Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity.

The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages (rs = -0.94, P less than 0.0005) and a positive correlation for peritoneal lymphocytes (rs = +0.97, P less than 0.0005) and spleen cells (rs = +0.76, P less than 0.005) existed.

The binding of lipopolysaccharide from Escherichia coli to mammalian cell membranes and its effect on liposomes.

The kinetics of the absorption of 32P- or 14C-labelled lipopolysaccharide from Escherichia coli NCTC 8623, serotype 0 125, chemotype XII, to erythrocytes, leukocytes, peritoneal macrophages and peritoneal lymphocytes was examined. Under variable conditions maximal levels of binding were found due to saturation of receptor sites on the cell membrane or steric hindrance by bound lipopolysaccharide. During adsorption slight leakage of haemoglobin was found but complete lysis of erythrocytes was ruled out after noting the effect of lipopolysaccharide on artificial lipid bilayers. The affinit of lipopolysaccharide to cell membranes revealed a consistent pattern of cyclic fluctuation between adsorption and desorption. A model was proposed to explain this cyclic fluctuation in binding based on membrane reorganization. It was significant that the cycle of lipopolysaccharide adsorption-desorption proceeded to completion even if the process was interrupted. The indication was that, once triggered, membrane reorganization occurred independently without influence from the test environment.

Procedures for large-scale antiserum production in sheep.

The increased cost of maintaining and purchasing small laboratory animals used in the production of large quantities of antiserum resulted in a search for a more suitable animal. This paper describes the procedures used to raise antisera in Scottish mountain sheep.

Adjuvanted oral vaccines should not induce allergic responses to dietary antigens.

Oral vaccines may contain adjuvants which might elicit allergies against dietary proteins. Four antigens were used to measure such an effect--ovalbumin, soya bean protein, lactalbumin and gluten. Neither guinea pigs nor mice showed IgE responses after oral administration of adjuvanted vaccines containing lactalbumin and gluten. No IgE responses were detected in mice with any of these antigens after oral immunization, but, in the guinea pig, nine out of 18 animals reacted to either ovalbumin or soya bean protein and none reacted to lactalbumin and gluten. It is concluded that the risk of allergy induction against normal dietary proteins is low but such tests should be applied to potential adjuvanted oral vaccines to measure any possible contraindication, especially with atopic individuals.

Studies on the Vibrio cholerae mucinase complex. III. Neutralisation of the neuraminidase activity by specific anti-neuraminidase IgG.

A partially-purified neuraminidase from the mucinase complex of Vibrio cholerae was used to prepare a specific anti-neuraminidase antiserum in rabbits. When the neutralising potency of this serum against V. cholerae neuraminidase was assessed in conventional tests, the enzymic activity, as measured by thiobarbituric acid, methoxyphenol-neuraminate and goblet-cell assays, apparently increased. These results are attributable to the presence of a sialylated glycoprotein substrate and small amounts of sialidase in the crude antiserum. However, a twice-purified DEAE-IgG fraction of the antiserum neutralised the enzymic activity of the V. cholerae neuraminidase.

Studies on the Vibrio cholerae mucinase complex. II. Specific neuraminidase activity measured histochemically in a goblet cell assay.

The activity of neuraminidase prepared from the mucinase complex of Vibrio cholerae was measured by a new, semi-quantitative goblet-cell assay. The counts of normal, alcianophilic, sialomucin-containing goblet cells (purple-stained) and neutral mucosubstance-containing goblet cells (magenta-stained) in serial sections of ileum were compared before and after neuraminidase treatment. The procedure provides a more natural assessment of the action of V. cholerae neuraminidase on the viscous intestinal mucus.

Studies on the Vibrio cholerae mucinase complex. I. Enzymic activities associated with the complex.

Mucinase enzymes were isolated and partially purified from the culture fluid of Vibrio cholerae grown in proteose peptone-colostrum medium. The mucinase complex contained neuraminidase, endo-beta-N-acetylhexosaminidase, nicotinamide-adenine-dinucleotidase and proteinases. Traces of phospholipase activity were detected but the complex lacked aldolase activity.

Factors affecting the lethality of Campylobacter fetus subspecies jejuni in mice.

Intraperitoneal injection of Campylobacter fetus ss. jejuni into HAM/1CR mice was lethal, but viable counts of bacteria from whole body homogenates, organs and blood indicated that death was not due to sustained bacterial multiplication. Heat-killed organisms (5 X 10(9) cfu) injected into 7-day-old mice caused death within 24 h and this was shown to be due to endotoxin. Both ferric iron and heterologous lipopolysaccharide enhanced virulence; the LD50 was lowered from 1.8 X 10(9) cfu to 2.7 X 10(7) cfu when both were used. Three-day-old or adult animals survived challenge with Campylobacter fetus without clinical symptoms when challenged orally or by intravenous or intraperitoneal routes.

Heat-labile and heat-stable haemolysins of Campylobacter jejuni.

During studies on the virulence mechanisms of Campylobacter jejuni clinical isolates it became apparent that some strains produced one or more haemolysins and some did not. There was no great difference between Group C (cholera-like) strains and Group D (dysentery-like) strains. The protein haemolysin(s) showed a spectrum of activity against erythrocytes from different animals; with maximum activity against rabbit and minimal activity against chicken erythrocytes. The results suggested a two-stage activation mechanism for haemolysis which involved a multi-hit lytic activity. It was concluded that the C. jejuni haemolysins were not identical to those described in other organisms and they may be involved in iron acquisition in vivo.

The use of adjuvants in experimental vaccines : I. Aluminum hydroxide gels.

How curious it is that we accept in therapeutic medicine the principle that non-metabolizable sutures, splints and prostheses are good but the employment of non-metabohzable vehicles in preventive medicine is evil Davenport [1968].

The Use of Adjuvants in Experimental Vaccines : II. Water-in-Oil Emulsions: Freund's Complete and lncomplete Adjuvants.

As mentioned in Chapter 9 , Freund's complete adjuvant (FCA), produced by the Statens Seruminstitut (Copenhagen, Denmark), was chosen as the standard adjuvant preparation because of its extensive use in experimental vaccines in animals, its strong adjuvant effect, and the considerable literature describing its activity. The combination of mineral oil (Bayol 55) and dead mycobacterial organisms produces a specific cellular reaction in experimental animals. The mycobacteria in FCA stimulate the formation of epithelioid macrophage cells and also the maturation of plasmablasts to plasma cells. The effect was greater when the oil and mycobacterial fraction were combined. Numerous studies have shown that the oil emulsion was responsible for the retention of the antigen at the site of inoculation. This depot provides a slow and prolonged antigenic stimulus to antibody-forming cells. FCA stimulates active humoral and cell-mediated immune responses but there may be concomitant adverse side-effects because of the reactogenicity of some types of mineral oil, particularly the formation of the epithelioid macrophage granuloma at the site of injection and local ulceration when the injection is given subcutaneously. In addition, other contraindications have been recorded, e.g., pyrogenicity, stimulation of experimental autoimmune diseases, and adjuvani. arthritis (reviewed in detail by Stewart-Tull[1, 2]).

The Use of Adjuvants in Experimental Vaccines : IV. ISCOMS.

The use of saponin in experimental vaccines has been known for more than 60 yr (1, Chapter 9 ) and generally it is more active as an adjuvant with strongly immunogenic antigens. A number of saponins are derived from the bark of the South American soaptree (Quillaja saponaria) as acylated triterpenoids each having quillaic acid as the aglycone but with different glycosidic side chains. From the crude preparations it has been possible to refine purer mixtures, e.g., Quil A (2) and QS-21 (3). These have been shown to exert an adjuvant effect at doses as low as 5.0-20.0 µg. Because a wide variety of foods contain saponins there is potential for their use in oral vaccines, but there has been some reluctance to use them parenterally because of their known membranolytic and hemolytic effects; ISCOMs have not been shown to possess hemolytm activity at the usual dose levels used in animals.

The Use of Adjuvants in Experimental Vaccines : III. Liposomes.

The use of artificial lipid bilayers in the form of vesicles has been described as an efficient means of presenting antigens (1, 2 and Chapter 9 ). A large variety of natural phospholipids or other polar amphiphiles can be used in an aqueous solution of an antigen to form either unilamellar or multilamellar spherules. The antigen(s) may either be lipid-soluble and insert into the artificial lipid bilayer, bmd to the bilayer, or become entrapped inside the spherule. In the multilamellar liposomes the antigen(s) may also be trapped in the aqueous compartments between the lipid bilayers. The surface of the liposome may also be positively or negatively charged by the addition of suitable charged amphiphiles. Many of the natural adjuvant substances are amphiphilic and may insert into the lipo-some through hydrophobic groups (3, 4). It has been my experience that the purity of the phosphohpid does affect the stability of the final preparation of liposomes and their leakiness. For instance, Gregoriadis (1) points out that plasma high-density lipoproteins will remove low-melting phospholipids from liposomes and cause leakiness. With high-melting phospholipids or with an excess of cholesterol, the lipid bilayers become rigid at 37°C, the result being a slower release of the antigen. There is not space here to record the many combinations that could be used; the preparation of a pure sample of phosphatidylcholine (ovo-lecithin) and positively and negatively charged liposomes will be given as examples.

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).