- Volume 3, Issue 4, 1970

The suckling NCS mouse proved to be a more sensitive and a more consistent subject for intestinal colonisation than the conventional suckling mouse.

An oral challenge dose with (5–10) x 106 coliforms, enteropathogenic or non-pathogenic, was followed by intestinal colonisation. The clearance of the introduced strains from the gut started at the weaning period in mice and was usually complete at 25–30 days of age.

However, the NCS mouse proved to be a suitable model for the differentiation of enteropathogenic from non-pathogenic coliforms on the basis of mortality, as only the enteropathogenic virulent E. coli serotypes caused deaths, apparently due to toxic effects.

Sections from the intestine of mice infected orally with doses of 106 E. coli 026:B6 or 0111:B4 showed no pathological changes.

Lung infections, following the oral challenge of mice with coliform strains, did not seem to be the sole cause of deaths as infections of the lungs via the intranasal route were not associated with mortality.

The dynamics of the implantation and of the clearance of intestinal coliforms were considered. Antagonistic activities involving the introduced coliforms and the resident bacterial groups such as coliforms, enterococci, lactobacilli and bacteroides were not observed.

Preparations of colicines A, B, D, E2, E3 and K were exposed in separate experiments to chloroform, heat, filtration, and ultraviolet light and the effects were examined quantitatively.

Chloroform at a concentration of 5 per cent. (v/v) sterilised preparations of each colicine within 5 min. and there was an acceptably low degree of inactivation. Exposure for longer periods was associated with progressive inactivation of B colicines and colicine K.

Millipore filtration and ultraviolet irradiation are slightly more effective but technically more exacting methods applicable to large and small samples respectively. Seitz filtration removed all detectable colicine activity.

Heating is not recommended as its effects on colicine activity are difficult to predict. The criteria of thermostability are discussed.

The effect of dietary lactobacilli on in-vitro deaminative and decarboxylative activities of the small-intestinal microflora has been studied in pigs in relation to weaning. Pigs were weaned abruptly at 21 days of age on to a diet of sterile milk or milk incubated with lactobacilli, and measurements of catabolic activity were made on small-intestinal digesta with and without supplementation with l-arginine, l-histidine, l-lysine and l-ornithine.

An increase in decarboxylative and deaminative activities was observed within 48 hr after weaning in animals fed on both diets. Decarboxylative activity was highest, and deaminative activity lowest, in digesta from pigs fed on milk without lactobacilli. During a later period, when diarrhoea was observed, there was a second peak of decarboxylative activity in the digesta of animals fed on both diets; but deaminative activity increased only in animals receiving dietary lactobacilli.

Continuous administration of dietary lactobacilli failed to induce any consistent changes in the small-intestinal microflora.

The extent of placental localisation of Vibrio fetus after experimental infection of pregnant ewes has been examined and a nutritional basis for the localisation has been investigated. After infection, V. fetus was found only in the uterine contents; the placenta and chorion were most consistently infected. In advanced infection, quantitative measurement revealed that the placenta, allantoic fluid and chorion together contained 80–90 per cent. of the total organisms present.

Erythritol, the factor responsible for the localisation of Brucella spp. in the bovine and ovine placenta, failed to stimulate the growth of V. fetus in a range of media. The rate of growth of V. fetus in extracts of foetal cotyledon was greater than that observed when the organism was grown in extracts of most other tissues; this finding supports the hypothesis that the tissue predilection is dependent on local nutrients.

The number of haemopbili in saliva was estimated by growth on a selective medium consisting of chocolate agar containing bacitracin (10 units per ml) and cloxacillin (5 µg per ml). The mean count of 100 saliva specimens was 31.79 × 106 haemophili per ml of saliva.

Area sampling on to selective medium from the cheek, palate and tongue often subjects gave mean counts of haemophili of 3870, 5675 and 7415 per cm2 respectively.

Examination of the colonies on ten randomly selected saliva plate counts showed that 92.8 per cent. were V-dependent and that the remaining 7.2 per cent. required V and X factors. No haemophili requiring only X factor were isolated. The V-dependent haemophili could be subdivided into five groups on the basis of growth in broth containing 0.2 per cent. Teepol, production of capsules, agglutination of human red blood cells, and beta-haemolysis on horse blood agar.

The mixed mass of oral bacteria when growing in culture will support satellite growth of H. parainfluenzae, but V factor could be demonstrated in saliva only when it had been heated at 60°C for 1 hr. Neither the saliva nor the bacteria in it was a source of X factor.

Pathogen-free CD1 mice were experimentally infected with mouse hepatitis virus (MHV3), and half of them were also given daily doses of cortisone. Ultrastructural examination of the splenic white pulp revealed viral particles, corresponding in size, shape and structure to MHV3, within small lymphocytes as well as extracellularly. Virus formation within the lymphocytes was associated with proliferation of the rough endoplasmic reticulum and budding of viral particles from these membranes. Intracellular viral particles were readily seen only in the cortisone-treated mice, possibly because of the stabilising effect of cortisone on intracellular membranes. It is suggested that the lymphoid necrosis of MHV3 infection is attributable to the virus replication within the lymphocytes.

A case of cryptococcosis is presented in which there was cervical and mediastinal lymphadenopathy with extensive caseation necrosis. A further unusual finding was the production of “ pseudo-hyphae ” by the cryptococci in the caseous nodes. There was a single subpleural pulmonary lesion, but only microscopical evidence of meningitis.

Accurate surface viable counts of Cl. tetani may be obtained by the method of Miles and Misra when horse blood agar containing commercial tetanus antitoxic serum to yield a final concentration of 40–60 units tetanus antitoxin per ml is used as the plating medium. The inhibition of swarming is probably due to agglutinating antibodies, which are known to be present in commercial tetanus antitoxic serum. Firm horse blood agar is much less satisfactory for this purpose.

Fowls were infected with avian-IBV and, subsequently, their tracheas were sectioned and examined in the electron microscope. The lining epithelial cells showed extensive loss of cilia, degeneration of microvilli, swelling of mitochondria, increased amount of endoplasmic reticulum and of other intracellular membranes, and the presence of electron-dense areas in the cytoplasm. Virus particles were first observed about 12 hr after infection within the electrondense areas and within spaces between the membranes of the endoplasmic reticulum. By 48–72 hr they were considerably more numerous and were present mainly within cisternae and vesicles formed from the membranes of the endoplasmic reticulum. There was little evidence of budding. The mature virus particle had a mean diameter of 81±1 nm and possessed both inner and outer electron-dense coats or shells separated by an electron-transparent layer. Virus particles grown in the embryonated hen egg and harvested from the allantoic fluid had a similar size and structure.

The potency of typhoid vaccine for mice could be increased 50-fold by incorporation in alginate. This adjuvance was evident only after intraperitoneal inoculation. It waned rapidly and had virtually ceased 26 days after vaccination.

Similarly, alginate increased the resistance to typhoid challenge of mice given a heterologous antigen, pertussis vaccine, provided this was injected intraperitoneally.

Alginate alone, when injected intraperitoneally, but not when injected subcutaneously, increased the resistance of mice to typhoid challenge.

It was concluded that the adjuvance of typhoid vaccine by alginate resulted from stimulation of antibacterial mechanisms in the peritoneal cavity and was independent of any specific immune mechanism.

An immuno-electrophoretic procedure is described for the detection of soluble bacterial antigens in culture extracts, and in material from ligated rabbit gut loops. All material examined in this study was derived from fully identified strains of Escherichia coli and rabbit gut loop exudates. A precipitin line, referred to as line 1, was produced when antisera (L sera) prepared with living vaccines were tested in the system against E. coli-induced gut exudates or extracts of cultures that evoked gut exudates. Rabbit antisera prepared in response to dead vaccines (D sera) of strains known to be enterotoxic to man did not produce line 1, nor was this precipitin band produced when L sera were tested against (i) the media used, (ii) normal rabbit gut washings, or (iii) extracts of E. coli that failed to dilate rabbit gut.

This study was extended to a survey of strains of E. coli isolated from healthy babies, from babies with diarrhoea, from urinary tract infections and other sources in man, and from various species of animals.

Line 1 was never produced by extracts of strains of E. coli isolated from the following human material: 39 faecal strains from healthy babies, 17 strains from infected urines, 2 strains from vaginal swabs and 1 from a sample of cerebrospinal fluid; nor was line 1 produced by 24 strains of animal origin. Ninety-nine of the 316 strains isolated from the faeces of babies with diarrhoea and 1 strain isolated from the faeces of a scouring calf produced line 1.

A case of staphylococcal empyema is presented in which resolution was slow in spite of apparently adequate therapy with cloxacillin. A relapse occurred when the drug was stopped after 2 wk because of the appearance of a rash. On two occasions while the patient was being treated with cloxacillin, large numbers of colonies of the bacterial form of Staphylococcus aureus appeared on plates of L-form medium but not on control plates without osmotic stabilisers. Relapse was associated with the re-emergence of staphylococci that could be isolated on conventional media, and a similar reversion of L-colonies made in vitro from the patient’s organism could be demonstrated on prolonged incubation. The infection was finally eradicated by erythromycin, novobiocin and drainage. It is suggested that cultures on L-form medium should be set up in any severe staphylococcal infection that fails to respond to chemotherapy and drainage, and that combined therapy with methicillin and an antibiotic that does not act on the cell wall should be considered.

Sensitivity of atypical mycobacteria to ethambutol is generally high, except in the two species Mycobacterium friedmannii and M. borstelense, in which at least 90 per cent. of strains are resistant to 25γ per ml or more. The character can be used as an aid to the identification of these species.

The single culture of M. thamnopheos examined was also resistant to ethambutol.

Forty-seven non-cholera vibrios (NCV) isolated from patients admitted to a Calcutta hospital with acute diarrhoea were investigated. Most of the strains belonged to Heiberg groups (HG) I, II and VII. A detailed biochemical study proved that organisms allocated to HG I and II are related to each other in sharing many biochemical activities, whereas NCV of HG VII are biochemically inactive. The HG-VII strains were characterised by their instability in saline or acriflavine or in both, lack of pellicle formation, and resistance to 25 γ of ampicillin; they gave negative reactions in lactose fermentation tests and in the ONPG, Simmons’ citrate, K-P organic acid fermentation, V-P, and haemolysin tests.

The authors wish to thank Professor J. B. Chatterjea, Director, School of Tropical Medicine, Calcutta, for his kind help during this investigation. The technical assistance of the late Mr B. R. Roy Chowdhury and Mr S. R. Sen Gupta is appreciated. Mr W. A. Cox of Allen and Hanbury Ltd, Ware, Herts, England, kindly supplied the sample of Vibriostatin 0/129.

Sera from 548 cancer patients and 500 control subjects were tested for the presence of complement-fixing antibodies against adenovirus type-12 tumour (T) antigen. Five of the cancer patients’ sera were positive, with titres ranging from 16 to 512. Eight of the control subjects’ sera were positive, with titres ranging from 2 to 16. No correlation was found with sex or socio-economic status, but all the positive sera were from patients 40 yr old or younger. The high titres of antibody were found only in some patients with acute lymphatic leukaemia or Hodgkin’s disease. Antibodies against SV40 T antigen were not found.

I am indebted to Mrs Diane MacAulcy for excellent technical assistance and to Miss Frances Cook for collecting some of the blood samples. Financial help was received from the National Cancer Institute of Canada and from the Medical Research Council of Canada.