A trial of an experimental live influenza B vaccine has been described.

The virus it contained was active and produced infections, antibody rises and clinical reactions.

Second and third vaccinations had much less effect than the first. Resistance to revaccination was only partially reflected in the serological response.

It seems that another factor, probably local antibody, exerts a considerable influence on resistance to infection with influenza viruses.

We are greatly indebted to Dr P. G. Higgins of the Public Health Laboratory, Cirencester, who went to much trouble to provide us with specimens from patients with influenza; to Dr H. G. Pereira of the National Institute for Medical Research for the antigenic analysis of the vaccine virus; to Messrs Sankey Ltd., Bilston, Wolverhampton, for their unfailing courtesy and forbearance throughout the trial; to the volunteers for their enthusiastic co-operation in the face of some discom forts; and to Messrs Pfizer Ltd., Sandwich, for originally providing facilities for the preparation of the vaccine.

In particular we wish to record our gratitude for the invaluable technical help of Miss Pamela Ball of the Common Cold Research Unit, Mrs Maria Gregory of the Bacteriology Department of Liverpool University, Mrs L. Johnson of the Virus Laboratory of New Cross Hospital, Wolverhampton, and Mr A. Westoby, an assistant in the practice of Dr Tyler.

No significant sex difference in the incidence of immunity to rubella at the ages of 20–25 was found in a comparative study of young adults from the Greater Athens area as well as from the rural department of Corinthia. The immunity to rubella of Greek adults on a national scale was then studied in a respresentative sample of 1200 males 20–25 years old. In 83·2% of them neutralizing antibodies to rubella were detected. A statistically higher proportion of immunes was found among those from urban areas. The factors responsible for the relative prevalence of immunity to rubella in various population groups are discussed.

The FH strain of Mycoplasma pneumoniae was studied in experimentally infected cultures of lung diploid fibroblasts. The multiplicity of infection was found to have a profound influence on the distribution of the cell-associated and extra-cellular mycoplasmas. At a high multiplicity there was very little difference between the ratio of cell-associated and extracellular mycoplasmas. In contrast, with a low multiplicity the cell-associated mycoplasmas predominated and with further reductions of the inoculum only the cell-associated mycoplasmas could be detected. The significance of these findings is discussed. It is suggested that caution has to be exercised in the determination of the potency of antibiotics against mycoplasma by methods employing acellular media alone, and it is desirable to utilize also appropriate cell culture models. A possible approach to designing such models is provided by this study.

We express our appreciation to the Tissue Culture Laboratory, Pfizer Limited, Sandwich, for kindly supplying us with cell cultures. We furthermore thank the Board of Directors, Pfizer Limited, for permission to publish this study. The helpful discussions with our colleagues Drs D. Jackson and G. M. Williamson in preparing the manuscript are acknowledged with thanks.

This paper describes an investigation into the successful preservation at room temperature of the bacteriophage H1 of Corynebacterium ulcerans U 103 which was extremely labile when in suspension at 4° C.

Cooling at a rate of 1° C./min. showed that the survival decreased logarithmically at temperatures between – 14 and – 45° C. Survival of broth suspensions of the corynebacteriophage were found to increase proportionally with an increase in the rate of cooling though there was a marked drop in survival at rates of approximately 900° C./min. The addition of peptone solutions was found to increase the survival over the range studied, whereas the addition of sucrose solutions had only a slight effect.

By avoiding freezing damage by cooling at rates of 450° C./min. in (a) 20% peptone solution, (b) 20% peptone and 10% sucrose, and (c) 20% peptone, 10% sucrose and 2% sodium glutamate, a study was made of the drying stage of the freeze-drying process. On drying at controlled temperatures it was found that there was no damage on rewarming to temperatures below – 21° C. after cooling to – 196° C., but that the survival immediately after drying in the absence of glutamate, showed a logarithmic relationship with the temperature of drying, lower temperatures giving better survival.

On storage for a period of 3 months at room temperature in vacuo and darkness, there was no appreciable loss in survival in the mixtures though suspensions in peptone alone showed a slight decrease. At higher temperatures this decrease in survival could be differentiated into two types of damage, each of which could be influenced by the presence of sucrose or glutamate.

We are grateful to Professor H. R. Carne for providing the initial sample of bacteriophage H 1 of Corynebacterium ulcerans U 103, and to Professor R. I. N. Greaves for his interest and encouragement during the course of this investigation.

A genital strain of Mycoplasma hominis was fractionated by differential centrifugation after disruption of the cells by alternate cycles of freezing and thawing, by gas cavitation under nitrogen, or by ultrasonic treatment.

Antigens of the cell membrane were distinct from those in the soluble cell contents, judging from metabolic inhibition (MI), indirect haemagglutination (IHA), gel-diffusion and immunoelectrophoresis tests, and by the antibody response in rabbits inoculated with these fractions. The antigens which gave rise to MI and IHA antibody were located in the cell membrane.

Extraction of whole cells of M. hominis by various chemical methods suggested that the active components were protein in nature and that there was no lipid hapten, as in M. pneumoniae, or polysaccharide, as in M. mycoides var. mycoides.

This work was partly supported by a Medical Research Council grant, which provided the remuneration of MRH.

We thank Professor R. A. Kekwick and Dr J. M. Creeth of the Biophysics Department of this Institute for their advice on the processing of plasma and density gradient centrifugation, and Mr F. C. Belton of the Microbiological Research Establishment, Porton, Wilts, for growing a 50 litre batch of M. hominis.

A modified Shigella—Salmonella medium in semi-solid agar was shown to inhibit the spread of most faecal organisms through a Craigie tube, while selectively allowing the passage of Salmonella.

A medium for routine use is described.

Over a period of 3 years, in this laboratory, 90% of specimens from which salmonellas were isolated gave positive results with this medium. Over 40% of these specimens would have been reported as negative if the method had not been in use. However, 10% would have been missed if this method alone had been used. It should therefore be used in conjunction with other culture methods, of which direct plating on SS agar was the most successful in this series.

Two cyclones for sampling dilute aerosols in the field are described. Their advantages are their robustness and reliability, the high degree of concentration achieved, and the ease with which they will provide a long uninterrupted sequence of samples.

Methods of correcting estimates of cloud concentration for transfer lag in the cyclone are derived.

The cyclones were constructed by J. Nott. We are indebted to G. J. Harper and E. J. Morris for allowing us to make use of their observations, and to Dr J. S. Paterson (Superintendent, Allington Farm) for providing the realistic aerosol at our convenience.

Investigations of the biological properties of tick-borne encephalitis viruses immediately after isolation from naturally infected Ixodes persulcatus from Western Siberia has revealed their different pathogenicity for monkeys and mice. Strains highly pathogenic for mice on extraneural inoculation and for rhesus monkeys on intracerebral inoculation have been isolated, but also strains with a low extraneural pathogenicity for mice and a complete non-pathogenicity or a mild pathogenicity for monkeys on intracerebral inoculation.

The viruses have been classified into three main groups by their virulence for mice and monkeys, and their ability to grow in tissue culture at 40° C. and the findings are summarized in Table 6. Group I included viruses highly virulent for mice by any route of inoculation, which caused fatal paralytic disease in monkeys, and which grew well in chick embryo culture at 40° C.

Group II strains also had a high virulence for mice, but in monkeys caused milder disease with a peculiar clinical course indicative of primary damage to the cerebellum. About half of the infected monkeys recovered completely or with mild sequelae (ataxia, pareses of the limbs). This group grew moderately well in chick embryo cultures at 40° C. Groups I and III differed essentially in their virulence for mice by subcutaneous inoculation, and virulence for monkeys decreased distinctly with transition from group I to group III. Group III strains showed a lower reproduction rate at 40° C. In chick embryo culture than group I. Groups I and II showed good correlation between extraneural pathogenicity for mice, pathogenicity for monkeys by IC inoculation, and growth rate in chick embryo cultures at 40° C.

Group I strains were isolated chiefly from foci with a high TBE morbidity and the strains of groups II and III in foci with sporadic cases. It is, however, highly probable that in any tick-borne encephalitis focus several variant strains of differing virulence exist.

Recent experience of vaccinating volunteers with naturally attenuated group III strains suggests that safe and effective live vaccines can be developed against tick-borne encephalitis. Vaccines against many other arbovirus diseases can probably also be derived from naturally occurring strains of virus isolated from arthropods in areas of low disease but high infection rate. This approach looks more promising than the usual approach of attempting to develop attenuated strains from highly virulent strains.

One hundred and twenty-nine samples of fresh cream collected in Worcestershire were examined bacteriologically. Sixty (46·5 %) creams had counts of over 100,000 bacteria/ml. The bacteria present were of many varieties, the commonest being Bacillus spp. (aerobic spore formers), Gram-negative bacilli, staphylococci and micrococci. Since most of the creams had been either pasteurized as cream or manufactured from pasteurized milk it was thought that the many bacteria were present because of contamination after pasteurization due to three main causes; unsatisfactory or unhygienic premises, unsuitable equipment, manual handling during the filling and capping process.

The methylene blue test results related well with bacterial counts but there were seven (5·4 %) anomalous results. Although the methylene blue reduction test therefore could serve as a simple and reasonable guide to the hygienic quality of fresh cream, 5·4 % of anomalous results would perhaps make it unsuitable as a statutory test.

We thank Mr R. Colenso, M.A.P.H.E., M.A.P.H.I., Chief Public Health Inspector, and Mr H. Beckett, Milk Sampling Officer of the Worcestershire County Council, for arranging the supply of samples.

1. Foot-and-mouth disease broke out on three pig farms of the Spetchley area of Worcestershire after the virus had been carried to them in skimmed milk.

2. A study of the natural features of the area and of the climatic conditions during the outbreak show that the wind could have carried the virus from one or other of these three to twenty-six others of the total thirty-two farms infected.

3. Bringing the cattle from the fields and keeping them in their sheds offered no protection. Nine farmers did so but the only effect was that the onset of infection was delayed for a day or so.

4. The direction in which cow-sheds faced likewise made no difference. Openings in the walls for ventilation obviously allowed virus to be carried inside.

Mr J. S. Needham, M.R.C.V.S., first showed me that the wind could have been responsible for the spread of the disease in the Spetchley outbreak. I am glad to be able, after my study, to support his view. I am indebted to Mr V. Harris, Officer-in-charge, R.A.F. Meteorological Station, Pershore, Worcestershire, for the climatic details.

Centrifuged deposits of washings of vegetables and fruits were plated on S.S. deoxycholate citrate and MacConkey agar for evidence of contamination.

Vegetables like beans, carrots, cabbage, celery, elabattu, leeks, kankum, pumpkin and spinach were found to be contaminated with Salmonella, Shigella and enteropathogenic E. coli. The extent of contamination of the vegetables which grew to a height of about 1 ft. above the ground was greater than that of the taller varieties. The rate of contamination among fruits was almost negligible.

The contamination might have originated from the water used for irrigation, from manure, or from the handlers of the vegetables.

Three groups of post-vaccination sera were studied for vaccinial antibody by precipitation, haemagglutinin-inhibition, complement-fixation and neutralization tests. All sera were negative by precipitation and many by haemagglutinin-inhibition and complement-fixation tests, but most showed neutralizing activity at serum dilutions of 1/10 or higher. The differences in antibody titres between the three groups of sera were most probably related to the past history of revaccination.

This investigation was supported in part by Public Health Service Grant AI–1632–16 VR from the National Institute of Allergy and Infectious Diseases, by the World Health Organization and by the Marcus T. Reynolds III Fund.

Hysterectomy-produced, colostrum-deprived pigs, reared in special isolation accommodation, were infected with enzootic pneumonia and later challenged with the same strain of the disease. Both the original infections and the subsequent challenges were made with intranasal inoculations of suspensions of ground pneumonic lung, but there was no evidence to suggest that any mycoplasma other than the J strain of Mycoplasma suipneumoniae was involved.

Pigs that had recovered from the disease were strongly immune to challenge, in that they developed virtually no lung lesions when inoculated with lung suspensions that produced extensive lesions of enzootic pneumonia in control animals. This was the case, even when the pigs were as young as 16 days old when first infected and were not challenged until up to 60 weeks later.

Sera from these pigs taken before infection, about 2–3 weeks after infection, at various times after natural recovery, and before and after challenge were examined using the metabolic-inhibition test, the indirect-haemagglutination test and the complement-fixation test.

The metabolic-inhibition test proved of little value, because non-specific inhibitory substances were present in the sera of some pigs both before and after infection: these substances inhibited the growth of Mycoplasma hyorhinis, Mycoplasma pneumoniae and Mycoplasma gallisepticum as effectively as M. suipneumoniae. Sometimes the non-specific inhibition was reduced by heating the sera at 56° C. for 30 min., but at other times it was not, which suggests that at least two types of non-specific inhibitors were present.

Apart from one pig, all the sera that were expected to be negative for antibodies against M. suipneumoniae proved to be so by the indirect-haemagglutination test. Titres of less than 1/5 were obtained in this test using the sera from pigs killed 12–22 days after infection, but high titres were obtained 16–60 weeks after infection. It was not possible to say whether these titres correlated with immunity.

All the pre-infection sera when examined by the complement-fixation test had titres of less than 1/10, but by 12–22 days after infection over half the serum samples had titres of 1/40 or more, and titres of 1/80–1/640 were obtained at 4 and 9 months after infection. There was some evidence to show that these titres declined more rapidly than the titres obtained in the indirect-haemagglutination test; for they were very low at 60 weeks after infection, at which time the indirect-haemagglutination titres were still high.

It seemed, therefore, that these two serological tests were measuring different aspects of the post-infection response. Also, because the complement-fixation titres were very low in two pigs that were shown to be powerfully immune, these titres did not appear to correlate with immunity.

Our work with the metabolic-inhibition test and the complement-fixation test has benefited from discussions with Dr D. Taylor-Robinson and Mr A. S. Wallis, respectively. We are grateful to Drs H. P. Chu, R. H. Leach and D. Taylor-Robinson for the reference sera and the culture mentioned in the text. Most of the expenses of this work, including the salary of two of the authors (R. G.H. and R.L.W.), were met by a grant from the Agricultural Research Council.

Two hundred and sixteen sera from 151 patients suffering from smallpox (non-haemorrhagic) were examined for antibody by precipitation in agar gel, by haemagglutinin inhibition (HI), complement fixation (CF) and neutralization tests. Most of the patients were adults and the majority had been vaccinated earlier in life. HI and neutralizing antibodies showed rising titres from the 6th day of illness while the majority showed precipitins and CF antibodies from the 8th day. The results of the precipitation-in-agar-gel tests are in marked contrast to the findings in healthy vaccinated and revaccinated individuals, none of whose sera gave a positive result for antibody by this technique. In unvaccinated patients the antibody response was frequently delayed and the titres lower than those attained by the previously vaccinated patients. There was no exact correlation in antibody titres obtained by the four methods of measurement, HI antibody, in particular, reaching in some cases relatively high titres when other tests showed low titres. It is suggested that with the methods and materials used, a positive precipitation test in agar gel, a CF titre of 1/20 or more and an HI titre of 1/80 or higher in a single specimen of serum would be suggestive of recent smallpox infections. Such a result might be of special value in the retrospective diagnosis of missed cases and in the detection of minimal or subclinical infections.

This investigation was supported in part by Public Health Service Grant AI–1632–16 VR from the National Institute of Allergy and Infectious Diseases, by the World Health Organization and by the Marcus T. Reynolds III Fund.

A serological survey for brucella antibodies has been carried out in Northern Ireland in an attempt to assess the effects of the brucellosis eradication programme in cattle which has been in progress since 1959. One thousand eight hundred and ninety four sera from blood donors, from farmer's lung tests, and from various groups occupationally exposed to cattle were examined by the serum agglutination test, the anti-human globulin (A.H.G.) (Coombs) test and the complement-fixation test.

The incidence of brucellosis in Northern Ireland is now less than it was 20 years ago, but is similar to that reported in a recent survey in England. It now appears that the principal method of transmission of brucellosis in the province is by occupational exposure rather than by drinking infected milk. Though it may be too early to make a serological assessment of the effects on human infections of brucellosis eradication in cattle, this survey provides a basis for future evaluation.

We thank Prof. O. L. Wade, Prof. P. C. Elmes and Dr W. R. Kerr for their encouragement and helpful advice; the Medical Officers of Health in Antrim, Armagh, Down and Fermanagh, Londonderry, and Tyrone, and in Belfast and Londonderry County Borough for help in obtaining blood samples; Dr M. C. Huth of the Northern Ireland Blood Transfusion Service, for sera; Dr D. W. R. Mackenzie and Miss H. E. Cairns for sera from the Mycology Diagnostic Laboratory; Dr J. H. Connolly for sera from the Virus Reference Laboratory, and for helpful advice; the farm workers and veterinary surgeons for volunteering blood samples; the technical staff of the Veterinary Research Laboratories for carrying out the tests.

A tryptone-yeast extract medium enriched with glucose and serum incubated anaerobically at 41° C. was found to give a good yield of Staphylococcus aureus from air samples while suppressing the growth of 70–80% of the other airborne bacteria.

Our thanks are due to Miss S. M. Taber and Miss Patricia Wall, the Sisters in the wards where the air samples were collected, for their help. The investigation was financed from the Ministry of Health grant to St Mary's Hospital for the support of clinical research.

In practically all acute fulminating smallpox infections—haemorrhagic type I cases—there is severe viraemia with 104 or more infective particles of virus per ml. of blood. In most of these patients soluble antigen can be demonstrated in serum by precipitation in agar gel tests, or by the complement-fixation technique. In late haemorrhagic cases (type II) the degree of viraemia is less and soluble antigen is less often demonstrated in the blood. Five of forty type II patients recovered. The majority of the 77 patients studied were adults and bore scars of previous vaccination. Thirteen were pregnant women and 10 of these suffered from type I infections.

The antibody response in patients who survived 6 days or longer as determined by the estimation of precipitins, CF antibodies and neutralizing antibodies in serum, was considerably less than that seen in non-haemorrhagic smallpox patients.

In acute fulminating smallpox infections, the finding of virus or soluble antigen in the blood is of value in establishing the diagnosis. Soluble antigen is usually found in the blood of patients suffering from severe viraemia and with the methods used has been demonstrated only in patients who are to die of their disease. Haemorrhagic smallpox represents a generalized virus infection of unusual severity in patients who show little resistance to their infection. The cause of this unusual susceptibility is unknown but there is little evidence that specific allergy to the virus is a feature of this form of the disease.

This investigation was supported in part by Public Health Service Grant AI–1632–16 VR from the National Institute of Allergy and Infectious Diseases, by the World Health Organization and by the Marcus T. Reynolds III Fund.

A bacteriological survey was made on the distribution and occurrence of coliforms and pathogenic indicators of pollution within the surf-zone and near-shore waters along a section of the Natal Coast, prior to the use of submarine outfalls. The distance covered measured approximately 47 miles. The waters sampled and assessed ranged from ‘clean’ beaches to heavily polluted areas; a single short run off an Eastern Cape coastal region was included for comparative purposes. In all cases, the bacteriological picture was related to sanitary features on the shore. The method is based on Escherichia coli I counts, parasite units, staphylococci, salmonellas and salinity, and provides an objective approach to the assessment of any future changes in water quality consequent on development.

The value of clean zones and of transfer areas in operating suites was assessed by comparisons of the amounts of contamination on floors, trolleys and footwear in suites with and without a clean zone and a transfer area; counts of Clostridium welchii were used as an index of bacterial contamination introduced into the aseptic zone from outside.

The mean counts of Cl. welchii on contact plates from the wheels of trolleys used to convey patients from wards to the operating suite (67·9 ± 7·68 per plate) were significantly higher than those from theatre trolleys (i.e. those used only inside a theatre suite provided with a transfer area) (3·13 ± 0·47 per plate); mean counts of total bacteria were only slightly lower on the wheels of theatre trolleys than on those of hospital trolleys. Other surfaces of hospital trolleys showed counts similar to those found on theatre trolleys.

Contact plates from floors showed significantly lower counts of Cl. welchii in the aseptic zone and the clean zone than in the hospital corridor, the protective zone and (when present) the transfer area.

The mean counts per 100 cm2 of Cl. welchii were approximately the same on the floor of a theatre with a clean zone and a transfer area (0·83) as in one with a clean zone but no transfer area (0·5). Counts of total bacteria were higher in the latter. A suite with no clean zone or transfer area showed a higher mean count of Cl. welchii on contact plates from the aseptic zone (operating theatre) (20·5 ± 12·33 per 100 cm2). These higher levels of contamination were due to sporadic high counts of Cl. welchii found near the door of the theatre with no clean zone; in another theatre with no clean zone the level of Cl. welchii on the floor was not higher than that in the theatres with clean zones.

Theatres with plenum ventilation had lower mean counts of airborne Cl. welchii than those ventilated by windows: there was no significant difference in the levels of Cl. welchii on the floors of theatres with the two forms of ventilation.

On sampling with contact plates, theatre footwear yielded fewer total organisms, Staphylococcus aureus and Cl. welchii than outdoor shoes removed before entering the clean zone.

The hygienic value of transfer areas and clean zones is discussed. Bacteriological support could not be obtained for the former, but the latter appeared to contribute something to the cleanliness of the theatre by preventing heavy sporadic contamination.

We wish to thank Mr M. Wilkins for valuable assistance, the staff of the operating theatres for their co-operation and Alne Engineering Limited, 57 High Street, Henley-in-Arden, Solihull, for supplying disposable contact plates.

The inactivation kinetics of CELO virus were studied in the presence of 1/4000 formaldehyde. Inactivation of the virus by formaldehyde at 4° C. was not complete after 14 days incubation. Formaldehyde inactivation at 36° C., however, was rapid and no virus was detected after 24 hr. incubation.

Neutralizing antibody to CELO virus was detected in 20–88% of sera tested from five flocks of hens. This suggested dissemination of the virus in England and Scotland. However, no CELO virus neutralizing antibody at a serum dilution of 1/8 was detected in 142 normal human sera or in 229 sera from persons who had been immunized with egg grown, inactivated influenza virus vaccine.

We would like to thank Professor C. H. Stuart-Harris and Drs J. E. Wilson, D. A. Martin and D. Breeze for their help and their criticisms of the manuscript.

Dr G. C. Schild kindly supplied a number of the human sera used in the study. The study was financed in part by the National Fund for Research in Poliomyelitis and other Crippling Diseases and by the British Empire Cancer Campaign.