Chicken Embryo Lethality Assay for Determining the Virulence of Avian Escherichia coli Isolates

Abstract

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

Microbial Infections Are Associated with Embryo Mortality in Arctic-Nesting Geese

“…Microbial infection can cause avian embryo mortality, and bacteria from eggshells and contents of nonviable eggs have been isolated from a range of avian species (10,(33)(34)(35)(36). Our identification of a Neisseria species as the predominant bacterium present in addled eggs appears to be novel, although species in the Neisseria genus have been associated with waterfowl on several occasions.…”

“…Inoculating doses were determined through serial dilution and CFU counts; five 10-fold dilutions, beginning with 100 CFU, were prepared from each isolate. Inoculating doses were within ranges reported to have been used by others (32,33,34). One-day-old fertilized White Leghorn chicken eggs obtained from Charles River Laboratories (Wilmington, MA) were incubated at 37°C with high humidity and mechanically tilted to a 45°angle every hour for 7 days prior to infection and throughout the experiment.…”

The BarA-UvrY Two-Component System Regulates Virulence in Avian Pathogenic Escherichia coli O78:K80:H9

“…The role of the BarA-UvrY TCS in the virulence of APEC strain 7122 was determined using an chicken embryo lethality assay, in which live embryos were infected with a controlled amount of bacteria and were scored as alive or dead by movement of the embryo when held close to a bright light source (12,16,42,69). Mutant strains of 7122 were generated by Red-mediated recombination, with modifications suggested for clinical isolates of E. coli (8,32,38) ( Table 2).…”

Xiang-Qi-Tang Increases Avian Pathogenic Escherichia coli-Induced Survival Rate and Regulates Serum Levels of Tumor Necrosis Factor Alpha, Interleukin-1 and Soluble Endothelial Protein C Receptor in Chicken

“…It has been shown that APEC proliferates significantly in 6 h postinoculation, causing acute systemic infection or death. 20,21) In our research, the death of infected chickens began from 12 h, and the most benefited doses of XQT decreased the mortality of infected chickens at the death peak during 24 to 48 h after inoculation of APEC. These data suggest that XQT can prevent the porliferation of APEC and the cascade of events induced by APEC or its toxins in vivo.…”