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.

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.

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.

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.

Attempts were made to demonstrate variola virus in the conjunctival exudate of 84 smallpox patients who developed conjunctivitis in the acute stage of the illness or during convalescence. Variola virus was isolated from 60 but not from the remaining 24. Of the 64 from whom virus was isolated the conjunctivitis developed from the onset up to the 15th day of illness. From conjunctivitis developing later virus was not recovered. In some patients who developed conjunctivitis early in the disease we failed to recover virus from the conjunctival exudate.

Of 55 close family contacts who stayed in hospital with smallpox patients four developed smallpox. In 21, conjunctivitis but no other illness developed. From 12 of these, variola virus was recovered from the conjunctival exudate and four of these 12, who were further investigated, showed after the appearance of conjunctivitis antibody titres similar to those seen in typical smallpox cases. From nine of the contacts who developed conjunctivitis virus was not recovered. One of these had antibody titres in serum collected before the onset of conjunctivitis which indicated recent smallpox infection. In another there was a marked antibody rise during her hospital stay although examination of conjunctival exudate on three separate occasions failed to yield variola virus. Twenty-six family contacts who developed no illness in hospital had antibody determinations made on sera collected soon after admission to hospital. In eight of these antibody titres were such as to indicate recent smallpox infection although there were no signs, in the form of scarring, or history of recent smallpox infection. These findings have been discussed in relation to the occurrence of minimal and subclinical infection in close family contacts of smallpox patients.

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.

White pock mutants of cowpox virus produce pocks in the CAM which ‘mimic’ those produced by the parent virus, in macroscopic and microscopic appearance, and in the amount of virus they contain. The proportion of such ‘atypical’ pocks was influenced by the age and source of embryo, incubation temperature and position of the CAM during incubation. Similar results were obtained with white pock mutants isolated from strains of vaccinia, rabbitpox and monkeypox. The appearance of pocks produced by variola, alastrim, ectromelia and vaccinia (Lister) was unaffected by changes in environmental conditions.

I would like to thank Professor K. McCarthy, Professor K. R. Dumbell, Dr C. H. Taylor-Robison and Dr C. R. Madeley for their interest and for kindly providing virus strains, and Miss M. Seyfang for technical assistance.

I would like to thank the Committee on Research, Liverpool University, for a grant towards the cost of the colour plate.

Studies were made of a 14-room isolation ward in which the bedrooms were provided with individual ventilation units to provide a slight positive pressure within the rooms. Airborne staphylococci were detected with sedimentation plates.

The median count of Staphylococcus aureus in the occupied bedrooms was 4·6 col. per 14 cm. plate exposed for 24 hr. or 13 col./m.2 hr. When the air count was high virtually all the staphylococci proved to be of the phage type harboured by the patient in the room; with low counts a substantial proportion were apparently derived from patients in other rooms.

The apparent rate of acquisition of S. aureus in the nose of the patients was between 2·7 and 4·2 per 100 patient weeks at risk. This is substantially lower than has commonly been observed in open wards.

We are very graetful to Miss M. A. Adams, Sister in charge of the Almroth-Wright isolation Ward, and all her staff for all their help and co-operation during this study, which was supported by funds provided to St Mary's Hospital by the Ministry of Health for clinical research.

The reconstruction of the ward was designed by Mr A. Stableford, Group Engineer to St Mary's Hospital; the ventilation units were designed and supplied by White Bays and White Ltd., London.

Nasopharyngeal secretions and cough/nasal swabs were taken from 111 children admitted to hospital in Newcastle upon Tyne with acute lower respiratory disease. A comparison was made between nasopharyngeal secretions and cough/nasal swabs as material for isolation of viruses in tissue culture. These results were, in turn, compared with those obtained by applying a fluorescent antibody technique to the exfoliated cells in the nasopharyngeal secretions for the rapid diagnosis of respiratory syncytial virus infection.

More viruses were isolated in tissue culture from nasopharyngeal secretion than from cough/nasal swabs. Further evidence for the superiority of nasopharyngeal secretions was obtained by comparing the virus isolations in the laboratory in 1967 with those in 1968. Respiratory syncytial virus was not only isolated more often but more quickly in tissue culture inoculated with nasopharyngeal secretions.

The fluorescent antibody technique not only provided a diagnosis on the patient's day of admission in 95% of those infected with respiratory syncytial virus but also proved to be as sensitive as the culture of nasopharyngeal secretions and considerably more sensitive than the culture of cough/nasal swabs for the diagnosis of respiratory syncytial virus infection.

We wish to acknowledge the invaluable help given us by consultants, medical officers, ward sisters and their staff in the paediatric wards of the Child Health Department. We also wish to thank the technical staff in the Department of Virology for their aid.

A large-volume sampler was used to recover virus excreted as aerosol by cattle, sheep and pigs infected with foot-and-mouth disease. Pigs were found to excrete virus to a maximum of 104.7 ID 50 per animal per hour and sheep and cattle to a maximum of 103.2 ID 50. Excretion from pigs totalled 106 ID 50 per animal over 5 days and from cattle and sheep 3 x 104 ID 50 per animal over 4 days. Maximum recovery occurred 41 hr. after infection in pigs and cattle when lesions had generalized and 17 hr. after infection in sheep before lesions had been observed. Sampling in a multistage liquid impinger showed that 65–71% of virus was excreted as an aerosol of size > 6 μ, 19–24% 3–6 μ and 10–11% less than 3 μ. The site of production of virus excreted as aerosol is suggested to be the upper respiratory tract. Under conditions of relative humidity greater than 70% and at low temperatures, survival of virus to a distance of 100 km. is likely to occur and because of the minute respiratory volume the aerosol would be sampled more efficiently by cattle than pigs or sheep and by large animals than by small. These findings are discussed in relation to spread of virus in the field.

Mr Dave Allen and Massey-Ferguson Ltd. are thanked for their generous donations, with which the large-volume sampler was purchased. We are grateful to Dr C. E. Gordon Smith, Mr G. J. Harper and Dr K. P. Norris of the Microbiological Research Establishment, Porton, for advice and help and for the loan of the multistage liquid impinger. The technical assistance of N. H. Cheale and C. W. Hawkins is gratefully acknowledged.

The effects of various salts on the viabifity of T4 and T4 Bo phages during freezing and thawing have been studied. The effectiveness of different salts in causing inactivation or protection of phage during freeze—thawing was of the same order as has been observed for their effects on the conformational stability of macromolecules. There were two important contributory factors causing inactivation of phage during freeze—thawing: (i) concentration of solutes during freezing, and (ii) lowering the temperature, without change in salt concentration.

The viability of phage following storage at subzero temperatures was dependent on the storage temperature, the species of ions in the suspending medium and the initial salt concentration. Viability was greatest when phage had been initially suspended in dilute solutions.

Survival of T4 Bo phage, following cooling to subzero temperatures and rewarming, was greater in D2O solutions than in H2O solutions.

It was found that 20% glycerol completely protected against inactivation of T4 Bo phage, suspended in 3·5 molal NaBr, which were cooled to subzero temperatures and rewarmed in the absence of ice. Without added glycerol the viability of T4 Bo phage suspended in 3·5 molal NaBr, which were cooled to −20° C and rewarmed was less than 1%. This protective effect of glycerol is in contradiction to the accepted views of its mode of action in prevention of freezing injury.

This work has been supported in part by grants from the Medical Research Council and the Office of Naval Research under contract F 61052–68-C-0041.

A local milk-borne outbreak of 85 cases of sore throat with cervical lymphadenitis, a third of whom later developed acute glomerulonephritis as a complication, is described. Renal involvement was shown by Volhard's criteria, filtered fraction data and renal biopsy findings. From lymph-node biopsies from patients, from the pharyngeal exudate of patients and carriers, and from three samples of cow's milk, 16 strains of beta-haemolytic streptococci of Lancefield group C, type zooepidemicus subtype lactose-positive, were isolated.

The aetiological role of the streptococcus isolated in this outbreak was confirmed by autoserodiagnosis (precipitation tests with Lancefield's antigens and patients' sera) which in this case proved more valuable than antistreptolysin-O titration as evidence of infection.

Two of the 85 patients, 7 months after the acute phase of illness, show nephrotic oedema and hypertension as chronic sequelae.

We are pleased to express our gratitude to Dr M. T. Parker and Dr W. R. Maxted for much advice, and confirmation of our serological and biochemical findings.

The ability of two live trachoma vaccines to protect against naturally acquired infection was tested in young Gambian children. With a mineral oil adjuvant vaccine prepared from a Gambian strain of trachoma (MRC–187) a barely significant measure of protection was demonstrable 6 months after the first dose, but not at 1 year, despite a reinforcing dose given 6 months after the first. In a later trial an aqueous vaccine prepared from the ‘fast-killing’ variants of strains ‘SA–2’ and ‘ASGH’ failed to induce immunity. Two years after vaccination, the proportion of vaccinated children progressing to cicatricial trachoma was less than in the controls, and the average severity of the disease in terms of clinical score was greater; vaccine-induced hypersensitivity may have contributed to this result.

Irrespective of whether they had received trachoma vaccine, children with completely normal eyes at the outset were less likely to acquire trachoma than those with slight conjunctival folliculosis or papillary hyperplasia. In children acquiring trachoma, there was a highly significant positive correlation between severity of the disease and the presence of conjunctival inclusions. The pattern of trachoma differed significantly in the two villages used in both trials; the prev alence, severity and proportion of inclusion-positive subjects were all higher in the village with the greater population density.

An efficient follow-up organization, use of a slit-lamp for clinical observations, and a scoring system for recording physical signs are all desirable for trachoma vaccine field trials.

We are highly indebted to Dr G. Turner (Lister Institute, Elstree, Herts) for his assistance in making the vaccine used for Trial II; to Dr N. M. Lam (Pfizer Ltd.) and Dr C. H. Smith (Evans Medical Ltd.) for making the Trial III vaccine; to Dr I. A. Sutherland (M.R.C. Statistical Unit) for his advice and help with the statistical aspects; to the Pennsylvania Refinery Co. Inc. for a generous gift of Drakeol 6 VR; and to Mr M. Race for his invaluable technical assistance in The Gambia. We are also grateful to the Director and staff of the M.R.C. Laboratories, The Gambia, for various facilities; and to The Gambian Government for per mission to undertake these trials.

Investigations were carried out at the desiccated coconut manufacturing ‘Mills’ in Ceylon to attempt to trace the source and sequence of salmonella contamination of desiccated coconut. It was found that one of the sources of contamination was animal excreta found on the mill premises—the yard where the nuts were stacked. Contamination was passed through the successive stages of preparation for manufacture and into the cutter which became the focal point of contamination within the dry section of the mill. The coconut appeared, in the majority of cases, to have been contaminated before drying. Some of the organisms survived the drying process. Two strains of S. senftenberg were found to be the most frequent contaminants of desiccated coconut.

These investigations were carried out with the assistance of the laboratory and field staff of the Ceylon Coconut Board, for whose co-operation many thanks are due; thanks are also due to Dr T. Velaudapillai formerly of the Medical Research Institute, Colombo for many helpful suggestions; to Dr Joan Taylor, Salmonella Reference Laboratory, Colindale, for identification of the salmonella serotypes; and to Dr Betty C. Hobbs, Food Hygiene Laboratory, Colinadale, for reading the manuscript.

The vaccination of sheep with one dose of the monkey-and mouse-attenuated tick-borne encephalitis virus (the Hy-HK 28 ‘2’ clone) causes seroconversion from negative into positive in 85% of animals. In sheep with pre-existing virus-neutralizing antibodies and increas of their titres was observed in 81%. The antibodies persisted for at least 12 months after the vaccination and during the summer period of grazing the number of serologically positive animals even increased.

The vaccinated animals, in contrast to the non-immune control sheep, developed no viraemia after challenge with the virulent louping-ill virus, performed 11 months after immunization.

Toxoplasma antibodies were absent from the sera of 32 adult Hong Kong women and were present in only two of 35 adult males and five of 46 meat workers, an overall incidence of 6·2%. This is the lowest incidence recorded in a large tropical city and lower than the incidence reported for most areas of the world, whether rural or urban. The reasons for this are unknown. Of 31 sera from swine imported from China and slaughtered in Hong Kong, 22 (71%) were positive for toxoplasma antibodies.

1. Response to selection was achieved with all strains of Myxoma virus used.

2. Heritability of resistance to myxomatosis was determined by intra-sire correlation using survival time as an index and by an all-or-none probit analysis. Both resulted in an estimate of heritability of about 35–40%.

3. The ability of animals to survive myxomatosis varied widely with environ mental variation in time.

I am indebted to W. Menzies for his very able assistance in all aspects of the work; to Dorothy Conolly for her critical assistance with data analysis, and to Dr J. M. Rendel for suggesting the probit analysis for determining heritability.