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.

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.

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.

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.

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 degree of protection provided by various air movement control systems, as applied to isolation and treatment rooms, has been assessed. The main factors influencing the efficiency of isolation were the air supply rate to the room and the time the door was left open. The effect of some other possible factors, such as temperature differences and method of air distribution, are discussed.

The type of facilities provided must depend on the type of patient and economics, but prediction equations, which allow the assessment of positive, negative and balanced ventilation systems, are presented. The ventilation requirements for isolation rooms should be assessed individually, but for a treatment room a balanced system of ventilation with between 500 and 1000 ft.3/min. should be very effective. Precautions should be taken to ensure the air quantities are balanced and temperature differences are minimized. An air lock should not be necessary for balanced systems provided these rooms are in a mechanically ventilated ward.

We thank Mr W. Carson and members of the Building Services Research Unit for their help and advice and Sister M. F. D. Muir and the staff of the experimental ward for their co-operation. This work was sponsored by the Nuffield Provincial Hospitals Trust.

Requests for reprints, or a fuller description of the ward unit and test results, should be addressed to George Baird, Building Services Research Unit, 3 Lilybank Gardens, Glasgow, W. 2.

Mice which carried the tapeworm Hymenolepis nana gave anomalous results when they were infected with tubercle bacilli. Males died earlier than females. The cause of death in some was intussusception or acute enteritis. More tapeworm eggs were excreted in the faeces of male mice, and, for each sex, egg counts were higher in BCG-vaccinated animals.

This work forms part of the studies on immunity to tuberculosis for a Ph.D. thesis, and was carried out while I was in receipt of a Harrison-Watson Studentship of Clare College and a Welcome Fellowship of the Animal Health Trust.

Mice immunized with a fraction from a culture filtrate of Ps. aeruginosa, then burned and infected with virulent serotypes of Ps. aeroginosa, were protected from septicaemia and death.

The immunogenic fraction killed mice at dosages 400 times those of the minimum protective dosages.

Treatment of the immunogenic fraction with 0·5% formalin reduced its lethal effect for mice and slightly improved its immunizing potency so that the difference between the smallest immunizing dose used and the smallest amount that killed mice was more than 80,000 doses.

In view of recent food-borne outbreaks of salmonella infection in which cooked-meat slicing machines have been implicated in the spread of organisms from contaminated meat products to other meats, experiments have been made to provide laboratory evidence that a contaminated slicing machine will easily cross-contaminate other products passing through it. Chopped pork inoculated with coagulase-positive staphylococci was cut on a slicing machine; staphylococci were isolated up to the 41st slice of various cooked meats cut on the same machine. The experiments were repeated with Salmonella oranienburg; this serotype was isolated up to the 31st slice of various cooked meats cut on the same machine, and from pieces of damp cloth wiped over the gravity feed, knife centre disk and cutting blade.

The importance of efficient and regular cleaning of slicing machines with hot water containing detergent/disinfectant, or detergent followed by disinfectant, applied with clean cloths or preferably disposable paper, is stressed.

I thank Dr Betty C. Hobbs, Director of the Food Hygiene Laboratory, and Dr J. C. Kelsey, Administrative Director of the Central Public Health Laboratory, for their help throughout the work and the compilation of the paper.

Investigations to define the epidemiological pattern of adenovirus infection in military recruits were carried out in a training centre at Ossendrecht, The Netherlands, during a 9-year period. Extensive outbreaks of adenovirus illness occurred in the winter and spring, and in some years in the late fall. The seasonal variation in total incidence of adenovirus infection as estimated by frequency of antibody rises in randomly selected recruits correlated well with that of adenovirus illness. In the cold season 20–60 of every 100 men were infected, whereas no or very little activity of adenovirus was found during the summer.

Type 7 was prevalent during the first 2 years of the study period, type 21 during the next 3 years and type 4 during the last 4 years. The proportion of adenovirus infection associated with clinical illness, the distribution of adenovirus illness by week of training and the immunity of incoming recruits against adenovirus infection as estimated by frequency of neutralizing antibody were compared for type 7, type 21 and type 4.

Adenovirus was responsible for about one third of all cases of acute respiratory illness. The next most common diagnostic category was influenza. Other respiratory agents (parainfluenza, RS and Coxsackie A-21 viruses, Mycoplasma pneumoniae and haemolytic streptococci) were of less importance.

This work was supported in part by the United States Department of the Army through its European Research Office.

We are indebted to the following persons who contributed to this study: C. P. A. van Boven, R. Brouwer, J. H. E. Custers, J. H. Dijkman, A. Prins and A. R. van der Werff, medical officers; Mr A. P. K. M. I. van Nieuwstadt, Miss M. C. J. van Nunen, Dr G. C. J. van der Ploeg and Dr H. J. A. Sonderkamp, members of the staff of our laboratory; and Misses C. H. Bronswijk, G. J. Hof, A. D. E. M. Leeuwenberg, W. T. C. J. Smulders and C. M. G. Stevens, and Mrs A. M. van Snik, technicians.

Major-General Dr H. J. van der Giessen and Brigadier General W. P. Blokpoel, former Directors-General of Medical Services of the Royal Netherlands Army, and Colonel Dr B. J. W. Beunders, former Head of the Division of Preventive Medicine, granted facilities for the conduct of this study.

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.

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.

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 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.

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.

The number of plaque-forming units is underestimated if plaques overlap. A simple model was developed to account for this bias, and tested by an extensive experimental analysis. It is shown that models of this type are inadequate, but can be modified to give objective methods for setting limits to the acceptable counting range, and for correcting the overlap bias of observed counts which fall within this range.

Where overlapping of plaques is a significant source of error, these methods will improve the efficiency of plaque assays and will render the statistical analysis of counting data more reliable.

The overlapping of plaques compresses their distributions and reduces observed variances. For a given distribution the reduction of the variance is substantially larger than that of the mean. To derive the error of a plaque assay from the assumption that the variance equals the mean may therefore lead to serious over-estimation of the precision of the assay.

Procedures for estimating the true error of plaque assays are developed, and their use is illustrated on experimental material.

A technique for neutralization tests using varicella-zoster virus propagated in primary human thyroid cells is described. The level of neutralizing antibody following chickenpox does not usually exceed a titre of 1/40 and in adults many years after infection it may be very low. After zoster a much higher and more persistent antibody response occurs. Contact with chickenpox also produced a rise in neutralizing antibody in one out of the five patients tested. One case who had had chickenpox but not zoster had a high level of neutralizing antibody and the possible reasons for this are discussed. No cross-neutralization with Herpes simplex virus was demonstrated but the rise in titre of complement-fixing antibody to HS occurring in herpetic subjects with chickenpox (Ross et al. 1965) was confirmed. Two samples of human gamma-globulin were shown to have high levels of neutralizing antibody to V-Z virus and one was known to have been found effective clinically.

We are indebted to Dr R. E. Hope-Simpson for the paired contact sera and also for many valuable discussions in the course of this work. We would also like to thank Dr D. Taylor-Robinson who collected the paired zoster sera, Dr R. M. Rawcliffe and Dr F. Whitwell who arranged the supply of thyroid tissue, and Dr G. C. Turner and Dr J. Eller for the samples of human gamma-globulin. We are grateful to Mrs M. Stewart for valuable technical assistance.

1. High titres of neuraminidase-inhibiting antibody were detected in convalescent human sera following natural influenza A2 infections.

2. Such antibody was encountered infrequently in acute serum samples. Antibody persisted only 5–6 months after infection in the four individuals from whom serial serum specimens were available.

3. Following immunization with killed influenza virus vaccines (with adjuvant) neuraminidase inhibiting antibody was detected in human sera. The titres were in general lower than those detected in convalescent human sera.

4. The specificity of the neuraminidase-inhibiting antibody in human and animal antisera was studied. Tests with convalescent human sera using purified neuraminidase preparations and with a recombinant virus containing A2 neuraminidase and haemagglutinin distinct from that of human influenza A viruses enabled the conclusion that the antibody detected was specific for influenza A 2 neuraminidase.

We wish to thank Dr D. A. J. Tyrrell of the Clinical Research Centre, Mill Hill, London, for human serum specimens, Dr D. Breeze of Evans Medical Ltd., Liverpool, for sera from an influenza vaccine trial and Dr M. S. Pereira of the Public Health Laboratory, Colindale, London, for serial serum samples from persons who had influenza in 1957. Dr M. S. Pereira carried out the haemagglutination-inhibition tests and complement fixation tests on these sera.

We are grateful to Dr H. G. Pereira and Dr D. A. J. Tyrrell for valuable discussions during the course of this study, and to Professor C. H. Stuart-Harris for his comments on the manuscript.

Oxoid dehydrated MacConkey broth was compared with laboratory-prepared Gray's improved formate lactose glutamate medium and with two dehydrated versions of it, by participants in ten laboratories. A variety of chlorinated and unchlorinated water samples was used. The superiority of the glutamate media over MacConkey broth for the detection of coliform organisms in water was again confirmed. The minerals-modified version, either dehydrated or laboratory-prepared, gave the best results and is the medium recommended for use in water examination. Care should be taken to ensure that the final pH after sterilization is 6·7.