Chicken embryo lethality assay for determining the virulence ofRiemerella anatipestiferisolates

Abstract

Riemerella anatipestifer is the causative agent of polyserositis and septicaemia in waterfowl. Twenty-one serotypes have been reported, and there is a strong variation in virulence between strains according to serotype or strain. However, little information is available to assess virulence, such as virulence-associated genes; thus, it is difficult to estimate the risk from field strains. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of R. anatipestifer strains. Three virulent strains (RA T1, RA T7, and V-1) and three avirulent strains (Av-1, Av-2, and Av-3), which were confirmed by duck challenge, were used to perform the ELA. Inoculating 10 2 to 10 4 colony-forming units into the allantoic cavity of 10-day-old embryos discriminated between virulent and avirulent strains based on mortality. Differences in invasion rates into embryonic tissues were found between the RA T1 and Av-1 strains. The maximum colony-forming units of the RA T1 strain were about 1000 times higher than those of the Av-1 strain in the tissue invasion rate for 4 days. We found that the virulent strains killed embryos at mortality rates ]50% during the first 3 days after inoculation and that the avirulent strains had death rates of 520% over 5 days. These results obtained by repeated testing suggest that the ELA could be used as a first-line screening method to determine the virulence of R. anatipestifer strains.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

“…In this work, we demonstrate that embryonated eggs are highly susceptible to P. insidiosum infection via the allantoic cavity in a dose‐ and age‐dependent manner. We observed that increased zoospore concentration causes a decrease in survival time, and infection of embryos at a later embryonic day (14) showed delayed mortality, which agrees with the observations of Jacobsen et al and Seo et al when they inoculated chicken embryos with A. fumigatus and Riemerella anatipestifer respectively. Cell‐mediated immunity in chicken embryos has been demonstrated to start by 12th‐15th day of development by T‐cell colonisation of the spleen .…”

SURVEILLANCE AND CHARACTERIZATION OF RIEMERELLA ANATIPESTIFER FROM WILD BIRDS IN SOUTH KOREA

“…We also detected RA in the cloacae of 2% of wild ducks. Cloacal isolates of RA have also been reported (Seo et al 2013), and Xie et al (2010) detected RA in cloacae by real-time PCR 3 h to 5 days after subcutaneous inoculation. This suggests that RA could be shed in feces of wild birds.…”

Chicken embryo lethality assay for determining the lethal dose and virulence of Enterococcus faecalis

“…However, relatively high costs, complexity and ethical considerations limit the application of such challenge tests. Therefore, it seems appropriate to use the chicken embryo lethality assay (ELA), which can provide an alternative tool to study the virulence of different strains in a faster, more sensitive, less expensive, more specific and relatively simple form (Wooley et al, 2000;Oh et al, 2012;Seo et al, 2013).…”

“…Chicken ELA has been proposed to determine the degree of virulence of isolated fungi (Jacobsen et al, 2010), and bacteria such as Escherichia coli (Wooley et al, 2000;Montgomery et al, 2005;Oh et al, 2012), Francisella spp. (Nix et al, 2006), Yersinia enterocolítica (Townsend et al, 2008), Campylobacter jejuni (Stewart-Tull et al, 2009), Staphylococcus aureus (Polakowska et al, 2012), Riemerella anatipestifer (Seo et al, 2013) and Listeria monocytogenes (Andersson et al, 2015). In the case of Enterococcus spp., Borst et al (2014) reported that the ELA is able to differentiate between virulent and avirulent strains, whose genotype has been closely related to each other through pulsedfield gel electrophoresis.…”

“…. The age of the embryos at inoculation, since strains' virulence appears to differ based on embryonic development (Seo et al, 2013). .…”