A Chicken Embryo Lethality Assay for PathogenicEnterococcus cecorum

Abstract

Pathogenic strains of Enterococcus cecorum cause outbreaks of arthritis and osteomyelitis in chickens worldwide. Enterococcal spondylitis (ES) is a specific manifestation of E. cecorum-associated disease of broilers and broiler breeders characterized by increased flock mortality, resulting from unresolved infection of the free thoracic vertebra by pathogenic E. cecorum. A study of 22 ES outbreaks in the southeast United States revealed that pathogenic E. cecorum strains isolated from spinal lesions were genetically clonal. Here, we compare the virulence of previously genotyped pathogenic strains (n = 8) isolated from spinal lesions and nonpathogenic strains (n = 9) isolated from ceca of unaffected birds in a chicken embryo lethality model. Strains were inoculated into the allantoic cavity of 12-day-old broiler and specific-pathogen-free (SPF) layer embryos; embryo survival was determined by candling eggs daily for 4 days. Significantly decreased survival occurred in both broiler and SPF embryos inoculated with pathogenic genotype strains compared with embryos inoculated with nonpathogenic genotype strains (broiler embryos, 23% vs. 60%; SPF embryos, 9% vs. 61%). Embryos infected with pathogenic strains were unable to control infection and consistently showed gross changes typical of sepsis, including hemorrhage and edema. After 48 hr, similar changes were not observed in embryos infected with nonpathogenic strains. This embryo lethality assay provides a useful tool for understanding the genetic basis of E. cecorum virulence.

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

“…As shown in Table 2, our results showed a clear relationship between infectious dose and EMR in accordance with the results obtained by Seo et al (2013) and Borst et al (2014), who observed higher EMR with higher infectious doses than lower infectious doses. The EMR produced with the infectious dose of 2500 CFU/ml used in the present study (93%) is consistent with the values reported by Rudolph (2004), who observed an EMR between 90% and 100% with the same strain and infectious dose.…”

“…Based on the revised literature (Landman et al, 1999b;Wooley et al, 2000;Rudolph, 2004;Montgomery et al, 2005;Townsend et al, 2008;Stewart-Tull et al, 2009;Jacobsen et al, 2010;Oh et al, 2012;Polakowska et al, 2012;Seo et al, 2013;Borst et al, 2014) an ELA was carried out as a diagnostic model to establish the pathogenicity of a single E. faecalis strain.…”

“…The results on the use of SPF embryos in the ELA obtained by Borst et al (2014) with Enterococcus cecorum showed that the SPF embryos are as susceptible as normal embryos, suggesting that SPF embryos have no innate resistance to bacterial infection. Therefore, in the present study no SPF embryos were used.…”

“…This result is the first to report the appropriate infectious dose of an E. faecalis strain to inoculate these chicken embryos lines. Borst et al (2014) reported that the lowest dose of E. cecorum strains able to produce around 50% embryonic mortality was 10 3 CFU/ml, and Nix et al (2006) observed that infectious doses of F. tularensis from 30 to 200 CFU produced 100% embryonic mortality. This observation suggests the importance to determine an appropriate infectious dose according to the type of isolate and probably the strain.…”

“…(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. Rudolph (2004) reported that different E. faecalis strains produced different degrees of mortality when the embryos were infected in the allantoic cavity (AC) with a single infectious dose (2500 colony-forming units (CFU)/ml).…”