Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104.

Commercial laying hens were infected with Salmonella typhimurium DT104 strain 16 alternatively via the crop (10(7) cfu per bird) or by an aerosol delivered directly to the beaks using a Collison nebuliser and Henderson apparatus (2 x 10(2) or 2 x 10(4) cfu per bird). Infection by both routes caused systemic infection and prolonged contamination of faeces. Contamination rates of eggs and muscle were much higher following the aerosol challenges despite the much lower doses given by this route. The frequency of Salmonella isolation from eggs rose from 1.7% following oral challenge to 14% and 25%, for each of the aerosol challenges respectively, and the frequency of isolation from muscle rose from 0% following the oral challenge to 27% following each of the aerosol challenges.

Characterisation of cytolethal distending toxin (CDT) mutants of Campylobacter jejuni.

In order to assess the contribution of cytolethal distending toxin (CDT) to the toxigenicity and pathogenicity of Campylobacter jejuni, the C. jejuni 81-176 and C. jejuni NCTC 11168 CDTs were inactivated by insertional mutation of the cdtB toxin subunit. Cell-free sonicates from isogenic C. jejuni 81-176 cdtB- strains were found to be greatly attenuated in HeLa cytotoxicity assays, whilst still retaining some toxigenicity. Sonicates from a C. jejuni NCTC 11168 cdtB- strain produced no detectable cytotoxicity. When orally administered to adult severe combined immunodeficient (SCID) mice, C. jejuni cdtB mutant strains were unaffected in enteric colonisation abilities but demonstrated impaired invasiveness into blood, spleen and liver tissues. These data suggest that CDT may be the principal toxin produced by this species and that some C. jejuni strains may generate additional toxigenic factor(s) distinct from CDT.

Experimental campylobacter infection and diarrhoea in immunodeficient mice.

The responses of previously untested immunodeficient mouse strains to campylobacter infection are described. Three strains of adult immunodeficient mice (SCID-Beige, C.B-17-SCID-Beige and RAG-2) were inoculated intragastrically with Campylobacter jejuni NCTC 11168. All mice became heavily colonised, but none developed clinical signs of disease. Immunocompetent BALB/c mice inoculated similarly had much lower colonisation levels. The co-administration of iron dextran had no effect on colonisation levels nor the development of clinical signs of disease. In contrast, C.B-17-SCID-Beige mice, when inoculated with one of a series of 10 clinical isolates of C. jejuni, were more heavily colonised for extended periods (up to 5 months) and approximately 10-20% of the mice became ill with diarrhoea. C. jejuni was detected in mouse faeces throughout at levels of 10(7)-10(9) cfu/g. All mice killed whilst ill with diarrhoea displayed histopathological lesions typical of human campylobacteriosis. Severe pathology was limited to the large intestine and was suggestive of an acute, bacteria-induced inflammation. Although blood was detected in the diarrhoeal stools, no evidence of mucosal epithelial cell invasion was found by immunohistology. No pathology was detected in tissue sections from any of the animals that had not developed signs of disease following C. jejuni inoculation. These immunodeficient mouse strains are readily, and heavily, colonised as adults by C. jejuni. The diarrhoea, although sporadic, was reproducibly produced, and could provide the basis for pathogenicity studies.

Enamel matrix and crystals--chicken and eggs?

This discussion is presented as an attempt to interrelate the matrix and mineral in terms of our existing knowledge as presented at this Symposium, and to relate it, where possible, to what is being accomplished in allied disciplines and research areas elsewhere. The present author is fully aware of the difficulties involved in this task and of his own inadequacies in attempting to perform it. As a general rule, with perhaps the morphologists being the exception to it, the matrix and crystal components have been investigated independently of each other. This, of course, is quite justifiable, as the presence of one can often interfere with the investigation of the other, and each needs to be examined initially in as pure a state as possible.

Remineralization of artificial caries-like lesions in human enamel in situ by chewing sorbitol gum.

The objective of the study was to determine quantitatively the effect on the potential for in situ remineralization of artificial caries-like lesions in human enamel when sugar-free gum containing mainly sorbitol as sweetener was chewed after meals and snacks. Artificial white-spot lesions were created in extracted human premolars and divided into three parts. One part was used as reference and the other two worn consecutively for two 21-day periods by 10 volunteers in a cast silver band cemented on lower molar teeth and covered with gauze to promote plaque formation. During the experimental periods, the subjects used fluoridated toothpaste twice daily, and consumed three meals (breakfast, lunch, and dinner) and two snacks (selected from chocolate bar, raisins, chocolate wafer, and iced cupcake). Sorbitol gum was chewed for 20 min immediately after each meal or snack during one of the experimental periods. The three parts of the enamel lesions were then sectioned (congruent to 80 microns) and examined together by means of quantitative microradiography and by polarized light microscopy. All estimates of mineral content indicated that significant remineralization occurred and was approximately doubled with gum-chewing. It is suggested that sorbitol gum stimulates salivation, which is responsible for the significantly enhanced remineralization, thus contributing to a therapeutic, caries-preventive effect. Because the gum was chewed immediately after meals and snacks, inhibition of demineralization may also have occurred.

Carbon load in aquatic ecosystems affects the diversity and biomass of water biofilm consortia and the persistence of the pathogen Campylobacter jejuni within them.

The influence of carbon load on autochthonous water microflora population distribution and diversity, and on the persistence of Campylobacter jejuni, was examined with a two-stage aquatic biofilm model. Serine was chosen since it is a carbon source utilised by C. jejuni and concentrations were chosen to reflect upper limits of amino acid load reported in surface water. The total viable count of the autochthonous biofilm microflora increased with increasing serine concentration (10-fold and 20-fold with 5 nM and 5 µM serine, respectively), as did the counts of the microflora in the planktonic phase. Differences in biofilm species distribution as determined by culture were small with changes in temperature or the addition of serine; but was markedly affected by serine as determined by light microscopy, becoming more luxuriant and dominated by long filamentous cells. The addition of serine to the water significantly and progressively reduced the persistence of C. jejuni, which decreased by 25% and 50% with serine concentrations of 5 nM and 5 µM respectively. We have demonstrated that carbon load affects the species diversity and density of both the planktonic and biofilm phase of aquatic autochthonous microflora. Although the survival of C. jejuni in water in a culturable form was sufficient for this to be an important vehicle for its transmission, carbon load significantly influenced survival; an increase in serine concentration significantly reduced survival.

An investigation of the effects of mellitic acid on hydroxyapatite.

The effects produced on the solubility and crystal growth of hydroxyapatite by the condensed phosphates and the diphosphonates are essentially physicochemical in nature. Other poly-anions, may, on this same basis, produce similar effects. Mellitic acid (benzene hexacarboxylic acid), in dilute solution, was taken up by hydroxyapatite in the presence of excess phosphate and was not significantly displaced by it. Mellitic acid treatment of hydroxyapatite inhibited its ability to "seed" metastable solutions of calcium phosphate. Mellitic acid, which has already been shown to be an effective inhibitor of dental calculus in experimental animals may also have some other properties that could be of advantage to its use in humans.

An ultrastructural investigation of the filamentous surface appendages of suspensions of plaque bacteria as revealed by negative staining.

The filamentous surface appendages of freshly-collected aqueous suspensions of plaque bacteria, obtained from 1 day old supragingival plaque have been examined in the electron microscope by the technique of negative staining with methylamine tungstate. Approximately half of the bacteria revealed surface appendages as either fimbriae (45%), flagella (13%) or both (3%). The appendages were distributed either polarly, intermittently or peri-trichously around the bacteria and varied in length from 0.2 micron to more than 20 micron. It was not possible from these observations alone to determine either the topology of the appendages or their role as they existed originally in dental plaque.

Bacterially catalysed N-nitrosation reactions and their relative importance in the human stomach.

Human exposure to endogenously formed N-nitroso compounds has frequently been suggested as a causative factor in carcinogenesis where this is related to chronic bacterial infection such as is seen in gastric achlorhydria. At least two distinct mechanisms of endogenous formation have been identified. The first, a direct chemical reaction between secondary amino compounds and nitrite, is strongly pH dependent and does not proceed rapidly at neutral pH even in the presence of chemical catalysts. The second depends on the direct bacterial catalysis of N-nitrosation. The data presented demonstrate that the bacterially mediated reaction is catalysed by bacterial enzyme systems and proceeds much more rapidly at neutral pH than the chemical reaction. This suggests a particular relevance to the in vivo situation where neutral pH, bacteria and elevated nitrite concentrations are found. Drawing on the kinetic information presented regarding the bacterially mediated nitrosation reaction, the known kinetics of the chemical reaction and the published values for the relevant substrate concentrations in both the colonised and the normal acid stomach the bacterial and chemical reactions have been compared. Using these criteria, and assuming the presence of bacteria with the appropriate metabolic activity, it may be predicted that N-nitroso compounds may be formed in the colonized stomach at much higher concentrations than in the normal acid stomach. The difference in yield may be by two to four orders of magnitude. Different bacterial species and different isolates of the same species show considerable variation in their abilities to catalyse N-nitrosation reactions. The most rapid catalysis is associated with those bacteria capable of reducing nitrate and nitrite by the process of denitrification. The most significant clinical corollary of these studies is that although bacterial catalysis of N-nitrosation has been demonstrated unequivocally, bacterial colonization of the stomach may not itself necessarily result in elevated endogenous N-nitroso compound exposure despite the elevated nitrite concentrations normally associated with such colonization. An increase in exposure to endogenously formed N-nitroso compounds would only be predicted in those individuals where a significant proportion of the colonizing bacteria expressed significant N-nitrosation activity. As a consequence the carcinogenic risk may be restricted to only a small proportion of colonized individuals depending on the prevalence of sustained infection by bacteria with significant N-nitrosation activity, particularly denitrifiers.