick and the lowest correlation in melanoma < or = 0.75 mm. In conclusion, the high accuracy of this technique in the preoperative staging of malignant melanoma would offer a basis for defining the surgical margins of > or = 0.76 mm thick lesions. The limited accuracy of sonometry in the preoperative staging of thin melanoma < or = 0.75 mm has emerged by applying adequate statistical methods.

Toxicity and activity of docetaxel in anthracycline-pretreated breast cancer patients: a phase II study.

Docetaxel has proven effective in advanced breast cancer. Myelosuppression and cumulative fluid retention syndrome are troublesome, potentially avoidable toxicities. In this consecutive cohort study, docetaxel (100 mg/m2 by 1 hour i.v. infusion, q3 weeks) activity and toxicity was explored in 56 anthracycline-pretreated patients (eligible: 55: median age: 51 years [range: 28-68 years]; median performance status: 0 [range: 0-3]) with metastatic breast cancer, using two different granulocyte colony-stimulating factor and steroid pre- and postmedication schedules. Twenty-nine patients (group A) received a 5-day oral prednisone premedication, and 26 (group B) received 4-day low-dose i.m. dexamethasone; group B patients also received prophylactic granulocyte colony-stimulating factor. All patients were evaluable for toxicity and 53 for response. Prophylactic granulocyte colony-stimulating factor significantly lowered the incidence of grade III-IV neutropenia and neutropenic fever (p = 0.0001 and 0.01, respectively). The incidence of moderate-severe fluid retention syndrome was lower in patients receiving i.m. dexamethasone (p = 0.08). Overall response rate was 53% (4 complete responses/24 partial responses, 95% confidence interval 39.4-66.2%); 32% have stable disease and 15% progressive disease. In 21 anthracycline-refractory/resistant patients, as well as in 10 paclitaxel-pretreated patients, the overall response rate was 50%. Docetaxel is highly active in anthracycline- and paclitaxel-pretreated metastatic breast cancer, with manageable toxicity. Optimal use of both granulocyte colony-stimulating factor support and steroid premedication deserves further investigation.

[Use of the in vitro enzymatic amplification method for the detection of Mycobacterium paratuberculosis in feces]. / Utilisation de la méthode d'amplification enzymatique in vitro pour la détection de Mycobacterium paratuberculosis dans les fèces.

A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.

Mycobacterium avium and Mycobacterium intracellulare infection in mammals.

Mycobacterium avium subsp. avium and M. intracellulare are ubiquitous organisms in the environment. The reservoir of M. avium subsp. avium is generally accepted to be environmental, in particular, water and soil are sources of the organism. In contrast to M. avium infection in wild and domestic birds, M. avium infection in mammals occurs only sporadically and is rarely transmissible. Generalised disease is usually uncommon, owing to the non-progressive, chronic character of the infection. However, some cases of disseminated disease have been reported, e.g. in captive non-domestic hoofed animals as well as in immunosuppressed dogs and cats. The majority of M. avium and M. intracellulare infections in livestock are detected at slaughter and the diagnosis is confirmed by bacteriological procedures. Condemnation of affected portions of the carcass can result in significant economic losses, although gross lesions are mostly restricted to lymph nodes close to the alimentary tract. Successful treatment with antibiotics in combination with surgery has been reported in some affected domestic cats, but is not considered to be effective or economical in other species. In the past, differentiation of M. avium bacteria from the closely related M. avium subsp. paratuberculosis was based on the mycobactin dependence and prolonged incubation period of the latter. More recently, amplification of the genomic insertion sequence IS900 has proved to be a powerful tool for identification of M. avium subsp. paratuberculosis. The potential zoonotic importance of M. avium infections has been indicated, but requires clarification.

[Pathogenic organisms in milk and milk products: the situation in France and in Europe]. / Les germes pathogènes dans le lait et les produits laitiers: situation en France et en Europe.

Milk and dairy products harbour a natural microbial flora and/or other micro-organisms, which vary within the wide range of products available on the French market. The origin of contamination by pathogenic bacteria varies with the type of product and the mode of production and processing. Contamination of milk and dairy products by pathogenic micro-organisms can be of endogenous origin, following excretion from the udder of an infected animal. Contamination may also be of exogenous origin, through direct contact with infected herds or through the environment (e.g. water, personnel). Treatment and processing of milk can inhibit or encourage the multiplication of micro-organisms. The authors describe the relevant aspects of bacterial physiology and ecology, the occurrence of bacteria in dairy products, and the public health significance for each of the principal micro-organisms found in such products. Bacteria most frequently involved are mycobacteria, Brucella sp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella). At present, systems of testing and surveillance are required for the control of pathogenic bacteria in milk and dairy products, as specified by regulations currently being developed for all countries in the European Union. Preventive measures should take into account the well-established facts concerning the potential microbiological impact of pathogenic bacteria on milk and dairy products. There should be increased recourse to risk analysis methods to assess the threat to the consumer with regard to the presence of pathogenic bacteria in food.

[Action of 4 percent sulfuric acid on various species of mycobacteria]. / Action de l'acide sulfurique à 4 p. cent sur les diverses espèces de mycobactéries

The action of treatment with 4 p. cent sulphuric acid for 10 minutes, was tested on 13 strains of mycobacteria. Rapidly growing mycobacteria, e.g. M. diernhoferi, M. chelonei M. abscessus, M. Fortuitum, proved much more sensitive than slowly growing mycobacteria, e.g. M. xenopi, M, scrofulacem, M. kansaii, M. avium, M. marinum, BCG, M. tuberculosis and M. Bovis. We observed the persistance of a certain number of viable bacteria in spite of a duration of contact of 120 minutes, Albumin in the culture medium used presented a protective effect together with the high concentration in bacteria.

[Mycobacteria identified in a centre for veterinary research between 1973 and 1979 (author's transl)]. / Mycobactéries identifiées dans un centre de recherches vétérinaires de 1973 à 1979.

During the years 1973 to 1979 the Central Laboratory of Veterinary Research in Alfort identified 425 strains of mycobacteria: 93 were pure cultures received from other laboratories, and the remaining 332 were isolated in this laboratory from 590 pathological specimens of different origin and from 12 samples from the environment. Sixteen species were identified, using biochemical criteria and drug sensitivity. "Tuberculous" mycobacteria (Mycobacterium bovis, M. tuberculosis and M. africanum) where the most frequent (58.5%), and "non-tuberculous" mycobacteria (41,5%) were identified as M. avium-intracellulare (136 strains), M. peregrinum (10 strains), M. paratuberculosis (5 strains), M. terrae (5 strains), M. chelonei (4 strains), M. fortuitum (2 strains), and M. aurum, M. vaccae, M. marinum and M. gordonae (1 strain of each). From the specimens received for isolation the more frequent organisms isolated were M. bovis (55%) and M. avium-intracellular (32%).

Isolation of Mycobacterium africanum from monkeys.

The first isolation of M. africanum from monkeys is reported. This finding leads the author to discuss the origin of the infection in monkeys, and to underline the potential public health hazard that animals may present for humans.

Relationship between Mycobacterium avium, M. paratuberculosis and mycobacteria associated with Crohn's disease.

Recently some mycobactin-dependent mycobacteria were isolated from patients with Crohn's disease. These mycobacteria should be very similar to M. paratuberculosis which is closely related to M. avium. The author has investigated the relationship between M. avium, M. paratuberculosis and the mycobacteria associated with Crohn's disease.

[Use of two dimensional immunoelectrophoretic method in the study of the antigenic relationships of "Mycobacterium simiae" and "M. habana" (author's transl)]. / Utilisation d'une méthode d'immunoélectrophorèse bidimensionnelle dans l'étude des antigènes de Mycobacterium simiae et M. habana

Following the cultural, biochemical and serologic studies reported elsewhere, it was thought of interest to investigate the antigenic structure of these strains. In the present work, sonicated suspensions of the bacteria were used as antigens, together with rabbit immune serum antibodies. The optimal conditions to obtain the largest number and the sharpest lines possible, were established. In this manner, we were able to obtain on two dimensional electrophoresis patterns were obtained. Using an anti-M. habana 4238 antiserum, 36 lines of precipitation were obtained with M. habana 4238, 41 lines with M. simiae 29 and 29 lines with for M. simiae 59-IX-7. Subsequently, identity tests were performed to verify the occurrence of were common antigens. These tests revealed such commons antigens in all the three strains. The comparison between M. habana and M. simiae 29 showed the occurence of at least one Precipitation line, that is characteristic of M. habana. These results appear to agree with those of Meissner, who obtained a small amount of anti-M. habana specific agglutinins. However, this worker did not believe that this was sufficient evidence to separate M. simiae 29 from M. habana. The antigenic differences between M. habana and M. simiae 59-IX-7 are more significant and appear to justify their differentiation into 2 serotypes: M. habana 4238 and M. simiae 29 being of serotype 1, M. simiae 59-IX-7 of serotype 2. The difficulties experienced in the separation of the antigenic fractions in the immunoelectrophoretic diagrams, led to consider the purification of the antigens and then to attempt to isolate the specific antigenic fractions.

Review of the occurrence of mycobactin dependence among mycobacteria species.

Most mycobacteria are able to make mycobactin for themselves. But, Mycobacterium paratuberculosis, M. avium atypical like wood-pigeon mycobacteria and some strains of M. avium typical for the primary isolation, lack this capacity and require mycobactin for growth in the laboratory.

[Comparative study of serotypes of "Mycobacterium avium" isolated from human and animal (author's transl)]. / Note sur l'étude comparative des sérotypes des souches de Mycobacterium avium isolées de l'homme et de l'animal.

The large number of avian strains isolated from pathological samples lead us to use serologic method in order to compare the serotypes of animal and human strains. The results show that the serotypes identified in strains from man (1, 2, 8, 9, 12, 14, 16 and 20) are more numerous than those identified in strains from animals (1, 2, 3, 4 and 8), and that the serotypes 1, 2 and 3 are found in animals more often (98%) than in man (47%). The distribution of the identified serotypes are similar to Piening et al's and Schaefer's but with different proportion, and inverse to Nemoto's and Tsukamura's. The transmission of M. avium from animal to man by the intermediary of the environment is discussed.

[Several strains of Mycobacterium paratuberculosis: biochemical characteristics and allergic activity]. / Etude de quelques souches de Mycobacterium paratuberculosis: caractéres biochimiques et activité allergique

Judged from its biochemical properties and antigenic structure, Mycobacterium paratuberculosis is related to M. avium. When M. paratuberculosis was compared to B.C.G., several essential differences, were found that allowed their clear separation, within Runyon's Group III, M. paratuberculosis is differentiated by its biochemical properties as shown in the following table : (formula: see text). The allergic response of a guinea pig infected with M. paratuberculosis was stronger when an avium sensitine was used that when a bovine tuberculine was used.

[Comparative study of mycobactin-dependent strains of mycobacteria isolated from the wood-pigeon with Mycobacterium avium and M. paratuberculosis: study of biological and antigenic characteristics]. / Etude comparative de souches de mycobactéries mycobactine-dépendantes, isolées de pigeon ramier, avec Mycobacterium avium et M. paratuberculosis: étude des caractères biologiques et antigéniques.

In this investigation three mycobacterial strains isolated in our laboratory from wood-pigeons were compared with one strain isolated by Matthews and another by Jorgensen from, respectively, a wood-pigeon and a roe-deer. The strains were also compared with various strains of Mycobacterium avium and M. paratuberculosis. The strains isolated from the wood-pigeons formed a relatively homogeneous group, which could be distinguished from M. avium and M. paratuberculosis. It was interesting to verify that most of the cultural and biochemical properties of the wood-pigeon mycobacteria were similar to those of M. paratuberculosis. The strains formed rough colonies and grew slowly in special mediums containing M. phlei extracts or mycobactin. However, one must recall that mycobactin dependence was also reported by Matthews for some strains of M. avium. The tween hydrolysis test (10 days), negative for M. avium, was positive for both the wood-pigeon mycobacteria and M. paratuberculosis. The trehalase test, which appears to be regularly positive for M. avium, was also positive for the wood-pigeon mycobacteria and M. paratuberculosis. In respect to drug susceptibility, no significant differences were observed. The organisms were resistant to most drugs studied, but were also resistant to cycloserine, in contrast to most M. avium strains. On the other hand, the organisms were not distinguished using sensitins, prepared from wood-pigeon mycobacteria and M. paratuberculosis using specifically sensitized guinea-pigs. However, the wood-pigeon mycobacteria could be clearly differentiated from M. paratuberculosis and M. avium using serology methods. Indeed, the wood-pigeon mycobacteria did not agglutinate in the presence of any of the sera defining the M. avium-intracellulare serovars (serovars 1 to 28); and by immunodiffusion in agar the wood-pigeon mycobacteria antigens formed two precipitation lines which were absent from M. paratuberculosis. Judging from our findings, the wood-pigeon mycobacteria are more closely related to M. paratuberculosis than to M. avium. It appears that biochemical and antigenic properties are not sufficient to completely differentiate these bacteria. Further studies are needed, and we plan to investigate in the near future their pathogenicity for rabbits, chicken and calves.

Specific surface antigens of SmT variants of Mycobacterium avium.

SmT variants of Mycobacterium avium ATCC 15769 formed novel antigens which were absent in SmD variants.