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Small colony (SC) strains of Mycoplasma mycoides subsp. mycoides from contagious bovine pleuropneumonia (CBPP) and from goats were compared with large colony (LC) strains of so-called M. mycoides subsp. mycoides from goats and sheep by means of a cross-protection test in which mice were challenged with M. mycoides subsp. capri. Of 13 LC strains, all gave partial cross-protection, and 11 were shown to be more closely related than four SC strains to subspecies capri. In a further experiment, six SC strains--three from CBPP and three from goats--all gave weak partial cross-protection against subspecies capri.

Sixty soil samples were collected from the redeveloped site of the former Metropolitan (Caledonian) Cattle Market, Islington, London. Of these, 15 (25%) contained Clostridium botulinum and no less than four types (B, C, D and E) were demonstrated. Early British soil surveys suggested that only 4--8% of samples contained Cl. botulinum (type A or B). Although there can be no absolute proof, it seems likely that the striking prevalence at the Market site was the result of faecal contamination by a small proportion of the many millions of farm animals brought there from elsewhere. The distribution of Clostridium tetani was uneven, but of 18 soil samples taken from one area of the Market site, 16 (89%) were positive.

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.

Mud samples collected during 1974 from a large proportion of the lakes and waterways of London were examined for Clostridium botulinum. Of 69 such sites, 50 (72.5%) contained at least one type of the organism. Of the 50 positive sites, 31, 12, 1 and 10 contained, respectively, types B, C, D and E. Most of the demonstrations of type B required trypsinization of culture filtrates. An examination of 7 lakes in Edinburgh, made for the purpose of comparison, showed that 4 contained type B and one type C. An analysis of the results gave quantitative information on the value of (1) resampling apparently negative lakes, (2) the use of both heated and unheated culture inocula, and (3) trypsinization of culture filtrates.

Nine caprine and ovine mycoplasma strains, said to be indistinguishable serologically from Mycoplasma mycoides subsp. mycoides (the causative organism of contagious bovine pleuropneumonia; CBPP) were examined in mice by (1) a mycoplasmaemia test, and (2) a cross-protection test. Of the nine strains, two from goats belonged to a small colony (SC) type; four caprine and three ovine strains belonged to a large colony (LC) type.The two SC strains - like a single SC strain examined in an earlier study - were indistinguishable from genuine M. mycoides subsp. mycoides as isolated from CBPP. They produced mycoplasmaemia readily. In a cross-protection test, the two SC strains and a CBPP strain immunized completely against each other.Of the seven LC strains, six - like six LC strains examined in an earlier study - were easily distinguished from genuine M. mycoides subsp. mycoides; except for one that was not tested, all were shown to lack the ability to produce mycoplasmaemia readily. In cross-protection tests all six strains immunized partially but not completely against a CBPP strain.The seventh LC strain (Mankefår 2833) was exceptional: it produced mycoplasmaemia readily, resembling the SC strains in this respect. Like other LC strains, in cross-protection tests it protected only partially against a CBPP strain. Strain Mankefår 2833 was isolated in ca. 1965 by Brack from a Barbary sheep (Ammotragus lervia) in a German zoo.The ability of Mankefår 2833 to produce mycoplasmaemia enabled it to be used as a challenge strain in cross-protection tests. For the purpose of such tests the collection of nine mycoplasma strains referred to above was augmented with six LC strains from an earlier study. Partial but not complete protection against Mankefår 2833 was produced by two caprine SC strains, one CBPP strain, and nine LC strains. Three further LC strains gave protection that may have been as strong as that produced by the homologous strain, but confirmatory experiments are needed. A strain of M. mycoides subsp. capri gave no protection against Mankefår 2833.

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.

In recent years, mycoplasma taxonomists have found that numerous mycoplasma strains from goats are serologically indistinguishable from Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia (CBPP), by routinely used tests, e.g. the metabolism- and growth-inhibition tests. As a result, such organisms are now openly referred to as M. mycoides subsp. mycoides. Seven of these so-called M. mycoides subsp. mycoides strains from goats were compared with two strains of M. mycoides subsp. mycoides from CBPP, and with one strain of M. mycoides subsp. capri, by means of two in-vivo tests, namely, (1) a test of the ability of each strain, injected intraperitoneally into mice, to produce mycoplasmaemia, and (2) a cross-protection test in mice. Of the seven strains, only one ('O goat') was indistinguishable from genuine M. mycoides subsp. mycoides; it also had small colonies resembling those of genuine M. mycoides subsp. mycoides. The other six were easily distinguished from genuine M. mycoides subsp. mycoides, and they produced large colonies. These six strains and others like them should no longer be given a name that fails to distinguish them from the causative agent of CBPP. Cross-protection tests showed that the seven goat strains referred to above differed from M. mycoides subsp. capri.

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.

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.

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.

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.

Soil samples from various parts of Britain were examined for Clostridium botulinum by a sensitive technique comparable with that recently used for mud samples from British aquatic environments. The results showed beyond doubt that in Britain the prevalence of the organism in soil is much lower than in mud. Of 174 samples from all sites examined only 10 (5 . 7%) could be shown to contain Cl. botulinum; this finding was consistent with the results of surveys made by less sensitive techniques in 1922, 1928 and 1942. No type other than B was found. The evidence suggested that in certain localized areas the prevalence was likely to be high. Three sites associated for many years with animals were included in the survey; at the Zoological Society's premises at Regent's Park and Whipsnade, and at the Market paddocks, Gorgie, Edinburgh, the prevalence of Cl. botulinum was either very low, or nil. In an earlier survey of the redeveloped site of the former Metropolitan Cattle Market, London, 25% of soil samples gave a positive result and no less than four types (B, C, D. and E) were demonstrated.

Mud samples from aquatic environments in many parts of Great Britain and Ireland were collected, mainly in 1975 and 1976, and examined for Clostridium botulinum. The samples were taken from lakes, ponds, reservoirs, marshes, mudflats, streams, rivers and canals, and the sampling areas included a number of bird refuges and reserves. Of 554 samples 194 (35.0%) were positive and 167 (30.1%), 19 (3.4%), 6 (1.1%) and 15 (2.7%) contained types B, C, D and E respectively; 13 (2.3%) contained more than one type. Each type demonstrated was found in both fresh-and salt-water environments. Type B was widespread; types C, D and E were demonstrated in widely separated areas in England and Wales, and type C was found in both the north and south of Scotland. The results were compared with those reported earlier in respect of surveys in the London area, the Norfolk Boads and the Camargue (France).

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.

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.

In 1975, 25 soil samples were collected from the London area. Of these, 20 were obtained 200-300 yards from 20 lakes that had been shown in 1974 to contain mud contaminated with one or more of types of B, C, D, and E of clostridium botulinum. By means of a technique comparable with that use for the examination of mud, the 20 soil samples were found negative. The remaining 5 soil samples, obtained from sites that were not in close proximity to lakes, were also negative except for one that contained type B.

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.

Mud samples collected in June 1975 from the lakes, marshes and waterways of the Camargue were examined for Clostridium botulinum. The Grand Rhône and Petit Rhône were shown to contain types B and E, but of 44 samples taken from well distributed sites on the Ile de la Carmargue, only two (4-5%) were positive and these contained type E alone. The survey indicated a much lower prevalence of Cl. botulinum than any encountered in recent surveys of inland aquatic environments elsewhere.

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.

Sub-lethal exposure of Aspergillus fumigatus spore suspensions to formalin resulted in prolongation by 1-22 days of the period of less than one day normally needed by spores to produce visible growth in Sabouraud's liquid medium at 37 degrees C.; the degree of delay depended on the concentration of formalin and the duration of exposure, and was due to an increase in the germination-time of spores. The formalin concentration could be adjusted so as to affect the germination-time of almost all spores in a suspension without reducing viability. The effect on germination was not abolished by thorough washing or treatment with sodium sulphite. The spores of four different strains of A. fumigatus and of cultures aged 3 to 14 days reacted similarly to formalin treatment. Although of greatly reduced virulence for mice, affected viable spores were still capable of producing infection and death following intravenous inoculation, provided they were not eliminated by the host before germination occurred.

Intravenous inoculation of Aspergillus fumigatus spores was used to study experimentally induced and natural resistance. Slight resistance resulted in increased survival time and higher resistance produced in addition a decreased infection rate.Sublethal doses of living spores gave significant protection against challenge 3 weeks later, but large doses of heat-killed spores had no demonstrable effect.Mice from one source showed a single, dramatic decrease in dose response to a deep-frozen strain of the organism over a period of 34 months. The dose response initially resembled that described by Scholer (1959) in which one million spores killed the majority of mice. The change was almost certainly due to an increase in resistance of the mice due to environmental factors, and the resistance was probably also effective against other strains of the organism. Although not proved, it seemed likely that the resistance was due to increased natural contact with A. fumigatus or related fungi. Possibly for a similar reason, mice of the same stock bred on different premises differed in their susceptibility to infection. The results indicated that environmental resistance-producing factors may have been operating simultaneously on a number of premises housing laboratory animals in south-east England. These findings may have significance in relation to the occurrence of natural aspergillosis of mammals and birds.Of five A. fumigatus strains, four were of closely similar virulence; the fifth strain grew more slowly in vitro and was somewhat less virulent. Isolates from mice which died sporadically after small doses of spores were of no greater virulence than the inoculated strain. Although the susceptibility of mice aged 3 weeks was not uniform under all conditions, such animals were less resistant than young adult mice. Mice from six different sources showed only slight differences in susceptibility between each other, or from mice known to have developed a natural resistance.