Complete nucleotide sequence of the Mycobacterium leprae 23 S and 5 S rRNA genes plus flanking regions and their potential in designing diagnostic oligonucleotide probes.

The complete nucleotide sequences of the Mycobacterium leprae 23 S and 5 S rRNA genes and their flanking regions are presented. As compared to other eubacterial homologous molecules the 23 S rDNA exhibits two insertions. A 16 nucleotide long insertion is almost unique to members of the genus Mycobacterium, while the second represents an extended version of helix 54. The potential of both insertions to serve as target for diagnostic oligonucleotide probes was proven by comparative sequence analysis of 23 S rRNA of several Mycobacterium species and by dot blot hybridization. In addition, a 19-mer oligonucleotide probe is described, which can be considered genus Mycobacterium-specific.

Doubling time of Mycobacterium lepraemurium in mouse footpads.

The doubling time of Mycobacterium lepraemurium (MLM) was measured in CBA/Ca mice. In eight experiments, 5 X 10(3) MLM were inoculated into the hind footpads of groups of mice, and the organisms were harvested from 1-45 days later. The harvested organisms were enumerated, and the doubling time was calculated assuming that MLM had multiplied without a lag phase and that multiplication continued at a constant rate from inoculation to harvest. Simultaneously, the proportions of viable organisms in the inocula were determined by inoculation of serially diluted suspensions into the footpads of other mice, harvesting 4 months later and calculating the most probable number. MLM were observed to multiply rapidly during the first several days, and more slowly thereafter; the mean initial doubling time was determined to be 0.5 days, a value much smaller than those previously reported by other workers.

Crossed immunoelectrophoretic analysis of Mycobacterium paratuberculosis.

Antigenic analysis of M. paratuberculosis revealed extensive cross-reactivity with M. avium; however, the number of cross-reactive antigens found was dependent on the strain of M. avium tested. One antigen was shown to be the common antigen while another appeared to be iron-regulated in its production. A commercial polyclonal antibody to M. paratuberculosis produced a CIE precipitin pattern comparable to that of the antibody produced for the present study. An antigen designated no. 6 was consistently precipitated by sera from cattle infected with M. paratuberculosis. This antigen exhibited complete cross-reaction with M. avium and partial cross-reaction with M. phlei. Among three commercially available complement fixation (CF) antigen that could be precipitated by M. paratuberculosis antibodies. A commercial antigen for use in an agar gel immunodiffusion test for Johne's disease diagnosis produced 12 precipitins with the M. paratuberculosis antibody, one of which was identical with antigen 6.

Cytokine-induced resistance to microbial infections in normal, immunosuppressed and bone marrow transplanted mice.

We studied the efficacy of in vivo and in vitro treatments with IL-1, IL-2, IL-3, and GM-CSF in the protection against bacterial (Salmonella typhimurium), fungal (Candida albicans) and viral (influenza virus A/PR8) infections, of normal, sublethally irradiated and lethally irradiated, bone marrow (BM) reconstituted mice. In parallel, the cytokines were tested for their ability to potentiate hematopoietic activity in vitro and in vivo. We demonstrate that, under the experimental conditions employed, IL-1 had the best protective activity against the three micro-organisms in both normal and immunocompromised mice when administered in vivo. Administration of IL-2 led to increased resistance in normal but not in immunodeficient mice, whereas GM-CSF had no beneficial effects. In contrast, preincubation of BM cells in these cytokines, singly or combined, prior to transplantation to lethally irradiated mice, did not confer protection against subsequent infection, although it increased the number of BM derived CFU-GM in culture (except in the case of IL-2). Administration of IL-1 or GM-CSF to BM transplanted mice facilitated WBC recovery, whereas IL-2 delayed it. Collectively, the data suggest that IL-1, alone or combined with other cytokines, may be beneficial in the prevention or treatment of microbial infections in immunocompromised and BM transplanted patients. It can also be concluded that enhanced hematopoietic recovery may not always coincide with the development of resistance to micro-organisms.

Development of sensitive methods for the detection of mycobacteria by DNA probes.

A sensitive method allowing the detection of mycobacteria by DNA probing has been developed. Besides the choice of a relevant probe encoding for part of the ribosomal RNA genes, the critical step allowing this high sensitivity was the method by which mycobacteria were lysed. Sonication of mycobacteria in the presence of chloroform followed by dot blot by this lysate gave the highest sensitivity in the detection of sequences homologous to the DNA probes.

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis.

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.

Identification and nucleotide sequence of Brucella melitensis L7/L12 ribosomal protein.

DNA sequencing of the gene encoding a Brucella melitensis 12-kDa protein revealed that this protein was the ribosomal protein L7/L12. The B. melitensis L7/L12 DNA sequence was identical to that of the corresponding B. abortus gene, showing the near identity of these two organisms. When comparing the sequence of this protein to that of other organisms some domains were highly conserved, especially the C-terminus, which contrasted with the lack of conservation of the sequences at the N-terminus. The finding that the ribosomal protein L7/L12 of Brucella is an immunodominant antigen provides a new rationale to explain the activity of ribosomal vaccines.

Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is an important animal pathogen that has also been implicated in human disease. The major proteins expressed by M. paratuberculosis were analyzed by two-dimensional gel electrophoresis, and a superoxide dismutase (Sod) was identified from this protein profile. The M. paratuberculosis Sod has a molecular mass of 23 kDa and an isoelectric point of 6.1. Sequence analysis of the corresponding sodA gene from M. paratuberculosis indicates that this protein is a manganese-dependent enzyme. We show that the M. paratuberculosis Sod is actively secreted, suggesting that it may elicit a protective cellular immune response in the host during infection.

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel.

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American from Israeli strains rejecting the possibility of an epidemiological link between S. iniae infections in the two countries. Israeli strains isolated from tilapia and trout could not be completely differentiated. The S. iniae ATCC 29178T (T = Type strain) strain, isolated from a freshwater dolphin belonged to a ribotype different from those of all the fish isolates.

Prevalence and correlates of diphtheria toxin antibodies among young adults in Israel.

The paucity of information about immunity against diphtheria of young adults in Israel prompted us to analyse sera from a random sample of 480 recruits (263 males and 217 females) aged 18-19 years. Antitoxin antibody levels were determined by means of ELISA. Of the recruits 58.1% had antibody values greater than 0.1 IU/ml; 38.5% had amounts between 0.01 and 0.1 IU/ml, which is considered low when using the ELISA method, and 3.3% had less than 0.01 IU/ml. The results of this study suggest that a booster dose of antidiphtheria vaccine should be given to adults in Israel in order to ensure adequate antibody levels.

Streptococcus agalactiae and Streptococcus difficile 16S-23S intergenic rDNA: genetic homogeneity and species-specific PCR.

Streptococcus difficile is a non-hemolytic Gram-positive bacterial coccus that causes septicemia and meningoencephalitis in farmed tilapia (Oreochromis sp.) and rainbow trout (Oncorhynchus mykiss). Recent studies have demonstrated S. difficile to be a group B, type Ib streptococcus with a whole cell protein electrophoretic profile indistinguishable from S. agalactiae and a biochemical profile similar to that observed for other group B, type Ib streptococci isolated from fish and frogs. The aim of this study was to expand on these findings by comparative nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of S. difficile and S. agalactiae. The 97.7% sequence homology identified in these studies supports the taxonomic relationship of these two organisms. The sequence data generated were also used to construct a pair of species-specific PCR primers for use in molecular detection and identification schemes.

Experimental streptococcal meningo-encephalitis in cultured fish.

In 1984 a disease of fish appeared in Israel which spread rapidly in cultured fishponds. The disease affected tilapia (Oreochromis aura x Oreochromis nilotica hybrids) and trout (Oncorhynchus mykiss). Common carp (Cyprinus carpus), although reared in community with tilapia were not susceptible to the disease. Various species of ornamental cyprinids and cichlids were also affected. Morbidity was high and mortality ranged between 50% (in trout) and 30% (in tilapia). Clinical and pathological findings indicated that the tilapia and trout suffered from meningitis and menigo-encephalitis. Two new streptococcal species, Streptococcus shiloi and Streptococcus difficile were isolated from diseased fish. The disease was reproduced experimentally in both trout and tilapia with the two streptococcal species. The LD50s of S. shiloi and S. difficile strains cultured in vitro (two to three passages on BHI medium) were 10(7)-10(8) cfu. The virulence of these strains was increased (LD50:10(2)-10(5) cfu) after three passages in vivo (brain to brain passage in fish without culture on agar plates). Highly virulent strains did not differ from low virulent strains by any identifiable extrachromosomal elements.

Yersinia enterocolitica among small wild mammals in France.

A survey of Yersinia enterocolitica was made in two French areas (Indre and Alsace) in several components of the terrestrial ecosystem. Y. enterocolitica has been isolated from several habitats and from numerous species of small mammals. The trap line method enabled us to estimate the density of the two abundant small mammal species, Microtus arvalis and Apodemus sylvaticus, and the densities of Y. enterocolitica carriers. The frequency of isolation was high when temperatures were low, but did not seem to be in direct relationship with animal densities. Infectious lesions were never found in spleens or intestines of 1893 captured animals. Samples of fresh water, fresh-water fish, soil and earthworms were occasionally collected. A total of 163 strains were isolated. Of these, 99 were of biochemical type 1; 20 different 0 serogroups were found.

Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout.

Formalin killed bacteria were used as a vaccine against Streptococcus iniae infections in farmed rainbow trout. A single intraperitoneal injection of this vaccine in trout resulted in specific antibody production detectable for 6 months. Trout vaccinated at 50 g were protected under laboratory (experimental disease) and field conditions (natural disease) for at least 4 months against S. iniae infection. Passive transfer of S. iniae specific antibodies conferred protection. Under field conditions, mortality of non vaccinated trout exceeded 50%, whereas mortality of vaccinated trout did not reach 5%. In addition, vaccinated trout under field conditions gained 20% weight when compared with non vaccinated fish.

Polymyxin B reduces total parenteral nutrition-associated hepatic steatosis by its antibacterial activity and by blocking deleterious effects of lipopolysaccharide.

Overgrowth of Gram-negative bacteria as a result of total parenteral nutrition (TPN) and bowel rest could be responsible for the release of a variety of hepatotoxic substances such as endotoxin or tumor necrosis factor (TNF) and the ensuing TPN-associated liver function derangements. Polymyxin B is an effective antimicrobial agent as well as a blocking agent for endotoxin (lipopolysaccharide) activity and TNF production. In the present study we compared the oral and intravenous effects of polymyxin in rats receiving TPN in an attempt to define these two possible mechanisms of action of polymyxin on TPN-associated hepatic steatosis. Both oral, as well as intravenous polymyxin B, significantly reduced total hepatic fat and triglyceride accumulation in TPN rats, more so in the intravenous group exhibiting close to control levels. Both polymyxin-treated groups exhibited significantly lower Gram-negative bacterial counts in the cecum, with the oral group exhibiting a lower count than the IV group. The spontaneous production of TNF by peritoneal macrophages was markedly increased in rats receiving TPN and very close to being undetected in both groups receiving TPN and polymyxin. We believe polymyxin B protects the liver during TPN by both its antimicrobial effect which prevents overgrowth of gut Gram-negative bacteria and the subsequent translocation of endotoxin, and by its specific antilipopolysaccharide activity which, in the present study, completely abolished hepatic steatosis and TNF production during TPN.

Antitumor necrosis factor antibodies reduce hepatic steatosis during total parenteral nutrition and bowel rest in the rat.

BACKGROUND: In previous studies, we demonstrated the overgrowth of gram-negative bacteria in the gut and an enhanced release of tumor necrosis factor (TNF) by peritoneal macrophages, suggesting that endotoxin, TNF, or both, may act as hepatotoxins to produce hepatic steatosis during total parenteral nutrition (TPN) and bowel rest. The present study attempts to better define the role of each of these two mediators. The first part examines the LD50 for various doses of endotoxin in TPN-treated rats compared with free-feeding and free-feeding saline-infused rats. In the second part we repeatedly administered anti-TNF monoclonal antibodies to rats subjected to TPN and bowel rest. METHODS: In the first set of experiments, 87 male Sabra rats were randomized into three groups: free-feeding, infused with normal saline, and infused with TPN. On day 7 of the experiment, all rats received an IV injection of endotoxin at various doses (1.5, 2.5, 5.0, 7.5, and 10 mg/kg). The LD50 in the three groups and at the various doses of lipopolysaccharide tested was determined at 24 hours postinjection. In the second set of experiments, 38 male Sabra rats were randomized into three groups: infused with normal saline and fed rat food ad libitum, infused with TPN, and infused with TPN but also receiving monoclonal antibodies against TNF. RESULTS: Lower endotoxin doses were required to achieve LD50 in the two IV-infused groups (2.5 to 5.0 mg/kg) compared with the free-feeding group (7.5 mg/kg) (p < .03). These findings suggest a moderate increase in susceptibility to the lethal effect of endotoxin in IV-treated rats. The total hepatic fat and triglyceride levels, which were markedly increased in TPN rats, were significantly reduced by using anti-TNF antibodies. Enhanced TNF production by peritoneal macrophages during TPN was completely eliminated by anti-TNF antibodies, probably the result of suppressed TNF production. CONCLUSIONS: The continuous translocation of endotoxin from gram-negative bacterial overgrowth in the gut during TPN and bowel rest results in enhanced release of TNF by macrophages. TNF causes hepatic dysfunction, portrayed in the present experimental model as hepatic steatosis. TPN-induced hepatic steatosis was significantly reduced by the administration of monoclonal antibodies against TNF-alpha.

Pentoxifylline and thalidomide fail to reduce hepatic steatosis during total parenteral nutrition and bowel rest in the rat.

BACKGROUND: We suggested that the continuous translocation of endotoxin from Gram-negative bacterial overgrowth during bowel rest and total parenteral nutrition (TPN) causes the release of tumor necrosis factor (TNF), resulting in liver damage and hepatic dysfunction. Because TPN-induced hepatic steatosis was significantly reduced by the monoclonal antibodies against TNF, we attempted a more clinically applicable approach using pentoxifylline and thalidomide. METHODS: A control group (group I) fed rat chow and four groups of rats receiving TPN were studied. Group II received TPN only; group III, TPN and 100 mg/kg/d pentoxifylline; group IV, TPN and 200 mg/kg/d pentoxifylline; and group V, TPN and 5 mg/kg/d thalidomide. On day 7, total liver fat was determined. RESULTS: Bowel rest and TPN resulted in a significant (p < .0005) increase in liver fat content that was unaltered by either pentoxifylline or thalidomide. CONCLUSIONS: Our results show no role for pentoxifylline or thalidomide in reducing TPN-associated hepatic steatosis.

Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection.

We isolated for the first time Streptococcus iniae strains associated with diseased marine fish. Diseased red drum Sciaenops ocellatus were lethargic, and presented external signs (exophthalmia and loss of orientation) resembling those of freshwater fish infected by S. iniae. Skin lesions, extending to a necrotizing myositis, were typical of S. iniae infection of red drum. Histopathological findings indicate that S. iniae infection in red drum produces a chronic disease with systemic involvement characterized by multiple necrotic foci. Molecular epidemiology (RFLP [restriction fragment length polymorphism] ribotyping) revealed that 2 different ribotypes were involved in a single outbreak. The first is the EcoRI 'Israeli' trout and tilapine ribotype (Hind III type a strains), while the second is the EcoRI 'American' ribotype (Hind III type b strains), typical of tilapines farmed in Texas and Idaho.

Haemophagocytosis and other haematological aspects of Mycobacterium lepraemurium disease of mice.

CBA mice inoculated i.v. with 2 x 10(8) viable MLM were observed at intervals for 5 months. In the bone marrow, haematopoietic cells were progressively displaced by MLM-laden phagocytes; depletion of erythroid cells began earlier and was more pronounced than that of myeloid cells. Transiently, mild anaemia and profound leucopenia were noted. The spleen was enlarged and the site of increasing histiocytosis and extramedullary haematopoiesis which was accompanied by displacement of splenic parenchyma. The liver was enlarged and its parenchyma contained scattered islands of haematopoietic elements. Lymph node cells had been largely replaced by MLM-laden macrophages by the end of the process. Thus, MLM infection exerts important effects on haematopoiesis of susceptible mice and is accompanied by active extramedullary haematopoiesis. In addition, the haemophagocytosis, observed most commonly in immuno-compromised patients infected with certain viral or mycobacterial pathogens, was observed late in the course of MLM disease of mice.

Mycobacterial infections in domestic and wild animals due to Mycobacterium marinum, M. fortuitum, M. chelonae, M. porcinum, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. simiae and M. genavense.

The epidemiology and the natural distribution of Mycobacterium marinum, M. fortuitum, M. chelonae, M. porcinum, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. simiae and M. genavense are described. In addition to the bacteriological, biochemical and genetic characteristics, the authors review the pathology of these species, including the natural and experimental diseases and the accompanying lesions, diagnosis, antibiotic sensitivities and treatment of animal infections caused by these mycobacteria.