Invasion of Salmonella into the cells of the intestinal epithelium is an important step in the infection process. This initial invasion is followed by colonization of other organs throughout the body. In an attempt to better understand this process, we moved defined mutations in several genes of the inv locus into Salmonella typhimurium UK-1 and two strains of Salmonella enteritidis. These mutant strains were evaluated for their oral and intraperitoneal virulence as determined by 50% lethal dose in 1-day-old white leghorn chicks. These inv mutants were also studied for their ability to colonize orally infected chicks. The invA, invB, and invC mutations all caused a reduction in oral virulence and colonization by UK-1 and the S. enteritidis strains. Mutation of the invH gene had little or no effect on oral virulence or colonization. None of the inv genes tested had any effect on virulence of these Salmonella strains when administered intraperitoneally.

We examined Bordetella avium for virulence factors common to Bordetella pertussis, including pertussis toxin, filamentous hemagglutinin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin. B. avium produced a dermonecrotic toxin and a tracheal cytotoxin. The dermonecrotic toxin of B. avium is a 155,000-molecular-weight, heat-labile protein which was lethal for mice, guinea pigs, young chickens, and turkey poults and produced dermonecrosis when injected intradermally into guinea pigs, chickens, and turkey poults. High-pressure liquid chromatography of B. avium culture supernatant fluid revealed the presence of a tracheal cytotoxin chemically identical to that produced by B. pertussis. B. avium isolates were negative for B. pertussis-like filamentous hemagglutinin and pertussis toxin when assayed with antibody against B. pertussis filamentous hemagglutinin and pertussis toxin. Furthermore, B. avium failed to induce the clustered CHO cell morphology characteristic of pertussis toxin. Adenylate cyclase assays indicated that B. avium does not produce an extracytoplasmic adenylate cyclase, even after passage through embryonated turkey eggs. Since production of virulence proteins by B. pertussis is regulated by growth in media containing nicotinamide or MgSO4 or by growth at reduced temperatures, we determined the eifect of these supplements and growth conditions on production of dermonecrotic toxin by B. avium. Production of dermonecrotic toxin in B. avium was not altered by growth in media containing 100 ,uM FeSO4 or 500 ,ig of nicotinamide per ml or by growth at 25 or 42°C, but production was significantly decreased by growth in media containing 20 mM MgSO4 and slightly reduced by growth in media containing 500 ,Ig of nicotinic acid per ml. These studies revealed that B. avium is similar to B. pertussis in that both species produce a dermonecrotic toxin and a tracheal cytotoxin and production of dermonecrotic toxin is regulated by nicotinamide and MgSO4. The presence of dermonecrotic toxin and tracheal cytotoxin in all Bordetella species indicates that these products may be important virulence factors in bordetellosis.

No abstract

The influence of infective dose on chicken immunogenicity was examined in 1-week-old chickens. Chickens were infected orally with various doses of chi 3761 or chi 3985. Fecal shedding, colonization of the cecum, and induction of Salmonella-specific serum immunoglobulin isotypes were analyzed over a 5-week period. The delta cya delta crp Salmonella typhimurium vaccine strain chi 3985 was used to assess the effect of vaccination dose on protection after oral vaccination of chickens at 1 day and 2 weeks of age. Wild-type S. typhimurium strain chi 3761 was used to challenge vaccinated and unvaccinated chickens at 6 weeks of age, and the recovery of Salmonella from the cecum was used as a measure of protection. Infection of 1-week-old chickens with chi 3985 was more effective in reducing fecal excretion and cecal colonization than was infection with chi 3761. Double vaccination with 10(8) or 10(7) CFU of chi 3985 at 1 day and 2 weeks of age protected vaccinated chickens against cecal colonization by the challenge strain chi 3761. Immunogenicity of Salmonella is dose and genotype-dependent.

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.