- Volume 3, Issue 3, 1970

Groups of mice were infected with two strains of Salmonella typhimurium. Those infected with the less virulent strain no. 8 were treated with niridazole in doses of 200 or 600 mg per kg. Those infected with strain no. 12 received either 100 or 400 mg per kg. Compared with non-treated control mice the treated groups showed significantly higher numbers of survivors. In the group infected with strain no. 8 no difference was found in the incidence of survivors between the 200 and 600 mg per kg treatment groups. Such a difference was noted, however, in the case of strain no. 12 between the 100 and 400 mg per kg groups. Bacterial counts of spleens of survivors showed that treatment with niridazole significantly reduced the numbers of bacteria as compared with control mice.

In-vitro testing with impregnated disks was found to be effective in determining sensitivity of salmonella strains. Of 212 strains of S. typhi 178 gave zone sizes of 13 mm or greater. Of 77 other miscellaneous salmonella strains 67 had zone sizes of this order. It is essential that disk tests be done with freshly prepared niridazole solutions.

It is suggested that niridazole may be worthy of trial in cases of chronic salmonellosis that are unaffected by chloramphenicol and ampicillin. It may also be worth while to evaluate its use in chronic enteric carriers.

Intestinal contents obtained at necropsy from 100 patients, 99 of whom died while in hospital, were examined for Ps. aeruginosa. The contents were taken from four sites; the organism was isolated from all sites in eight patients. The over-all carriage rate was 36 per cent., but the rate was increased to 43-2 per cent, if oral antibiotics had been administered, and to 52-6 per cent, if gastro-intestinal surgery had been performed.

Ps. aeruginosa was isolated from the faeces of 13-6 per cent. of 103 out-patients, 18-3 per cent. of 240 in-patients and 4 per cent. of 50 normal persons, and from 73 per cent. of 108 ileostomy fluid specimens.

The sampling method for detecting intestinal carriers of Ps. aeruginosa is discussed.

Pregnant sows were immunised with hen-egg albumin and intradermal tests were carried out on their piglets after birth. Colostrum-deprived piglets from immunised sows were hypersensitive to egg albumin, and it is postulated that these piglets may have been sensitised through the transference of small amounts of antibody across the placental barrier. Antibody present in colostrum also conferred passive sensitisation. The symptoms and lesions that developed in the piglets after intravenous challenge were characteristic of anaphylactic shock, and were compared with those found in naturally occurring E. coli infection.

Two enterotoxins formed by enteropathogenic strains of Escherichia coli of pig origin were studied. One of them (LT) was heat-labile and antigenic and the other (ST) was heat-stable and apparently non-antigenic. They were identified by their ability to dilate ligated segments of pig intestine.

The ST component was produced by all of 40 enteropathogenic pig strains examined and the LT component was produced only by those strains that naturally possessed, or had possessed, K88 antigen.

Both enterotoxins were found to be controlled by transmissible plasmids (Ent). In transmission experiments with donor strains that produced both LT and ST, it was not possible to isolate recipient strains that produced only one or other of these two enterotoxins; they all produced both. As a group, strains that had received Ent (Ent+) from donor strains that produced only ST dilated ligated segments to the same extent as did similar strains that had received Ent from donor strains that produced both ST and LT.

The dilating effect of LT was neutralised by antisera prepared against live organisms of strains that produced LT and ST and, when larger doses were used, by antisera prepared against strains that had been shown to produce ST only. None of the antisera examined neutralised ST.

Antisera prepared against living organisms neutralised the dilating effect of these organisms. This neutralising effect was bactericidal, not antitoxic, in character and it appeared to be largely strain-specific.

ST and LT preparations of Ent+ strains produced a similar type of severe diarrhoea when given by mouth to piglets. Similar proparations of the corresponding Ent− strains did not.

It is concluded that LT and ST are probably two forms of essentially the same enterotoxin.

The relatively labile (LT) form of the enterotoxin produced by pig-entero-pathogenic strains of Escherichia coli strongly dilated ligated segments of rabbit intestine; the more stable (ST) form had a weaker effect. Dilatation was also produced by LT-type preparations of some non-enteropathogenic pig strains.

Similar preparations of cultures of bovine and ovine enteropathogenic strains failed to dilate rabbit and pig intestine; those of the human strains tested also failed to dilate pig intestine.

LT-type preparations of many of the human enteropathogenic strains dilated rabbit intestine; so did a smaller proportion of the non-enteropathogenic human strains tested. The dilating ability of these preparations was destroyed, or markedly reduced, by exposure to heat at 65°C for 15 min. The activity was also neutralised by antiserum in a serotype-specific or strainspecific manner.

The effect of a tubercle bacillary lipid, lecithin and some proteins on the activity of tetanus toxin was measured by injecting them into mice with graded doses of the toxin and noting the survival times of the animals.

When the tetanus toxin was given as a set volume of a dilution in saline containing lipid or protein, it gave shorter survival times and was lethal at lower concentrations than when it was given as the same dilution in saline alone. However, the addition of lipid or protein to the toxin already diluted in saline alone had no influence on the survival times of the mice. Injection of the corresponding amount of toxin as a measured volume of the initial dilution gave even shorter survival times.

Tetanus toxin is adsorbed or inactivated at glass surfaces and is inactivated at gas-liquid interfaces. The losses from these causes are reduced in the presence of lipid or protein in the diluent or by treating the glass surfaces with these materials before they are brought into contact with diluted toxin.

The apparent activity of the lipids or proteins in activating the toxin is due to the reduction of such losses. No evidence was found of any direct effect on the activity of the toxin.

The clinical, morphological, biochemical and bacteriological characteristics of two methods for inducing a cholera-like condition in rabbits were evaluated. The infection in suckling rabbits was considered analogous in all essential respects to the natural disease in man, and the infection in ileal loops of adult rabbits duplicated the local pathology closely until excessive luminal pressure introduced artefacts. It appears that the loop method is better suited for immunological studies, but that the baby-rabbit infection will give more information on host-parasite interactions. An enterotoxin reproducing the manifestations of cholera was demonstrated.

Parenterally vaccinated rabbits and guinea-pigs, and passively immunised baby rabbits, were challenged intra-intestinally with graded doses of a cholera vibrio strain. All vibrio vaccines, possibly including one made from an avirulent water vibrio, appeared fairly protective. Live and heat-killed vaccines seemed equally effective, and were superior to a formolised vaccine. The immunity seemed antibacterial, but possibly independent of the agglutinating antibodies. Suitability of a vibrio strain for vaccine production was not reflected in its specific serotype. Development of maximum resistance seemed to take several weeks after adequate dosage, and repetition of doses during this period showed no immediate advantage. The duration of immunity was, however, not ascertained.

Lactic dehydrogenase, alkaline phosphatase and a magnesium-independent adenosine triphosphatase were assayed in normal, diarrhoeal and cholera stools. The diarrhoeas, examined bacteriologically, included bacillary dysentery, salmonellosis, amoebiasis and non-specific gastro-enteritis.

Alkaline phosphatase levels were found to be similar in normal and cholera stools, whereas lactic dehydrogenase was 30-fold higher and adenosine triphosphatase four-fold higher in cholera. The distribution of activity of lactic dehydrogenase was higher, and of the phosphatases lower in cholera than in the stools of bacterial infections showing evidence of tissue necrosis. The levels of activity of all three enzymes in cholera stool were found to reach a maximum between 9 and 17 hr after onset of purging.

It is suggested that these findings indicate an increased permeability of the intestinal epithelium in cholera, leading to a leak of cell enzymes into the intestinal lumen.

Electron microscopy of short-term cultured blood lymphocytes from spontaneous cases of “classical” (neural) Marek’s disease and some clinically normal chickens from the same flock showed a herpes-type intranuclear virus in a high proportion of transformed cells. In a few instances virus particles were closely associated with intranuclear filaments, which were thought to represent the product of aberrant viral replication. Some non-transformed cells showed granule-lamellar arrays in the cytoplasm, thought possibly to represent the inner structure of the filaments.

The addition of cultured lymphocytes to healthy chick kidney monolayers induced a typical transmissible cytopathic effect associated with nuclear inclusions. Similar results were obtained using fresh lymphocytes, or tumour cells, from cases of the “acute” disease. A spontaneous cytopathic effect often developed in monolayers prepared from clinically normal chickens in a flock in which the disease was endemic.

Indirect immunofluorescence tests with either classical or acute Marek’s disease antisera indicated the presence of viral antigen in a high proportion of transformed lymphocytes and in the kidney cells in the region of the cytopathic effect.

A herpes-type virus was also demonstrated in cultured transformed blood lymphocytes in cases of acute Marek’s disease with gonadal tumours.

The indirect fluorescent antibody technique was used to determine the titre and immunoglobulin class of antibodies to Candida albicans in sera from 65 subjects. In patients with candidosis, antibodies were detected in the three main classes of immunoglobulins; significant titres of IgG were found in 78 per cent. of them, of IgM in 51 per cent., and of IgA in 30 per cent. Some differences were also found in incidence, titre and immunoglobulin class between the four types of candidosis. Serum fractionation by DEAE cellulose chromatography confirmed that antibodies to Candida albicans are found in the three immunoglobulin classes. The concentrations of serum IgG, IgA and IgM were also determined, but these failed to show significant differences between patients and controls. There was no correlation between the fluorescent antibody titre of a particular immunoglobulin class and the corresponding immunoglobulin concentration.

Eight recently isolated strains of Bacteroides corrodens, seven of which originated in Britain, were compared with two strains of this species from the National Collection of Type Cultures.

The principal characteristics of B. corrodens were: Gram-negative, nonmotile ovals and rods; usually grows as depressed or pitting colonies on agar media, but also produces non-pitting variants; prefers CO2 -enriched atmospheres or anaerobic conditions on primary isolation, but becomes a facultative anaerobe on subculture in the laboratory; catalase-negative; oxidase-positive; nitrate reduced to nitrite; arginine dihydrolase not produced, lysine and ornithine decarboxylase produced; acid not produced from carbohydrates; otherwise biochemically inert.

The DNA-base composition was determined by the melting-temperature method and was found to range from 56.2 to 58.2 per cent. GC.

The sequential changes occurring during bacillary division of Mycobacterium leprae in infected foot-pads of mice and human skin and nerves were studied with the electron microscope and found to be as follows: (1) a slight concavity develops in the plasma membrane, (2) two new electron-dense cell-wall layers are found between the concavity and the original cell wall, and an electrontransparent cell-wall layer appears between them, and gradually separates them, and (3) the annular ingrowth of the plasma membrane and cell wall proceeds until division is complete. There is no essential difference between the behaviour of Myco. leprae and that of other mycobacteria, nor between the behaviour in bacteria obtained from human biopsy specimens and bacteria from infected mice. Although Myco. leprae multiplies slowly the actual process of cell division may be relatively rapid.

The fate of three freshly isolated strains of N. gonorrhoeae following phagocytosis by human or guinea-pig polymorphs was studied in vitro. Less than 1 per cent. of gonococci phagocytosed by the guinea-pig cells survived at 100 min., but some 20 per cent. of gonococci associated with the human polymorphs were not killed even after 3 hr. The persisting gonococci were destroyed when the polymorphs were exposed to penicillin and this indicates that the surviving bacteria lay outside the polymorph membrane.

Cationic proteins prepared by fractional ethanol precipitation of acid extracts of the granules from human and guinea-pig polymorphs rapidly destroyed gonococci in vitro and, presumably, are partly responsible for the ability of polymorphs to kill gonococci.

Although these findings require confirmation in studies with gonococci grown in vivo, they strongly suggest that man’s susceptibility to gonorrhoea is not explained simply by the survival of gonococci in polymorphs.

In-vitro tests with homogenates of rat intestine showed that cephaloridine, cephalexin and 7-(thienyl-2′-acetamido)-3-methylceph-3-em-4-carboxylic acid were degraded more quickly in the caecum and rectum than in other parts of the alimentary tract; even after 4 hr at 37°C at least 57 per cent. of the low dose of cephalosporin used remained intact in other portions of intestine. The extent of breakdown in the caecum varied with the structure of the cephalosporin. Cephalexin was more stable than cephaloridine to biological degradation.

Different parts of rat intestine were assayed for β-lactamase and acylase activity and the effect of proteolytic enzymes on the cephalosporins was studied. The only degrading activity found was due to β-lactamase-producing bacteria in the caecum.

Cephalexin, cephaloridine and 7-(thienyl-2′-acetamido)-3-methylceph-3-em-4-carboxylic acid (TMC) were injected directly into the alimentary tract of rats and their subsequent fate was studied. The three antibiotics showed respectively good, poor and intermediate degrees of absorption from the alimentary tract. These differences are attributable to the rate at which each antibiotic is absorbed from the small intestine and not to the amount of degradation that occurs in the gut.

Cephalexin is much more rapidly absorbed from the small intestine than are cephaloridine and TMC. Cephalosporins that are not absorbed in the small intestine pass down the alimentary tract and are destroyed when they reach the caecum, from which there is little absorption.

Polymyxin B, polymyxin E, and colistin sulphomethate sodium are fungicidal in vitro for Candida tropicalis. Four hundred random isolates of Candida from two hospital laboratories were tested for sensitivity to colistin: 25 (6.2 per cent.) were sensitive, and 22 of these were C. tropicalis. One hundred and twelve isolates of eight species of Candida were screened for sensitivity to colistin and, of the 14 that were sensitive, 12 were C. tropicalis.

Polymyxins B and E liberate intracellular material from C. tropicalis, which suggests that the drug acts on the yeast cell-membrane.

Colistin sulphomethate was successfully used to treat a urinary-tract infection with this species of Candida. The potential therapeutic value of polymyxins for C. tropicalis infections is discussed.

Colonies of T-mycoplasmas isolated from a monkey, a dog, a bull and man lysed erythrocytes suspended in an agar overlay. Haemolysis was of the α′ (alpha-prime) type, and was usually detectable only with the low power of the microscope. As compared with M. pneumoniae, T-mycoplasma haemolysis occurred inconsistently in repeated tests. Crowding of colonies inhibited haemolysis. The addition of catalase also inhibited haemolysis indicating that the T-mycoplasma haemolysin is a peroxide. This haemolysin is probably produced in only small amounts. Protection of erythrocytes against lysis in the close proximity of the colonies of T-mycoplasmas and M. pneumoniae was also observed. This phenomenon occurred as frequently as haemolysis, and may have contributed to the difficulty experienced in detecting weak haemolysis. It too was inhibited by crowding of colonies. The protective effect was not due to catalase production, and it is suggested that a peroxidase might be responsible.

When tested by intracerebral injection in mice, a strain of Staphylococcus aureus containing antigen 17 was found to be much more virulent than a variant that lost this antigen and contained antigen 1 in its place. Since 85.7 per cent. of the mice challenged died between 7 and 11 hr after inoculation it is suggested that a toxin was the cause of death.

A standardised test by intracerebral inoculation is considered to be a useful test of virulence in mice. Out of 100 recently isolated coagulase-positive strains assessed by this method, only 18 were considered to be avirulent. The degree of virulence was correlated with the clinical source of the strains.