- Volume 11, Issue 3, 1978

The microtechnique of Galton et al., (1965) for the routine screening of blood samples for leptospiral antibodies was used to examine more than 8000 serum samples from animals that had aborted and from herds in which leptospiral antibodies had previously been demonstrated. The method gave consistently accurate results. In respect of ease and speed of performance, it was superior to the microscopic agglutination test and it required only minute volumes of serum and antigen suspension. Its most important advantage was that the agglutination reaction could be read directly on the plate, and the removal of samples for microscopical examination was therefore unnecessary.

The cervical and high vaginal flora were studied by quantitative culture methods applied to 356 specimens from 53 healthy non-pregnant women, 102 pregnant women and 57 women with cervicitis. Specimens were obtained with a specially designed “slimesucker”. In all groups the total viable counts ranged from 108-1010 bacteria per ml of fluid from the cervix and posterior fornix. There was only a slight difference between the flora of cervix and fornix.

In healthy women the means of the number of bacterial species per cervical and high vaginal specimen were 1.5 and 1.9 respectively. Lactobacilli predominated in 72% and 80% of cervical and high vaginal cultures respectively. Staphylococcus albus, non-haemolytic and α-haemolytic streptococci, diph-theroids, peptostreptococci, peptococci and Bacteroides spp. could be isolated in high numbers, but less commonly than lactobacilli. Members of the Enterobacteriaceae and yeasts also occurred less commonly than lactobacilli, and in lower numbers (105-108 organisms per ml). It is postulated that the Enterobacteriaceae and yeasts are not part of the normal flora. In specimens from pregnant women the viable bacterial counts were comparable with those in specimens from healthy non-pregnant women. The flora was even more homogeneous during pregnancy, the means of the number of species being 1.5 and 1.7 in cervical and high vaginal cultures respectively; lactobacilli occurred in 92% of the cervical and 86% of the high vaginal samples. In women with cervicitis the means of the number of species obtained from the cervix and fornix were 2.2 and 2.5, with lactobacilli predominating in 65% and 84% respectively. Bacteroides spp. and peptostreptococci were more commonly encountered in women with cervicitis than in those without, but the viable counts of these organisms were similar in all women. The role of bacteria in the production of cervicitis is discussed.

A method was developed to study virulence of coagulase-negative staphy-lococci. Our results showed that coagulase-negative staphylococci injected into adult mice by the intracerebral route did not give rise to lethal infections, whereas mice aged 2 days were much more susceptible.

Novobiocin-resistant strains of Staphylococcus saprophyticus were more virulent than strains of Staphylococcus epidermidis. Strains of S. saprophyticus biotype 3 of Baird-Parker’s classification varied in virulence according to novobiocin sensitivity. In the classification of Kloos and Schleifer, S. saprophyticus biotype 3 can be subdivided into four distinct staphylococcal species, namely S. saprophyticus, S. cohnii, S. haemolyticus and S. warned. S. cohnii and S. saprophyticus were equally virulent for mice aged 2 days, but novobiocin-sensitive S. haemolyticus was less virulent. On epidemiological grounds, however, it would seem that S. saprophyticus has some undefined advantage in invading the urinary tract.

Naegleria fowled, strain HB-1, caused a destructive cytopathic effect (CPE) in secondary mouse-embryo (ME) cells. No evidence was found to suggest that cell-free cytotoxic factors secreted by the amoebae play a part in ME-cell destruction. In culture systems designed for the study of cytopathic factors, mammalian-cell damage seemed to occur only as a result of direct contact with active amoebae. This was confirmed when the progressive destruction of individual ME cells was observed continuously by direct microscopy and time-lapse cinemicrography. The cytoplasmic shrinkage characteristic of naegleria-induced CPE appeared to be associated with phagocytic activity of trophozoites. Adjacent ME cells remained undamaged until they themselves were physically attacked. The apparently intracellular location of amoebae seen in fixed and stained preparations was considered to be an artefact created when trophozoites and ME cells were superimposed.

The controversy regarding the possible role of genital mycoplasma infection in human reproductive failure gave rise to a study in which pregnant rats, previously shown to be of normal fertility, were inoculated 7-11 days after conception with either Mycoplasma arthritidis or an equal volume of sterile broth medium. Repeated observations during the course of pregnancy revealed several pathogenic effects.

A statistically significant decrease was observed in the mean litter size of infected mothers. Furthermore, the offspring showed low viability at birth and, still more, at 10 days after birth. Our data are compatible with the hypothesis that genital mycoplasma infection plays a role in reproductive failure.

The production of neuraminidase (EC 3.2.1.18) by a range of clostridial species was investigated with techniques previously developed to distinguish neuraminidase-negative and neuraminidase-positive strains of Clostridium perfringens (welchii). Large amounts of extracellular neuraminidase were produced by representative strains of C. perfringens and C. septicum in the test media. Under similar conditions, two strains each of C. chauvoei and C. tertium were found to produce small amounts of the enzyme. All of 12 strains of C. sordellii were clearly shown to produce neuraminidase, often in large amounts, but none of five strains of the closely related but non-pathogenic C. bifermentans had demonstrable neuraminidase activity. No neuraminidase was produced by C. novyi (oedematiens) types A-D (10 strains), C. tetani (6), C. botulinum types A, B, C or E (4), C. sporogenes (4), C. histolyticum (4) or by single strains of five other clostridial species.

Clostridial neuraminidase was predominantly extracellular and was not calcium-dependent. The investigation took account of variations in growth and enzyme production in different media. It was necessary to prolong the neuraminidase-assay reaction time to 24 h and to monitor for the presence of NAN-aldolase (EC 4.1.3.3) to define true negatives. It is suggested that neuraminidase production may be of value in taxonomic studies and that its production by several pathogenic species of clostridia may be of interest in studies of pathogenicity and virulence.

The effects of various treatments on the immune response of chickens to vaccine prepared from Pasteurella multocida strain PI059 were investigated. The treatments, given on or before the day of hatching, consisted of surgical bursectomy (SB), hormonal bursectomy (HB), surgical thymectomy (ST) and X-irradiation (X).

In chickens subjected to SB, SBX or HB before vaccination, the production of agglutinin and immunoglobulin was impaired, but the production of resistance against challenge was not. In chickens subjected to ST or STX before vaccination, the production of agglutinin and immunoglobulin was unimpaired, but the production of resistance against challenge was diminished. Protection was therefore thymus-dependent.

Mice were given an intravenous dose of formalinised C. welchii type-D epsilon prototoxin and an immunoperoxidase technique was used to demonstrate this antigen in the tissues. The antigen was found to bind to the luminal surface of the endothelial lining of certain blood vessels, to the luminal surface of the cells lining the loops of Henlé and distal convoluted tubules in the kidney, and to the hepatic sinusoids. As it has been shown previously that formalinised epsilon prototoxin and epsilon toxin can compete for the same receptor sites it is postulated that the binding sites demonstrated represent the location of the receptors for C. welchii type-D epsilon toxin.

Intradermal injection of Clostridium welchii type-D epsilon toxin increased the permeability of blood vessels in guinea-pig skin to Evans blue dye by a mechanism not dependent on the release of histamine. The toxin was also found to raise the plasma concentration of cyclic adenosine 3′, 5′-monophosphate in mice. It is concluded that epsilon toxin is an enterotoxin capable of causing widespread damage, after binding to receptor sites on the surface of certain cells, through a mechanism mediated by an adenyl cyclase-cAMP system.

Epsilon toxin, at relatively low concentrations, killed guinea-pig peritoneal macrophages in vitro. The cells became swollen, the nuclear and cytoplasmic membranes “blistered” and discontinuous, and the cytoplasm appeared structureless. Formalinised epsilon prototoxin was shown to bind closely to the outer surface of the cells and it is concluded that this site represents the location of the receptors for epsilon toxin.

In addition the toxin at higher concentrations killed rabbit peritoneal macrophages after increased periods of incubation, but had no demonstrable effect on other cells from guinea-pigs, rabbits, mice and sheep.

Suspensions of erythrocytes from certain marine fish (cod, saithe, haddock and mackerel) were 4-16 times more sensitive than human or horse erythrocytes to staphylococcal δ-haemolysin. Cod erythrocytes were lysed rapidly by δ-haemolysin and the titres of haemolysin were independent of the temperatures at which the tests were incubated, over the range 0-37°C. Fish erythrocytes were not lysed by purified α- and β-haemolysins and no synergistic action was found between β- and δ-haemolysins. In blood agar-overlay plates, the zones of lysis that developed in cod blood agar were larger and clearer than those that developed in human or horse blood agar.

Fluorescein-labelled lectins of known specificities for different sugars were used in an attempt to identify fungi in paraffin sections of surgical and postmortem material. Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Paracoccidioides brasiliensis and Rhizopus oryzae have been studied with five fluorescein-labelled lectins and with fluorescein-labelled aprotinin. The fungi were readily distinguishable on the basis of differences in their reactions with these stains. The results accord well with what is known of the chemistry of the organisms and the method offers promise to practising histopathologists.

A serial study of rotavirus infection in gnotobiotic piglets is described. They were infected when 7 days old and the course of infection was followed for 21 days, by immunofluorescence and histological examinations of the intestinal epithelium and by titration of the virus content of the gut lumen. There appeared to be two stages of recovery of the intestinal wall, the earlier stage involving non-immune mechanisms and the later involving antibody.

Thirty-eight strains of a Gram-positive bacterium identified as Bacillus licheniformis var. endoparasiticus (Benedek), referred to as BLE, were isolated in various stages of reversion from the L-forms, from 28 out of 100 samples of whole blood or erythrocytes from normal healthy subjects, after prolonged incubation. Similar results were obtained from 100 samples from hospital patients with conditions not usually associated with blood infection. BLE was isolated from only one of 125 samples of plasma, including those separated from infected erythrocytes.

Isolates from cultures incubated for up to 4 months were usually in the form of spheroplasts or diphtheroid bacilli; the fully reverted phase, resembling B. licheniformis, with the capacity to form endospores, was isolated occasionally from cultures aged 1-6 months, and it constituted about half the isolates recovered from cultures aged 6-25 months.

BLE was isolated in subculture, and with the usual frequency, in previously unopened, primary cultures. It did not occur in 1200 subcultures of 150 control cultures made with autoclaved or irradiated blood cells; it was not detected in the environment of the laboratory or blood-sampling areas, or on the skin or in the respiratory passages of the operators and other persons associated with the laboratory, where typical, saprophytic B. licheniformis was very rare.

It is concluded that this Bacillus species exists as an L-form, associated with the erythrocytes of a large proportion of normal persons, as previously recorded by several observers. Some of the morphological variants associated with the L-cycles have in the past been described as different organisms, for example L-forms of various bacteria or mycoplasmas, and the diphtheroid stage has been thought to belong to the genera Corynebacterium and Listeria. The sporogenous stage, although frequently described, has normally been discounted as a contaminant.

These observations do not admit of any conclusion in respect of the claims that such bacteria may have a role in arthritis, cancer or other diseases.

When suspensions of Yersinia enterocolitica were stained with triphenyl-tetrazolium chloride (TTC) their antigenic specificity, as determined by tube and microtitre-plate agglutination tests, was altered.

Thus, a TTC-stained suspension of serotype 05,27 detected antibodies to nine O serotypes of Y. enterocolitica in sera from experimentally infected animals but did not cross-react with antisera to organisms of five other unrelated genera including Bruceila. The same suspension did, however, cross-react weakly with antisera to four serotypes of Yersiniapseudotuberculosis.

The value of this antigen preparation in detecting antibodies to the wide range of Y. enterocolitica serotypes likely to cause infection in man should now be assessed.