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.

One hundred and eleven consecutive deaths in seven mental hospitals during the months of February to October 1967 were investigated histologically and bacteriologically.

Bacteria were present in the lung tissue significantly more frequently when inflammation was present than when it was absent and the differences were significant for both Escherichia coli and Staphylococcus aureus; E. coli was isolated from 22(34%) of the 65 patients with inflamed lungs compared with two (4%) of 46 control patients and the corresponding figures for Staph. aureus were 20(31%) of 65 patients compared with seven (15%) of 46 control patients.

Some strains of E. coli were serotyped using antisera against 0 antigens 1, 4, 6, 18ab, 18ac, 25, 50, 65, 69 and 75 and the strains most commonly found in the inflamed lungs, spleens and other inflammatory lesions of 25 patients were 06, 018 and 075. These strains were isolated more frequently from these sites than from the faeces of a group of 19 patients with no such lesions.

In this series E. coli was the commonest organism to be associated with terminal bronchopneumonia and the possible reasons for this are discussed.

The dose—response relationships between two strains of lymphocytic choriomeningitis virus (WE3 and Armstrong) and two hosts (mice and L cell tube cultures) were determined. The statistical analysis showed that, if infection was regarded as the response, and in the case of Armstrong virus in mice also death, the shapes of the empirical dose—response curves did not deviate from expectation which was based on the zero term of the Poisson distribution. Hence, the hypothesis that individual infectious units are capable of initiating infection and that co-operation is not required, was not contradicted. Furthermore, the units of assay were found to be equally susceptible under the experimental conditions applied.

By way of contrast, the relationship between WE3 virus and mice dying after intracerebral inoculations was found to be more complex. In this case the empirical curve did not run the expected sigmoid course at all, but rather was bell-shaped with a maximum of mortality (92%) at approximately 50 ID 50.

The work was supported by a research grant from the Deutsche Forschungs-gemeinschaft.

Between August 1964 and November 1965 Salmonella senftenberg was isolated from poultry, sheep and cattle on eight farms in England and Scotland. From an analysis of the case records its presence would appear to be incidental; but it may contribute to poultry mortality by acting in conjunction with other intercurrent infections or following stressful events such as severe debeaking or cold brooder conditions.

I should like to thank the officers of the veterinary field investigation and research services who made available their records and notes for this work, and who visited the farms and hatcheries, but especially Mr R. Duff, Veterinary Laboratory, Lasswade, for his reports on the turkey poults from farm A; also Dr Betty Hobbs and Mr B. Hanson for their valuable criticisms.

Influenza vaccines prepared with a mineral oil adjuvant induce a substantial and durable antibody response. However, vaccines containing mineral oil in simple emulsion (water-in-oil emulsion) sometimes produce persistent local reactions (Medical Research Council, 1964). The development of a redispersed ‘multiple’ emulsion in which antigen is incorporated as an oil-in-water emulsion starting from an original water-in-oil emulsion was described by Herbert (1965). Such multiple emulsion vaccine has a lesser viscosity than simple emulsions and might therefore produce a substantial antibody response with fewer reactions. In this investigation a comparison has been made of the antibody response and short-term vaccination reactions after aqueous influenza vaccine, influenza vaccine containing a mineral oil in simple emulsion, and influenza vaccine containing mineral oil in multiple emulsion.

During the 1968 epidemic of meningococcal meningitis in Greece, 90% of the meningococci recovered from the spinal fluid of patients belonged to serological group A. In one army barracks and in one Naval Training Centre, where cases occurred, a rate of 52% and 38·9% respectively of healthy nasopharyngeal carriers of group A meningococci was found, despite repeated courses of sulpha-prophylaxis given to all the population of these military establishments shortly before the swabs were taken.

The susceptibility to sulphadiazine of 64 group A meningococci isolated from patients in different regions of the country and from healthy carriers was tested in vitro. It was found that only one strain was sensitive to a concentration of 0·1 mg./100 ml. of sulphadiazine and another to 1 mg./100 ml. For the remaining strains, the minimal inhibitory concentrations of sulphadiazine ranged from 5 mg./100 ml. to more than 20 mg./100 ml.

Thirty-six strains of group B meningococci were isolated from 34 healthy carriers and from two patients. Some of these strains were resistant to sulphadiazine. Only three strains of group C meningococci were isolated from healthy nasopharyngeal carriers in one army barracks.

We wish to express our thanks to Dr R. Faucon, Director of the International Reference Centre for meningococci, for the valuable help he gave to us during our investigation, as well as for the supply of the group-specific agglutinating antimeningococcal sera.

Experiments have been carried out on the extent to which movement of persons in a room ventilated by a horizontally directed uniform air velocity can transport airborne bacteria from one position to another. More than 107 particles approximately 13 μ in diameter (settling rate in air about 1 ft./min.) carrying spores of Bacillus subtilis var. niger were liberated in each experiment and the numbers reaching the different parts of the room were estimated by those recovered on exposed settling plates. At air velocities of 35 ft./min. and over, no particles could be certainly found to have moved against the direction of air flow except into areas actually entered by persons, and the numbers found in these areas were between 100 and 1000 times less than would have been expected in rooms turbulently ventilated with the same volume of air. There was some transport of particles transversely across the air-flow lines but at 4 ft. distance from the area where movement of persons was taking place the numbers had fallen substantially below 1/100 of those to be expected in a turbulently ventilated room.

The results recorded at an air velocity of 22 ft./min. were significantly less satisfactory.

Our thanks are due to the N.W. Metropolitan Regional Hospital Board, to the Hospital Management Committee of St Charles' Hospital, London, W10, and especially to the hospital engineer for accommodating the experimental room and facilitating its operation.

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.

Phagocytosis prevents the multiplication of Streptococcus suis in blood samples from conventionally reared adult pigs. It is mediated by antibody, probably IgM, present in the serum of adults and specifically directed against the capsular polysaccharide of S. suis. The blood of young pigs reared in a ‘pathogen-free’ environment allows multiplication of encapsulated S. suis. Of 160 piglets conventionally reared, the blood of 92(58%) did not permit multiplication of S. suis. Blood from the remaining 68(42%) allowed multiplication but became bacteriostatic when the animals reached the age of 6 to 8 weeks. Serum from adult pigs confers bacterio static activity on blood from susceptible piglets in vitro and passively protects such piglets against experimental S. suis infection in vivo.

We thank Dr T. J. L. Alexander, formerly of Ontario Veterinary College, and Mr P. C. Trexler of the Royal Veterinary College, London, for providing us with serum from gnotobiotic pigs.

The work described in this report was carried out by one of us (K. K. A.) during the tenure of a Commonwealth Scholarship and was incorporated in a dissertation submitted for the Ph.D. degree of the University of Cambridge.

Some effects of freeze-thawing on the T 4 bacteriophage have been studied. The results indicated that most of the inactivation of rapidly thawed samples was due to osmotic damage, whereas inactivation of slowly thawed samples appeared to be correlated with the eutectic temperature of the suspending medium. Peptone significantly increased the survival of slowly thawed T 4 phage. The particular constituents of peptone largely responsible for this protection were separated as two distinct peptide fractions, using G 25 Sephadex.

This work has been supported in part by grants from the Medical Research Council and the Office of Naval Research under contract F61052–68-C-0041.

Studies of the chemical reactions of the haemagglutinins and neuraminidases of eight strains of influenza viruses have been made by the use of chemical reagents reacting with chemically active groups in the protein molecule. The results indicate a close resemblance between the active centres of the haemagglutinins and neuraminidases in all the strains tested. In all cases the activities were unaffected by reagents reacting with the —SH group of cysteine, the —CH3S group of methionine, the amino group of lysine, the guanidyl group of arginine, or the indole ring of tryptophan. In all cases both the haemagglutinating and enzymic activities were reduced or destroyed by agents reacting with amide groups or reacting with both tyrosine and histidine.

By the use of iodine under conditions in which tyrosine reacts but not histidine, and fluorodinitrobenzene under conditions in which histidine reacts more strongly than tyrosine, it was possible to detect a number of different active centres.

(1) An active centre containing histidine and an amide group but not containing tyrosine was present in all the virus strains and was the only centre detectable in A1 and A2 strains. This type of centre appeared to possess both haemagglutinating and neuraminidase activity.

(2) Active centres containing tyrosine and an amide group were detected in strains of A and B viruses. There was some evidence suggesting that tyrosine-containing centres were of two types: one possessing both haemagglutinating and enzymic activity while the other was a haemagglutinin without neuraminidase activity.

The results could be explained by supposing that the presence of histidine in the active centre was essential for neuraminidase activity and that enzymically active tyrosine-containing centres also contained histidine, but that tyrosine could substitute for histidine, but that tyrosine could substitute for histidine in haemagglutinating centres.

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.

The infection of a child with Salmonella paratyphi B phage type Battersea caught from his pet terrapins is recorded. In a small subsequent survey a relatively large proportion of these animals was found to be excreting salmonellas. The danger of these seemingly harmless pets is emphasized.

It is a pleasure to acknowledge the help and encouragement given by Dr E. H. Gillespie at all stages in the production of this manuscript. We also thank Dr Joan Taylor and Dr J. Wallace for final identification of some of the organisms isolated and Dr E. S. Anderson for phage typing the Salmonella paratyphi B strains.

The results of treatment of influenza virus strains with chemical reagents acting on the higher-order structure of protein molecules shows that both the haemagglutinating and enzymic activities are susceptible to these agents but there are considerable differences between the different strains and the neuraminidase activity is more sensitive than the haemagglutinating activity.

The neuraminidase activity of A and A1 strains is destroyed by urea, guanidine, urea+dithiothreitol and mercuric chloride. The haemagglutinin of the PR 8 and SWINE strains is resistant to urea and mercuric chloride but destroyed by guanidine and by urea+dithiothreitol. The haemagglutinin of the DSP strain of virus A and the A1 strains is resistant to urea, guanidine and mercuric chloride but is destroyed by urea+dithiothreitol.

The neuraminidase activity of the A2 strains is more resistant than that of the A and A1 strains. It is resistant to mercuric chloride and partially resistant to urea but is destroyed by guanidine and by urea+dithiothreitol. The A2 haemagglutinin is resistant to urea, urea+dithiothreitol, and mercuric chloride but is destroyed by guanidine.

The LEE virus neuraminidase is resistant to urea and partially resistant to guanidine but is destroyed by urea+dithiothreitol and mercuric chloride. The LEE haemagglutinin is resistant to urea, guanidine and mercuric chloride but is destroyed by urea+dithiothreitol.

It is suggested that the surface projections of the virus particle are protein polymers each made up of three or four monomers which are the components of the V antigen complex. Antigenic activity is a function of the primary or secondary structure of the monomers, haemagglutinin activity is a function of the tertiary structure of the monomers, while neuraminidase activity is a function of the quaternary structure of the polymer.

From studies of the chemical reactions of their haemagglutinins and neuraminidases strains of influenza virus A can be classified into groups. These groups are very similar to but not precisely identical with groupings made by serological methods.

Primary kitten kidney cultures are frequently contaminated with wild feline infectious enteritis (FIE) virus and this led the authors to develop feline embryo diploid cell lines. Monolayer cultures were prepared from the lungs or from eviscerated and decapitated carcases of embryos obtained by Caesarian section from healthy pregnant queens. At about the 30th passage, these cells lost their fibroblastic morphology to become polygonal. After a further thirty passages the monolayers exhibited foci of low cell density circumscribed by bands of cells stacked in disorganized arrangement. All the developed cell lines were susceptible to infection with FIE virus and produced intranuclear changes resembling those described by Johnson (1965) in primary kitten kidney monolayers.

On four occasions the cell lines became contaminated with Mycoplasma and although there was evidence that the virus could infect the cells, there was no production of infective virus.

A simple karyotype was devised in which the 38 chromosomes were arranged in three groups according to the arm-length ratio and the percentage mean index length. After the 50th passage many of the nuclei of lung-derived cultures exhibited abnormal chromosomes resulting from ring formation or translocation, whilst those of embryo culture demonstrated a new modal chromosome number of 37.

We are grateful to Mrs L. Hitchcock and Mrs P. Waller, A.I.S.T., for technical assistance, to Mr E. A. Jones, A.I.I.P., for the photomicrographs and to Dr R. H. Johnson, University of Bristol, for supplying the leopard and mink enteritis viruses.

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.

In a herd affected with bovine mastitis, dwarf colony variants of Staphylococcus aureus, auxotrophic for Ca-pantothenate, were isolated from the milk of four cows with mastitis. From one of these cows, a dwarf variant with an unknown metabolic disorder was isolated during the same period. Infections with auxo trophic staphylococci were always chronic and proved to be refractory to anti biotic treatment, though the causative micro-organism was highly susceptible to the drugs used as judged by in vitro tests.

The authors acknowledge with appreciation the skilful help of Miss Nili Abramova.

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.

The toxicity and palatability of coumatetralyl (3-(α-tetralyl)-4-hydroxycoumarin) to rats (Rattus norvegicus Berk.) were investigated in the laboratory by means of feeding tests. Animals resistant to warfarin (3-(α-acetonylbenzyl)-4-hydroxy-coumarin) and warfarin-resistant rats from infestations refractory to coumatetralyl, as well as non-resistant animals, were employed in the tests.

Medium oatmeal containing a concentration of 0·1% coumatetralyl was not markedly less palatable than the same food unpoisoned. In comparison warfarin at 0·05% but not at 0·025% was significantly less readily eaten than the plain food. Coumatetralyl at 0·05% and 0·005% was about as toxic as 0·005% warfarin is reported to be to non-resistant rats. Warfarin-resistant rats were significantly less susceptible to coumatetralyl than were non-resistant rats. Warfarin-resistant rats from an infestation refractory to coumatetralyl were significantly less susceptible to coumatetralyl than were animals from other sources.

It is considered that coumatetralyl at concentrations of the order of 0·05% in bait would be a good alternative to warfarin against non-resistant rats. While it would be expected that, at this concentration, coumatetralyl would often give good results against warfarin-resistant infestations, this use might eventually produce an increase in the incidence of resistance to both anticoagulants.

We thank Baywood Chemicals Ltd., for supplying the coumatetralyl used in this work. We are also indebted to several colleagues for supplying wild rats and information on field investigations and to Messrs G. Snell and P. Romer for technical assistance.

A retrospective survey of Coxiella burnetii infection in North-Western England and North Wales was made in five Public Health Laboratories for the years 1962–6 inclusive. Groups of the normal population and of persons occupationally exposed to infection were tested for the presence of antibody and the incidence of infected milk was investigated in seven counties.

Forty-one cases of C. burnetii infection were diagnosed in 7046 patients investigated serologically for the presence of viral and rickettsial antibodies. In just over a quarter of the patients there was evidence of occupational hazard or a history of drinking raw milk. The remainder gave no relevant history.

We would like to express our thanks to Dr C. M. Patricia Bradstreet of the Standards Laboratory, Colindale, for the supply of antigen, and to the numerous clinicians who allowed us access to their case records.