Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults

Authors: Sylte, Matthew; CHANDRA, LAWRANCE - Orise Fellow; Looft, Torey

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2016
Publication Date: 2/16/2017
Citation: Sylte, M.J., Chandra, L.C., Looft, T.P. 2017. Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults. Poultry Science. 96(7):2412-2420. doi: 10.3382/ps/pex022.

Interpretive Summary: Germ-free animals are born and reared without any contaminating microbes, and are a valuable tool to help study the host-microbe interaction. In the case of birds, their eggs are in contact with intestinal bacteria prior to being laid; therefore, the outer layers of the egg must be sterilized in order to hatch germ-free birds. The use of chemical antiseptics and disinfectants have been used to sterilize turkey and chicken eggs. Often, these chemicals are toxic to the developing embryo, which can affect the number of hatched germ-free poults or chicks. In this study, we define conditions to safely sterilize turkey eggs and hatch live, germ-free poults. A combination of disinfectants and an iodine containing antiseptic sterilized the surface of turkey eggs, did not cause damage to the developing turkey embryos, and hatched poults were free of detectable bacteria. This protocol will be useful to researchers to maximize numbers of germ-free poultry, and will be used to study methods to limit turkeys from becoming infected with foodborne pathogens.

Technical Abstract: Bird eggs and are in contact with intestinal microbiota prior to oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was detected in the cecal microbiota of hatched poults, suggesting that poults may acquire spore-formers by exposure to shell contents during hatching. Generating gnotobiotic poults for research requires sterilization of the egg’s surface without damaging the developing embryo. The ability of different disinfectants and antiseptics to sterilize the egg shell surface without harming the developing embryo was tested. Different classes of disinfectants and antiseptics (halogens, biguanidines and oxidants) were selected to target spores and vegetative bacteria likely present on the egg’s surface. Eggs were treated by fully immersing in heated antiseptic (betadine or chlorhexidine) or disinfectant (sodium hypochlorite, acidified sodium hypochlorite, chlorine dioxide, Oxysept-333 or Virkon S) solutions for up to 15 minutes. Shells aseptically harvested for aerobic and anaerobic culturing of bacteria. Toxicity to the developing embryo was assessed by gross evaluation of developmental changes in treated eggs incubated up to 27 days of embryonation. Halogen disinfectants acidified sodium hypochlorite, chlorine dioxide and oxidants Oxysept-333 and Virkon-S sterilized egg shells. However, addition of oxidants, alone or in combination with other treatments, produced significant (P less than 0.05) embryotoxicity, compared to eggs treated with phosphate buffered saline (PBS). The combination treatment of acidified sodium hypochlorite, chorine dioxide (CLD) and betadine produced minimal embryotoxicity and sterilized whole turkey eggs, and produced hatched poults in a gnotobiotic isolator. Eggs treated with PBS were incubated and hatched under germ-replete conditions. After hatching, poults were euthanized and tested for sterility of intestinal contents. Gnotobiotic poults had no detectable bacterial growth or qPCR amplification of 16S rRNA gene, demonstrating that acidified sodium hypochlorite, CLD and betadine safely hatched gnotobiotic poults. Generation of germ-free poults is an important tool and will be used to evaluate the host-pathogen interaction by food-borne pathogens such as Campylobacter spp.