1. In an endemic area of Q fever in south-east England, two flocks of sheep, with serological evidence of infection by Rickettsia burneti, were examined for excretion of the rickettsia at parturition.

2. R. burneti was isolated from two out of ninety-six placentas collected from 118 ewes which lambed in one flock. The organism was also isolated from ‘wool tags’ obtained from one of the ewes which voided an infected placenta.

3. Infectivity titrations showed that the two placentas contained respectively 104·5 and 101·5 guinea-pig infective doses per gram of cotyledon. The wool tag from the sheep which voided the more highly infected placenta contained 103·25 infective doses per gram.

4. Except for one doubtful result, which could not be confirmed, no isolations were made from 194 placentas collected from 199 ewes in the other flock.

Details of the design of the microblender were supplied by Dr A. P. Goffe and Mr T. Nash of the Central Public Health Laboratory, Colindale, to whom we are much indebted. A gift of Triton X-100 was kindly given to us by the Rohm and Haas Corporation, Philadelphia 5 Pa.

C. botulinum types A and B toxins required larger amounts of antitoxin for neutralization when fractions of the test dose of toxin were used than would be expected.

The degree of this dilution effect, i.e. the dilution ratio, varied with different sera. The dilution ratios with type A sera could be improved by allowing a greater time for combination to occur, or by mixing toxin and antitoxin in high concentration and diluting these mixtures.

The dilution ratios of type A sera from horses rose as immunization proceeded, the greatest rise occurring during the 3-month rest period following three small doses of toxoid.

A type A serum could be separated into fractions of different dilution ratio by precipitation with increasing concentrations of ammonium sulphate.

Type A antitoxins prepared by immunization of groups of guinea-pigs with two doses of toxoid, had dilution ratios far lower than those found in hyperimmune horse sera.

The dilution ratios of sera were lower when tested against a toxic toxin than against a more toxoided one.

A number of type A sera were tested at one level of test against a number of test toxins. There was no evidence of heterogeneity of the toxins and antitoxins tested.

The serum ratios were found to be correlated with the dilution ratios except where the serum ratios exceeded 0·64. Flocculation time was slow and varied from 6 to 48 hr. with different sera tested against the same toxoid.

I am greatly indebted to Miss M. Barr for her constant advice and encouragement in the execution of this work and in the preparation of this paper. I am grateful to Dr M. Sterne for the supply of C. botulinum type A and B toxins and for the benefit of his experience of animal botulism.

An investigation into the classification of strains of Shigella sonneiby the method of phage-typing (using unadapted bacteriophages) is described.

829 strains, obtained during 3 years from twelve regions in England (including Monmouthshire) were examined by a standard technique. There were fifty-two outbreaks (involving 587 strains) from which two or more strains were isolated.

Results confirmed the finding by Hammarström in Sweden that such classification was possible, strains falling into twenty phage-types of which thirteen were found more than once. All strains were typable, 74% belonging to the same type. In forty-seven outbreaks the phage-type was uniform, the commonest type occurring in thirty-eight of these. In five outbreaks the phage-type was not uniform.

It is concluded that this method of phage-typing, because of evidence of type instability and because the majority of strains belong to the same phage-type, has little practical value as a routine procedure in the control of Sonne dysentery in this country.

Soil from a sugar-cane farm in North Queensland was infected with leptospirae. In some instances a culture of Leptospira australis A was added to the soil; in others it was infected by rats, Rattus rattus, known to be excreting leptospirae in their urine.

After periods ranging from 8 to 43 days, the soil was flooded with rain water which was subsequently examined for leptospirae by treating guinea-pigs according to the ‘subcutaneous stream’ technique.

Leptospirae survived in culture-infected soil for 43 days and in urine-infected soil for 15 days prior to the addition of the rain water. They were recovered from the water at intervals ranging up to 24 days after the flooding of the soil.

The infected soil was of pH 6·1–6·2, its moisture content where determined was 34 and 37%, and the prevailing temperature 20–29° C. The reaction of the infected water samples ranged from pH 6·6 to 7·6.

The possible significance of these findings in relation to the epidemiology of leptospirosis in North Queensland is briefly discussed.

Equipment for the sterilization of air by heat with capacities of 140 l./min., and 1700 l./min. respectively is described. An improved method for testing sterility is given.

Mice and rats which are normally resistant to histamine become more susceptible to its lethal action after an injection of H. pertussis. This so-called sensitization to histamine is not an anaphylactic phenomenon. It is due to the action of a component of H. pertussis, the histamine-sensitizing factor (HSF), which in some unknown way overcomes the physiological mechanism in rats and mice that makes them more resistant than other species to histamine. Guinea-pigs which appear not to possess this mechanism and are about 200-fold more susceptible, weight for weight, than rats and mice, were not made more sensitive by H. pertussis.

After a stated dose of vaccine, sensitization was detectable in 48 hr., reached a maximum in 3–4 days, remained at this level for about 2 weeks and gradually disappeared. The effects of dosage, route of injection, and weight and sex of mice have been examined.

The HSF was found in strains of H. pertussis; it was not found in H. parapertussis, H. bronchisepticus or H. influenzae. It was only slightly affected by heating at 70° C. for 1 hr. but was destroyed at 80° C. in ½ hr. It was destroyed when bacteria were disintegrated by shaking with glass beads, or by grinding after being freeze-dried. It was found in the supernatant fluid of a partially autolysed vaccine.

HSF was antigenic. Antisera were prepared in rabbits. Anti-HSF combined with HSF and neutralized its histamine-sensitizing activity. Bacteria treated with antiserum in vitro absorbed anti-HSF and did not thereafter sensitize mice.

Antiserum protected mice passively against the sensitizing action of vaccine, presumably by combining with HSF. After sensitization had developed the sensitive state was not affected by antiserum.

Although HSF is an antigen there was no indication that histamine-sensitization was due to its antigenicity.

The HSF was differentiated from heat-labile and heat-stable toxin, haemagglutinin, capsular material and agglutinogen.

The preparation V 17, which is a small fraction of the disintegrated bacteria, adsorbed on red cell stromata (Pillemer et al. 1954) had a high histamine-sensitizing value. Compared with whole bacterial vaccine it caused little production of agglutinin in mice; in rabbits it caused a slower and smaller production of agglutinin and a faster and greater production of anti-HSF. For this reason immune rabbit sera may have a high agglutinin titre and a low anti-HSF value or vice versa. Anti-HSF rabbit serum protected mice against sensitization by V 17.

Vaccines could be graded according to their histamine-sensitizing activity. This did not always correspond to their grading by agglutinin production in mice. The relation of HSF to the immunizing antigen of H. pertussis and the use of histamine-sensitization to indicate the immunizing potency of pertussis vaccines are discussed.

We wish to thank Glaxo Laboratories Ltd. for the supply of reference vaccine, and the Whooping Cough Immunization Committee of the Medical Research Council for vaccines and sera.

1. Rickettsia burneti has been isolated from the tick Haemaphysalis punctata collected from sheep in an endemic area of Q fever in south-east England.

2. Isolation from one batch may have been due to contamination from the wool of sheep excreting R. burneti, but evidence is presented that isolation from another batch was due to the presence of the rickettsia in the tick tissues.

We should like to express our gratitude to Mr R. D. Brown, M.R.C.V.S., Mr F. L. J. Kett, F.R.E.S., and Mr J. Carey, who helped to collect the specimens. We are also indebted to Dr A. D. Lees (Agricultural Research Council Unit of Insect Physiology) for his help with the identification of the ticks. Finally we wish to acknowledge the valuable laboratory assistance given by Mr P. C. Collings, Miss Z. Page and Miss K. Arnold.

1. Direct conglutination has been demonstrated with Rickettsia burneti.

2. Sera from human volunteers inoculated with killed suspensions of R. burneti and from persons who had recovered from an attack of Q fever as well as sera from a small number of healthy blood donors were all examined by the direct conglutination reaction, the haemolytic-complement fixation test and the direct agglutination test. A good correlation was found between the titres of the direct conglutination reaction and the haemolytic complement-fixation test but the results with the direct agglutination test were unreliable.

1. Particles of antu of 5 μ diameter were significantly less toxic to domestic rats than particles of 50–55 μ.

2. In each of three bio-assays they were less toxic than particles of antu of 100–110 μ.

3. It is concluded that the specification for antu for use in poison baits against R. norvegicus should not require it to be in the form of fine particles.

This work was initiated at the suggestion of Dr E. E. Turtle of this Division, and his advice during its progress is gratefully acknowledged. Thanks are also due to Mr L. E. Hammond for much help in the preparation of the test doses, and to Dr H. Egan and Mr E. I. Johnson of the Government Chemist's staff who carried out a number of the many micro-weighings involved.

1. Strains of Proteus derived from animals produced septicaemia in mice when given in large doses intravenously, but smaller doses caused necrotic lesions in the kidneys. The organisms failed to produce kidney lesions when administered intra-peritoneally or subcutaneously but did cause septicaemia.

2. Killed suspensions of Proteus failed to induce kidney lesions.

3. Strain differences in pathogenicity were found, some strains producing naked-eye lesions in the kidney, others giving microscopic changes only in this organ; of those strains which caused no changes in the kidney tissue, some persisted for several days and others were eliminated rapidly.

4. The mechanism of this localization in the kidney is discussed.

The writer wishes to thank Dr A. Wilson Taylor and Prof. R. Lovell for their advice and helpful suggestions and Mr R. Hood for the photographs.

1. Requirements for a heat-stable smallpox vaccine are suggested.

2. Sheep lymph dried from the frozen state retains its potency at 22 and 37°C. better than aqueous, glycerinated or lanolinated suspensions of lymph. Dried crude lymph is not, however, sufficiently stable for use in tropical areas; it is also liable to vary in stability from batch to batch. No better results were obtained by prolonging secondary desiccation.

3. Phenol in low concentration is deleterious to vaccinia virus stored in the dried or liquid state at 22 and 37° C.

4. Dried vaccinia virus stored under nitrogen loses potency more rapidly than when sealed in vacuo, even when precautions are taken to rid the nitrogen of traces of oxygen and moisture.

5. A number of suspending media were examined for their preservative influence on purified vaccinia virus. None was effective in the liquid state, but all protected the virus against the lethal influence of freeze-drying. In the dried state, these media varied in their ability to protect the virus during storage at 22 or 37° C. The best results at both temperatures were obtained with virus freeze-dried in 5% peptone. This medium also protects the virus well at 45° C. Different types of peptone were equally effective.

6. Virus dried in 5% peptone still gave a full quota of successful primary vaccinations of children after storage for 12 months at 22° C., or for 4 months at 37° C.

7. Using partially purified sheep virus dried in 5% peptone, a vaccine can be produced which shows a high degree of resistance to heat, is relatively free from bacterial contamination, and is easy to reconstitute after prolonged storage. These results are highly reproducible.

In an investigation of impetigo among troops, carried out in 1941, nearly half of the strains of Staphylococcus aureus isolated from the lesions had the ability to inhibit the growth of corynebacteria on solid media. A much smaller proportion of strains from other superficial lesions and from nose and throat swabs had this ability, and strains from deep suppurative lesions were uniformly negative.

Three-quarters of Staph. aureus strains isolated from schoolchildren with impetigo in Lancashire in 1953 and early 1954 were of one variety, which could be denned by its susceptibility to typing phages (‘type 71’).

Nearly 90 % of ‘type 71’ staphylococci, and very few others, produced a narrow, sharp zone of inhibition of Corynebacterium diphtheriae mitis on solid media.

A small number of other staphylococci, mainly non-typable or unclassifiable strains, produced a wider, hazy zone of inhibition.

The majority of the impetigo staphylococci were penicillin-resistant, and most of the resistant strains were members of ‘type 71’. However, ‘type 71’ gave rise to only a small proportion of the penicillin-resistant hospital infections occurring in the same district at the same time.

Three-quarters of the Str. pyogenes strains from impetigo lesions belonged to one of two groups of closely related serological types, one of which was rarely encountered in other situations.

1. Gastro-enteritis caused by certain varieties of Bact. coli is a highly infectious disease among babies, and it has been suspected that the infectivity is associated, partly at least, with the very large numbers of pathogens excreted in the faeces. Very large numbers were found in the faeces, but large numbers of pathogens were also found in dysentery, salmonella food-poisoning and in some cases of paratyphoid fever.

In cases of gastro-enteritis the dilution of faeces containing 5 to 50 pathogenic Bact. coli per ml. was usually 10−8; in Sonne dysentery and salmonella food-poisoning the corresponding dilution was 10−6. It is doubtful if even this difference can account for the differences in infectivity.

2. When normal Bact. coli outnumber pathogenic Bact. coli, shigellae or salmonellae by as little as 10: 1 it is difficult to identify the pathogens unless a selective culture medium can be used.

3. Ordinary nutrient broth is generally able to act as an enrichment medium for the isolation of pathogens from faeces.

Influenza A virus labelled with radiophosphorus was introduced as a primary inoculum into the allantoic sac of fertile eggs. Virus so introduced enters the cells of the chorio-allantoic membrane, and a study has been made of the chemical state of 32P present in the membranes 1½ hr. after inoculation.

It was found that on entry into the host cell the virus particle disintegrates probably as a result of destruction of its phospholipid. Radiophosphorus derived from the virus phospholipid can be recovered from the infected membranes by extraction with physiological saline in the form of compounds of small molecular weight which are not precipitated by protein precipitants and are not sedimentable at a centrifugal force of 100,000 g.

Saline extracts of the infected membranes also contain labelled nucleic acid which appears to be derived from the virus nucleoprotein.

A large part of the nucleoprotein phosphorus of the inoculated labelled virus cannot be recovered from the infected membranes by extraction with physiological saline but can be recovered if the cell nuclear material is dissolved in molar sodium chloride. The 32P in such molar chloride extracts is partly precipitated with the deoxyribonucleoprotein on dilution with water, and is partly present as free nucleic acid.

It is suggested that on entry into the host cell the virus particle is broken down, the phospholipid being destroyed and the nucleoprotein disintegrated with the release of free nucleic acid which enters into a close relation with the cell nuclear material.

The addition of glucose considerably reduces the amount of glycine required to protect complement against heat-inactivation.

A similar effect has been demonstrated with other sugars.

The cytoplasmic inclusion bodies produced by fowl-pox virus in the chorioallantoic membrane of the egg consist of a mass of virus particles, frequently surrounding an apparently normal component of the cytoplasm. Finely granular or filamentous material is seen in the centre of the inclusion and round the more central virus particles into whose structure it appeared to be incorporated. The inner virus bodies consisted of a granular core surrounded by a single membrane. The outer virus bodies consisted of a cortical layer 20 mμ thick, enclosing a space in which lay a core with a granular centre.

A series of comparative assays of two pertussis vaccines was carried out by the mouse intracerebral method, using litter-mates and placing one member of each litter on each dose.

A statistical analysis of the results by the method described by Irwin (1950, p. 231), was carried out, making due allowance for the use of litter-mates. A slight increase of accuracy was found in five out of six assays, but this did not reach the 5% level of significance. Any real increase of accuracy must have been small. The results show that, as far as these tests are concerned, there is no advantage in the use of litter-mates, particularly when one takes account of the large amount of additional work necessary in the animal house.

This paper describes efforts to determine the LD50 for mice, 24–48 hr. old, of pools of four strains of Herpes simplex virus, using the intraperitoneal route of inoculation.

The results have been analysed in an attempt to account for the very irregular relationship between dose of virus and mortality which was observed.

It is clear that, in addition to those difficulties which beset the interpretation of the results of any biological assay, titrations in very young animals raise difficulties of their own. The principal sources of difficulty are the high and uncertain non-specific mortality, the tendency of the mothers to eat their babies and individual variation between the litters. The latter, it is suggested, is mainly a reflexion of differences between the mothers, at least when a closely inbred strain of test animals is used.

Interesting and unexpected correlations were observed between the dose of virus and the proportion of mice eaten by the mothers and between the strain of virus and the proportion of mice eaten by the mothers.

It is emphasized that the results of titrations in very young animals must be interpreted with the greatest care and that in carrying out titrations the litter rather than the individual baby mouse should be regarded as the unit. Mice eaten by the mothers are probably best ignored altogether, although there are grave theoretical objections to doing this.

I should like to thank Professor H. R. Dean for his advice and encouragement. It is a pleasure to acknowledge the technical help which I have received throughout this investigation from Miss D. Sutton. I am also indebted to Dr M. G. P. Stoker for advice and for making available facilities in his laboratory; to Dr W. L. Smith and Mr T. H. Hollingsworth of the Department of Human Ecology, Cambridge, for advice on some of the statistical problems raised; to Miss M. E. Skinner and her staff on the ‘Animal Floor’ of this department, without whose help it would not have been possible to ensure that the large number of litters used were available when required; and to those of my colleagues who have been good enough to offer advice and criticism, especially Mr Douglas Annear.

Previous workers (Stuart-Harris, Pownall, Scothorne & Franks, 1953; May, 1953)have reported studies on the bacterial flora of the sputum in cases of chronic bronchitis. Both pneumococci and Haemophilus influenzae have been reported frequently in the sputum but the source of these organisms, which might be either the patient's own upper respiratory tract or the environment, has not been traced.A long-term study of individuals with and without chronic bronchitis and of their families was therefore begun in an attempt to discern the role of exogenous infection in this disease. Observations were limited to the pneumococcus because serological identification of individual strains was readily possible in this species.

The evaporation rates of small particles in aerosols of the following substances have been measured: 4−n−hexyl-1:3-resorcinol, 1, 1, 1-trichloro-2, 2−di (p−chlorphenyl)ethane (‘DDT’), 1, 1, 1−trichloro-2-p−chlorphenyl-2-o−chlorphenyl-ethane (‘op′-DDT’), γ-hexachloro-cyclo-hexane (‘BHC’, ‘Lindane’, ‘Gammexane’), ‘Dieldrin’ and azobenzene.

The slit ultra-microscope method was used; a consideration of the experimental conditions leads to the conclusion that the results are of reliable quantitative significance.