Departments of Mi(crobiology ancd Foods anid Nitr ition, Tl/e University ojA/bAlbert Edmontoo,. Alber-tti, Canadai T6G 2M8

The first chick to hatch and dry in each of a series of incubators was fed a suspension of Campylobacter jejuni via a stomach tube and returned to the incubator. Subsequently, all hatched chicks were taken out of the incubators and housed in standard transport boxes for a further 24 h, after which they were killed by carbon dioxide inhalation. The intestinal tracts of all hatched birds were excised, enriched in liquid media and then plated on media selective for C. jejuni. Campylobacters were cultured from up to 70% of the chicks but this percentage varied with the strain originally fed to the initial chick. The spread of poultry-derived strains was as extensive as that of some human-derived strains, while other human strains showed little tendency to spread amongst chicks. A significant number of hatched, healthy chicks had distended intestinal tracts and showed abnormal gross liver pathology. This symptom was typical of those strains of C. jejuni known to be invasive or toxigenic. However, the gross pathology occurred more frequently than did the incidence of viable C. jejuni in the intestine of the same chicks, suggesting either that the C. jejuni were now mainly unculturable or that some cellular product was responsible for the intestinal pathology. The serotype of each of the strains isolated, was identical to the serotype of the strain initially fed to each banded bird. This laboratory study shows that one infected chick in an incubator potentially can infect other chicks before the birds reached the farm in transport boxes, so accounting for a further source of C. jejuni contamination of poultry.

Campylobacter jejuni will grow in egg yolk and in yolk-albumen melanges reaching populations in excess of 108 CFU/ml. Albumen alone is highly toxic, with D10 values of 2.4 h in vitro at 42°C. Exposure to albumen in vitro is not reversed by later exposure to yolk; rather a substrate accelerated death effect is seen in the presence of added yolk. C. jejuni was generally more sensitive to gelatinous albumen than to less viscous albumen, although both forms of albumen inhibited motility after 6 h of incubation at 42°C. Sensitivity to albumen was only partially due to the individual effects of pH or lysozyme. The major factor in the sensitivity of C. jejuni to egg white was the conalbumen as demonstrated by in vitro culture.

Fertile chicken eggs were infected in our laboratory with Campylobacter jejuni suspensions by using temperature or pressure differential methods of inoculation. After 2 days of incubation, over 90% of the eggs carried C. jejuni when iron was present in the inoculum. This percentage declined rapidly until by day 8, less than 10% of the eggs were detectably infected. However, up to 11% of hatched, healthy chicks carried C. jejuni in their intestinal tracts. The isolated organisms were of the same serotype as the initial inoculum. C. jejuni was recovered without difficulty when the intestinal tracts of chicks were enriched, but recovery from early dead-in-shell or infertile eggs was poor. This poor recovery and the rapid decline of C. jejuni after 2 days of egg incubation suggest that the vibrio is sensitive to some part of the incubating egg or to the temperature of prolonged incubation. It was impossible to predict which eggs would yield infected chicks on the basis of the number of organisms taken up by each egg, and no correlation existed between the number of organisms taken up and the efficiency of the hatch, i.e., the hatch ratio. If iron was omitted from the inoculum broth, the egg infection rate at day 2 was lower.

SUMMARYGraded doses of live and heat-killed cells of Campylobacter jejuni were injected into the yolk-sac of 5-day-old chick embryos, and the 50 % lethal dose (LD50) was determined 7 days later. A strain dependent virulence was seen. In the diluted series of cultures the LD50 values for live campylobacter ranged from 106 C.f.u.beyond the last dilution showing growth, that is to less than one organism per embryo. When the 22 strains were tested as heat-killed cells, the chick embryo LD50 values retained the same relative order of toxicity obtained with viable cells, but the LD50 values were increased by + 1 to + 4 log units. Heat-killed cells from strains known to be invasive, but non-toxigenic, were still lethal for the embryos, suggesting that viability was not solely necessary for virulence. Semi-pure lipopolysaceharide from a non-virulent strain of C. jejuni was not toxic to the embryos, but semi-pure and ultracentrifuge-purified lipopolysaceharide from the most lethal campylobacter strains gave LD50 values in the order of 3 0 jug lipopolysaceharide per ml (0 6 ,tg per embryo) in the yolk-sac assay. No relationship between serotype and lethality was seen. Injection into the yolk-sac appears to be an easy, rapid and reprodueible in vivo assay of the virulence of C. je,junt. INTRODU"CTIONCampylobacter jejuni is a major cause of acute enterocolitis, but there is no one laboratory animal in which all of the clinical symptoms of human campylobacteriosis can be reproduced. The two clinically severe presentations are described as bloody diarrhoea and watery (secretory) diarrhoea which, respectively, suggest invasive and toxigenic virulence factors. Laboratory evidence supports the existence of a variety of putative virulence factors but only a toxin, immunologically related to Escherichia coli LT and cholera enterotoxin, has been isolated in a semi-pure form (1). Neither the cytotoxin (2) nor the invasive factor have been characterized, although the outer membrane proteins and lipopolysaccharides are under investigation for their possible implication as virulence factors (3)(4)(5). Despite the large number of C. jejuni isolated and reported in the literature, few strains have been assayed for invasiveness. Toxigenicity assays were carried out on isolates from Mexico and the USA by Klipstein and co-workers (6). Subsequently this group showed that of 40 strains obtained from one