At 46 days of age, the chickens in each group were challenged i.v. with 0.5 mL of a bacterial suspension containing 108 CFU/mL of E. coli O78 strain J46, which harbors the iss, tsh cvaC, and papC genes. Z-VAD-FMK clinical trial The LD50 value of this challenge strain for i.v. infection against 5-week-old chickens is 2.9 × 107 CFU /bird. The challenged chickens were observed for 7 days, and their clinical signs scored as follows: none = 0, reluctance to walk = 1, mild depression or ataxia = 2, depression or astasia = 3, death = 4. Dead chickens were necropsied immediately on the day of death. Seven days after challenge exposure, the surviving chickens

were killed and necropsied. Macroscopic lesions were recorded and scored separately for each organ as follows: heart and pericardium (normal = 0, turbid with excessive or cloudy fluid in the pericardial cavity or partial pericarditis = 1, marked pericarditis = 2, severe pericarditis or death = 3); liver (normal = 0, small amount of fibrinous exudate = 1, marked perihepatitis = 2, severe perihepatitis or death = 3). Samples for bacteriologic examination were taken from the liver and heart of each chicken at necropsy. Twenty 19-day-old embryonated eggs

were allotted to two equal groups and immunized with AESN1331 or sterile PBS. Each egg was oriented with ICG-001 supplier the large end up and a hole punched in its top with an 18-gauge needle. Using a 21-gauge needle, an inoculum of 10 μL (103 CFU) of AESN1331 per egg (or an equivalent volume of PBS) was injected into the amniotic fluid. All inoculated eggs were then hatched in the same incubator. Hatching was assessed after 21.5 days of incubation. Until exposure to challenge, the hatched chickens were monitored daily for signs of illness and for death. At 28 days of age, all chickens were challenged and assessed as described above. Fisher’s exact test was used to compare the number of dead chickens and the number of organs positive for the challenge Casein kinase 1 strain in each group. Student’s two-tailed t-test was employed to compare the clinical and the lesion scores between experimental groups. A P value of < 0.05 was considered significant. We compared the in

vitro and in vivo properties of the mutant strain to those of the parent; results are summarized in Table 1. As with the parent, E. coli O78 antiserum agglutinated AESN1331. Colonies of the mutant were smaller than those of the parent. AESN1331 colonies were colorless on MacConkey agar, demonstrating an inability to ferment lactose. AESN1331 also was unable to ferment D-mannose, D-sorbitol, L-rhamnose, sucrose and D-melibiose, but could still ferment glucose and L-arabinose. Although the mutant had lost tryptophan deaminase activity and indole production, the strain resembled its parent in harboring β-galactosidase, lysine decarboxylase, ornithine decarboxylase, and oxidase activities while lacking arginine dihydrolase, citrate production, H2S production, urease, acetoin production, gelatinase, and ability to reduce NO3− to NO2−.