RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.

The aim of this study was to assess whether interleukin-10 (IL-10) and/or transforming growth factor beta-1 (TGFbeta1) downregulate HLA-DR expression using the HT29 cell line as a model of colonic epithelial cells. HLA-DR expression was induced in HT29 cells with gamma-interferon. The effects of IL-10 alone, TGFbeta1 alone, and IL-10 and TGFbeta1 in combination were studied. HLA-DR expression was assessed using flow cytometric analysis. Gamma-interferon induced HLA-DR expression in a dose-dependent fashion. In the absence of gamma-interferon, neither IL-10 nor TGFbeta1 induced HLA-DR expression. In isolation, neither IL-10 nor TGFbeta1 downregulated HLA-DR expression. When IL-10 and TGFbeta1 were added in combination, small (6-30%) statistically significant reductions in HLA-DR expression were seen. The biological significance is unclear.

Carrion is a major source of botulinal toxin for animals. A type D strain of Clostridium botulinum differed from type A and B strains in producing (1) much higher concentrations of toxin in putrefying mouse carcasses at several different temperatures over a period of 35 days, (2) toxicity that sometimes persisted in mouse carcasses for at least a year, and (3) mouse carcasses with exceptionally high oral toxicity. Fish carcasses were much less favourable than mouse carcasses for type D toxigenesis. The study, together with earlier studies on types C and E, indicated that carrion contaminated with C. botulinum type C or D is likely to be particularly dangerous for animals that may ingest it. This accords with the observation that carrion-transmitted botulism in animals is usually caused by type C or D.

Botulism due to Clostridium botulinum type C causes considerable mortality in gulls in the UK, and refuse disposal sites are suspected as a major source of toxin. C. botulinum types B, C and D were each found in 12 (63.2%) of 19 landfill sites examined. Type E was detected in only one (5.2%) and types A, F and G were not found. The prevalence of type C spores was much higher than that demonstrated in the UK environment by earlier surveys. The presence of these spores, together with the rotting organic matter and generated heat associated with landfill sites, undoubtedly leads to bacterial proliferation and toxigenesis. This is likely to result in botulism in scavenging gulls unless skilled landfill management prevents the ingestion of toxic material. Type D spores were previously shown to be rare in the UK environment and their high prevalence on landfill sites was therefore surprising. Four composite samples of refuse collected before distribution on a landfill gave negative results for C. botulinum and it seems likely that the gulls themselves play a major role in introducing contamination.

Necrobacillosis occurs in man and animals. The typical forms of the disease in animals are caused by Fusobacterium necrophorum biovar A; biovar B strains are much less pathogenic. In this study the pathogenicity for mice of eight human isolates of F. necrophorum was compared with that of animal biovar A and B strains. By subcutaneous inoculation seven of the human strains differed from biovar A but resembled biovar B in (1) producing, at the most, mild local lesions that rapidly healed, and (2) showing no enhancement of infectivity when suspended in sub-lethal doses of Staphylococcus aureus broth culture. The eighth human strain (A2433) resembled biovar A but differed from biovar B in (1) producing severe lesions, and (2) showing greatly enhanced infectivity in the presence of S. aureus. Nonetheless, strain A2433 differed from biovar A, both in the nature of the lesions produced and in its failure to cause severe general signs of illness and rapidly fatal infection. By intravenous inoculation one of two biovar B strains and all except one of the eight human strains produced purulent lesions, often severe, in the liver and elsewhere, but infection was not usually associated with general signs of illness. In contrast, intravenous injection of a biovar A strain gave rise to a rapidly fatal infection, with severe lesions in the liver or elsewhere. The results suggest that the term 'necrobacillosis' as used in human and veterinary medicine refers to diseases that differ in important respects.

Oral pretreatment of mice with either a mixture of kanamycin and erythromycin or metronidazole to modify the gut microflora greatly enhanced the faecal excretion of Fusobacterium necrophorum biovar A given by mouth. This lends support to the suggestion that disturbance of the gastrointestinal microflora in animals such as cattle, which often carry the organism in the rumen, may lead to intestinal multiplication and faecal excretion, thereby providing a source of infection that may lead to necrobacillosis of the body surface.

Only a small proportion of animals tested were found to be excreting Fusobacterium necrophorum biovar A, the causative organism of necrobacillosis, in the faeces (3 of 69 wallabies, 1 of 66 deer, 2 of 81 cattle). The two positive cattle belonged to a single group of calves on a farm with a history of necrobacillosis and the litter underfoot also readily yielded biovar A organisms. All attempts to demonstrate biovar A in litter on other farms and in soil from an area populated by wallabies and deer failed. Ruminal contents from young beef cattle proved a fertile source of F. necrophorum biovar A, 15 of 18 animals giving a positive result. It is suggested that disturbance of the gastrointestinal microflora leads to intestinal multiplication and faecal excretion of the organism, which may then give rise to necrobacillosis of the body surface.

It had already been shown with a single virulent strain (A42) of Fusobacterium necrophorum that suspension of the fusobacteria in sub-lethal doses of broth cultures of other bacteria reduced the minimum infective dose (greater than 10(6) organisms) for mice by subcutaneous inoculation, sometimes to less than 10 organisms. The present study extended the known range of bacteria with strong infectivity-enhancing properties to include Bacillus cereus, Klebsiella oxytoca and Staphylococcus aureus; and those with weaker effect to include Bacillus subtilis, 'Bacteroides melaninogenicus', Clostridium sporogenes, Pasteurella haemolytica, and Proteus mirabilis. The study also showed that five further virulent strains of F. necrophorum closely resembled A42 in respect of striking susceptibility to infectivity enhancement by Escherichia coli. Actinomyces (Corynebacterium) pyogenes and S. aureus. One further strain (A6) of F. necrophorum resembled A42 in respect of strong infectivity enhancement by A. pyogenes, S. aureus, B. cereus and K. oxytoca but differed from it and the other five strains in being only slightly affected by E. coli. This work was a necessary prelude to the development of a method, based on infectivity enhancement, for the detection and isolation of F. necrophorum present in small numbers in heavily contaminated material such as faeces.

The isolation of Fusobacterium necrophorum present in small numbers in heavily contaminated material such as faeces or soil is hampered by the lack of an efficient selective medium and by the high minimum infective dose of the organism. A sensitive method for the detection and isolation of faecal strains of F. necrophorum type A was based on the subcutaneous injection of faeces, suspended (5% w/v) in broth culture of Actinomyces (Corynebacterium) pyogenes or Staphylococcus aureus to increase fusobacterial infectivity, into mice pretreated with clostridial antitoxins. When necrobacillosis developed F. necrophorum was identified microscopically in tissue from the advancing edge of the lesion and isolated on a partly selective medium. The enhancement of fusobacterial infectivity produced by A. pyogenes and by S. aureus was high, but the latter was slightly the more efficient, enabling as few as 80 F. necrophorum organisms/g of faeces to be detected. Use of the method showed that 3 of 16 wallabies had F. necrophorum in their faeces at the time of examination. Numerous epidemiological applications are suggested.

Earlier studies showed that the minimum infective dose (greater than 10(6) organisms) of a virulent strain of Fusobacterium necrophorum could be greatly reduced by suspending the fusobacteria in sub-lethal doses of cultures of other bacteria such as Escherichia coli before inoculating mice subcutaneously. In the present study the infective dose of the same strain of F. necrophorum was reduced by a factor of greater than 10(3) by suspending the fusobacteria in sub-lethal doses of 5% homogenate of gaur or wallaby faeces. Sterile faecal filtrate had no such effect. The sites of low grade infection produced by the prior subcutaneous injection of E. coli culture or gaur faecal suspension were susceptible to superinfection by doses of F. necrophorum far below those required to infect normal tissue. This work helps to explain the production of necrobacillosis by the faecal contamination of small wounds. It proved impossible, however, to produce necrobacillosis in mice by the subcutaneous injection of faecal suspensions from 33 farm cattle. This suggests that the proportion of cattle with virulent F. necrophorum in their faeces is low.

The study arose from the recent finding that sub-lethal numbers of certain bacterial species greatly enhanced the infectivity of Fusobacterium necrophorum. A severe F. necrophorum infection in mice, cured with metronidazole, produced significant though slight resistance, which was demonstrable by challenge with a minute dose of F. necrophorum (less than 20 organisms) suspended in a sub-lethal dose of Escherichia coli (300 x 10(6) organisms) to enhance fusobacterial infectivity. In an earlier comparable experiment, challenge with F. necrophorum alone, in necessarily large doses (greater than or equal to 3 x 10(6) organisms), failed to demonstrate that a single cured fusobacterial infection gave rise to resistance; such an infection neither protected against the fatal necrobacillosis produced by challenge nor prolonged survival. A sub-lethal E. coli infection was also shown by challenge with a minute dose of F. necrophorum (less than 10 organisms), suspended in a sub-lethal dose of E. coli (152 x 10(6) organisms), to produce significant though slight protection against necrobacillosis. The degrees of resistance demonstrated were too slight to give any encouragement to the prospect of an effective necrobacillosis vaccine.

Necrobacillosis is caused by Fusobacterium necrophorum (FN), but other organisms are often present in the lesions. Their possible role was studied in experiments made with a virulent FN strain which, by itself, produced fatal necrobacillosis in mice provided that large doses (greater than 10(6) organisms, subcutaneously) were given. Mice were inoculated subcutaneously with FN suspended in sub-lethal doses (0.1 ml) of undiluted or diluted broth cultures of other bacteria. Undiluted culture of a strain of Escherichia coli reduced the infective dose of FN to less than 10 organisms; in the necrobacillosis lesions that developed, fusobacteria greatly outnumbered E. coli. A heat-killed preparation or sterile filtrate of E. coli culture had little if any effect on FN. Citrobacter freundii and comparatively small numbers of Corynebacterium (Actinomyces) pyogenes produced effects similar to that of E. coli. An alpha-haemolytic streptococcus, Pseudomonas aeruginosa, Bacteroides fragilis and Fusobacterium nucleatum also enhanced the infectivity of FN, though less strikingly than E. coli. FN increased the persistence in vivo of the alpha-haemolytic streptococcus and B. fragilis, and enabled the latter to multiply profusely.

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Mice, birds (chicks, quail) and fish (rudd, goldfish) killed shortly after receiving 1300-2000 spores of Clostridium botulinum per os were incubated, usually at 23 degrees C for 7 days. A 10% (w/v) homogenate of each rotting carcass was then prepared, sterilized by membrane filtration, and assayed for toxin. In mouse carcasses a type C strain of C. botulinum usually produced greater than 2 X 10(5) mouse intraperitoneal LD/g; in fish carcasses it usually produced less--often much less--than 2 X 10(4) LD/g. Avian carcasses appeared to be intermediate between those of mice and fish in their ability to support toxigenesis. A type E strain of C. botulinum, unlike type C, produced toxin equally well in fish and mouse carrion, usually at a concentration of between 2 X 10(4) and 2 X 10(5) LD/g.

Mice killed shortly after receiving c. 2000 spores of a type E strain of Clostridium botulinum per os were incubated at one of five chosen temperatures together with bottles of cooked meat medium seeded with a similar inoculum. After incubation the rotting carcasses were homogenized. Sterile membrane filtrates of the homogenates (10%, w/v) and pure cultures were then titrated for toxicity. Some of the main findings were confirmed with two further type E strains. Toxicity produced at 37 degrees C was poor in both carcasses and cultures (200-20,000 mouse intraperitoneal LD/g or ml). It was good in both systems at 30 and 23 degrees C, usually reaching 20,000-200,000 LD/g or ml, and in carcasses occasionally more; at 30 degrees C maximal toxicity was reached more quickly in carcasses than in cultures. Prolonged incubation (36-118 days) at 30 or 23 degrees C resulted in complete loss of toxicity in virtually all carcasses but not in cultures. At 16 degrees C the development of toxicity in carcasses was strikingly greater than in cultures. At 9 degrees C neither system produced more than slight toxicity after prolonged incubation. Trypsinization increased the toxicity of cultures but not usually of carcasses. Unfiltered carcass homogenate (10%, w/v) with maximal intraperitoneal toxicity was harmless for mice by mouth in doses of 0.25 ml. These findings differed in important respects from those made earlier with a type C strain.

Mice killed shortly after receiving 1300-3000 spores of Clostridium botulinum type C per os were incubated at one of four chosen temperatures together with bottles of cooked meat medium seeded with a similar inoculum. After incubation the rotting carcasses were homogenized. Sterile membrane filtrates of the homogenates (10-20.8%, w/v) and pure cultures were then titrated for toxicity. A temperature of 37 degrees C produced less toxicity in most carcasses than in cultures. At 30 degrees C, however, toxicity (often 2 X 10(5) to 2 X 10(6) mouse intraperitoneal LD/g or ml) was roughly similar in both systems, and some carcasses and cultures were still toxic (2 X 10(4) to 2 X 10(5) LD/g or ml) after 349 days. Surprisingly, at 23 degrees C, through greatly reduced in the cultures, toxicity was high in many carcasses for at least 28 days. Little or no toxin was produced in either system at 16 degrees C. Unfiltered homogenates (17.8-22.5%, w/v; dose 0.25 ml per os) of toxic carcasses incubated at 30 degrees C for 7 days invariably produced death from botulism, often within as little as 4 h, but a 1 in 10 dilution produced less than 100% mortality.

In two trials the efficacy of inactivated vaccines against contagious bovine pleuropneumonia was tested by exposing vaccinated cattle to droplet infection provided by close contact with experimentally infected 'donors'. Complete protection was given by an extreme form of vaccination in which a heavy suspension of killed Mycoplasma mycoides subsp. mycoides emulsified with Freund's complete adjuvant was given in two large doses. 'Mouse-protective antibody' (MPA) was also produced, i.e. serum transferred to mice 2-4 h before intraperitoneal challenge prevented the development of mycoplasmaemia. However, the study did not answer the question 'Is MPA protective for cattle?'. No protection was given by a milder form of vaccination in which a lighter suspension of killed mycoplasmas emulsified with Freund's incomplete adjuvant was given in a comparatively small dose on a single occasion.

Dilution had an adverse effect on the infectivity of 24 h cultures of a strain of Fusobacterium necrophorum, which became apparent at or near the minimum lethal dose. Thus in mice inoculated subcutaneously the mortality produced by 0.01 ml of undiluted culture was almost invariably greater than that produced by 0.1 ml of a 1 in 10 dilution. The explanation appeared to lie in the increased physical separation of bacterial cells that was the inevitable consequence of dilution.

Three of four extreme methods of immunization completely failed to protect mice against challenge with the homologous strain of Fusobacterium necrophorum. Unsuccessful vaccines included (1) broth culture killed by mild heat and emulsified with Freund's complete adjuvant, and (2) a homogenate of heavily infected mouse brains, inactivated by mild heat and given in two doses. Also unsuccessful as a method of immunization was the production of a severe subcutaneous infection with F. necrophorum, followed by curative treatment with metronidazole. Slight but significant protection against subcutaneous challenge resulted, however, from two such infections given in rapid succession. It would appear that the main virulence factors of F. necrophorum are only weakly immunogenic, and the experiments give little encouragement to the prospect of an effective necrobacillosis vaccine.

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The ELISA and an immunoblotting technique were used to study F38-type mycoplasmas - an important cause of contagious caprine pleuropneumonia - and a number of related mycoplasma species, subspecies, types or serogroups. Two-way ELISA cross-reactivity was demonstrated between five mycoplasmas, namely strain F38, Mycoplasma mycoides subsp. mycoides (LC strain), M. equigenitalium, M. primatum and bovine serogroup 7. In addition one-way cross-reactivity was demonstrated between F38 and each of the following mycoplasmas: M. mycoides subsp. mycoides (two SC strains), M. mycoides subsp. capri, and bovine serogroup L. F38 and M. capricolum did not cross-react. Immunoblot analysis, unlike ELISA, revealed that F38 and M. capricolum were closely related. At least four major protein antigens were shared between F38, M. mycoides subsp. mycoides (SC and LC strains), M. mycoides subsp. capri and bovine serogroup 7. The ELISA cross-reactions (above) shown by M. equigenitalium and M. primatum with each other, with F38 and with other mycoplasmas were not apparent by immunoblotting.

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In vivo methods were used to study the F38-type mycoplasma in parallel with related mycoplasmas. Three of five strains of 'bovine serogroup 7' with an unknown history of subculture produced mycoplasmaemia in mice inoculated intraperitoneally. A strain of 'bovine serogroup L' also produced mycoplasmaemia, but no evidence of similar ability could be found for single strains of Mycoplasma capricolum, M. equigenitalium and M. primatum, or for two strains of the F38-type mycoplasma. In cross-immunization tests a bovine serogroup 7 strain (NCTC 10133) and a strain ('Blenheim') of the SC (small colony) type of M. mycoides subsp. mycoides were used for the purpose of challenge. Cross-protection was described as 'complete' or 'partial', depending on whether it was as great as, or less than, that produced by homologous vaccine. Although strain NCTC 10133 protected strongly, possibly completely, against Blenheim, and Blenheim gave partial protection against NCTC 10133, challenge with NCTC 10133 and Blenheim gave strikingly different results. Thus (1) F38-type strains, M equigenitalium and M. primatum all gave partial cross-protection against NCTC 10133 but not against Blenheim, (2) NCTC 10133, unlike Blenheim, was seldom susceptible to partial cross-protection by LC (large colony) strains of M. mycoides subsp. mycoides, and (3) three SC strains - which would have protected completely against Blenheim - protected only partially against NCTC 10133. NCTC 10133 and Blenheim were similar, however, in that M. capricolum and M. mycoides subsp. capri failed to cross-protect against them both.

Intracerebral inoculation was more effective than intraperitoneal, intravenous or subcutaneous inoculation as a means of producing lethal infections with Fusobacterium necrophorum in mice. Strains varied in virulence but, of five examined, two had LD50 values as low as ca. 8000 and 14000 viable organisms. Profuse bacterial multiplication in the brain was demonstrated. Intravenous vaccination with a single large dose of heat-killed whole culture or washed bacterial cells failed to protect against intracerebral challenge. Intracerebral injection of other fusobacteria (F. nucleatum, F. varium and F. necrogenes) and of 22 strains belonging to 10 Bacteroides spp. was without apparent effect on mice, except for a slight transient illness in some animals given B. fragilis. This organism (five strains) differed from the other Bacteroides spp. tested, which included eight strains belonging to the fragilis group, in being eliminated more slowly from the mouse brain--a point that may be relevant to the special pathogenicity of B. fragilis in endogenous infections in man. There was no evidence that B. fragilis multiplied in the brain or that intravenous vaccination with a large dose of heat-killed homologous culture affected the rate at which it was eliminated.

Much evidence of immunogenic heterogeneity among the LC strains of Mycoplasma mycoides ssp. mycoides emerged from cross-immunization and -hyper-immunization experiments in mice in which three LC strains (Vom/Plum Island, 74/2488, and Mankefår 2833) were used for challenge purposes. All heterologous LC-strain vaccines cross-immunized against the three challenge strains, but protection was usually only 'partial', i.e. significantly less than that given by homologous vaccine. Cross-hyperimmunization with all heterologous LC but not SC strains produced protection against challenge with Vom/Plum Island that was virtually 'complete', i.e. similar to that produced by homologous vaccine. Challenge with 74/2488 gave generally similar results; but against Mankefår 2833 six heterologous LC vaccines gave complete protection and six did not. Vaccines prepared from the Smith (1423) strain of M. mycoides ssp. capri gave some protection against Vom/Plum Island but none against 74/2488 or Mankefår 2833. The cross-immunizing ability of three further M. mycoides ssp. capri strains appeared to resemble that of Smith (1423). In a cross-hyperimmunization experiment, vaccines prepared from SC strains of M. mycoides ssp. mycoides varied greatly in their ability to protect against challenge with strains 74/2488 and Mankefår 2833.

So-called LC strains of Mycoplasma mycoides subsp. mycoides and, where appropriate, SC strains, were examined, together with M. mycoides subsp. capri strains, by growth, fermentation, and antibiotic-sensitivity tests. Growth curves in BVF-OS medium showed that, from the 6th until at least the 19th day of incubation, strain Mankefår 2833 had a viable count strikingly higher than that of any other LC strain. Its robust growth properties may explain its ability--unusual among LC strains--to produce mycoplasmaemia readily in mice. Strain 143-A66 Conn, also shown by earlier experiments in mice to possess unusual properties, lost viability more rapidly than any other LC strain between the 13th and 19th days of incubation. The viable count of a subsp. capri strain was considerably lower than that of any LC strain for much of the period between days 6 and 19. In fermentation tests with 27 substrates and sensitivity tests with 11 antibiotics the strains gave results that, in all but the following respects, were uniform. Sorbitol was fermented to varying degrees by all the LC and subsp. capri strains tested but was unaffected by the SC strains. The LC and subsp. capri strains were in general more resistant than the SC strains to streptomycin. The growth of the LC strains, much more than that of the other strains, was greatly stimulated by the presence of fermentable substrate.

Nineteen of 98 samples of mud or sand taken from the Mersey estuary in 1981 contained Clostridium botulinum type C, the organism almost always responsible for botulism in water birds. In the Dungeon and Score Bank areas, where many dead and dying birds were found during the period September-December 1979, almost half the samples contained type C. Most of the positive samples were essentially muddy rather than sandy. The findings do not prove that botulism contributed to the 1979 mortality but are nonetheless thought-provoking, particularly because type C--unlike type B--is by no means ubiquitous in Britain. Type B was present in 12.2% of samples from the Mersey estuary.

The so-called SC (small colony) and LC (large colony) strains of Mycoplasma mycoides subsp. mycoides are said to be indistinguishable by the in vitro serological tests of generally used in mycoplasmology. In mice the immunity given by a single dose of killed LC-strain vaccine against challenge with SC strains is - unlike that given by SC-strain vaccine - only partial. When multiple doses of killed or living vaccines were given, the majority of 13 LC strains still failed to immunize completely against a SC strain. This suggests that, although some protective antigens are shared between both types of strain, at least one of importance is present in the SC strains but absent from the majority of LC strains. The difference between the protective-antigen content of SC and most LC strains is thus qualitative, and not merely quantitative.