Characterization of virulent netB+/tpeI+ clostridium perfringens strains from necrotic-enteritis-affected broiler chicken farms

Authors: GU, CHANGQIN - US Department Of Agriculture (USDA); Lillehoj, Hyun; SUN, ZHIFENG - US Department Of Agriculture (USDA); LEE, YOUNGSUB - US Department Of Agriculture (USDA); ZHAO, HONGYAN - US Department Of Agriculture (USDA); XIANGYU, ZHEZI - US Department Of Agriculture (USDA); YAN, XIANGHE - US Department Of Agriculture (USDA); LIN, SHUDAI - US Department Of Agriculture (USDA); LIU, LIHENG - US Department Of Agriculture (USDA); Li, Charles

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/6/2016
Publication Date: 5/9/2019
Citation: Gu, C., Lillehoj, H.S., Sun, Z., Lee, Y., Zhao, H., Xiangyu, Z., Yan, X., Lin, S., Liu, L., Li, C.Z. 2019. Characterization of virulent netB+/tpeI+ clostridium perfringens strains from necrotic-enteritis-affected broiler chicken farms. Avian Diseases. https://doi.org/10.1637/11973-092018-Reg.1.
DOI: https://doi.org/10.1637/11973-092018-Reg.1

Interpretive Summary: Necrotic enteritis (NE) is one of the top priority enteric diseases in poultry, and negatively impacts the poultry industry and food safety worldwide. NE is caused by a bacterium called Clostridium perfringens (CP), which produces gut-damaging toxins in the bird that results in body weight loss and underperformance of poultry growth, and affects animal welfare and human food safety, with estimated annual economic losses of $6 billion worldwide. During the past several decades, NE has been well controlled using in-feed antibiotics (called antibiotic growth promoters); however, in response to public concerns over the spread of multidrug-resistant bacteria, governments have adopted strict regulations that reduce or even withdraw antibiotics from the feed. NE incidence has increased in Europe in parallel with the policy changes for banning on the in-feed antibiotics as growth promoters. To effectively control NE without in-feed anitbiotics, the disease infection mechanism of the bacterial pathogen needs to be elucidated. A simplified NE disease model should be established to test the efficacies of alternatives to antibiotics. Nineteen CP strains were isolated from the tissues derived from birds that were infected with NE during 2003 and 2004. These strains were characterized microbiologically and molecularly, and several of these strains were further tested for their pathogenicity in birds. Molecular amplification revealed that all the strains tested were harboring a gene called netB toxin, a pore-forming toxin which can puncture a hole on cell membrane to cause the release of cell contents and cell death. Five out of 19 strains contained gene called tpeL toxin (LLY_Tpel 13, _Tpel 15, _Tpel 17, _Tpel 18, _Tpel 19). A protein band of 200 kDa was detected in the culture supernatant of strains Tpel 17 and Tpel 19 by Western Blotting analysis, a protein detection assay with specific antibodies. An animal experimental study using a multiple oral CP inoculations, combined with a high protein diet, showed that the Tpel 17 strain was the most virulent in inducing NE lesions, followed by the Tpel 19 as the next virulent strain in broiler chickens. Furthermore, Tpel 17-infected birds showed the slowest growth rate in terms of body weight at Day 5 post-infection. Availability of the virulent netB+tpel+ CP strains will facilitate the development of CP-alone NE disease challenge model that will provide increased insights on the CP pathogenesis and vaccine development.

Technical Abstract: Clostridium perfringens (CP) Type A and newly created Type G strains are the key etiological factors in the induction of necrotic enteritis (NE), an important enteric disease which is responsible for the annual loss of $ 6 billion in worldwide poultry industry. Several CP toxin genes were found to be critical in the NE pathogenesis in chickens, but limited information is available on CP lethal toxin tpeL gene. In this report, 19 CP strains isolated from NE-afflicted birds were characterized microbiologically and molecularly, and evaluated for their pathogenicity in broiler chickens. Toxinotyping by PCR revealed that all the strains tested were netB toxin gene-positive, but only five strains were tpeL toxin positive (LLY-Tpel 13, -Tpel 15, -Tpel 17, -Tpel 18, -Tpel 19, simplified as Tpel 13, Tpel 15, Tpel 17, Tpel 18, Tpel 19). A protein band of 200 kDa was detected in the bacterial culture supernatant of strains Tpel 17 and Tpel 19 by Western Blotting analysis. Quantitative PCR showed that strains Tpel 13, 15, 17, 18, and 19 harboring the high copies of tpeL genes, while Tpel 19 showed the highest copies of netB gene among the all CP strains tested when normalized with copy numbers of 16S rRNA gene as a house-keeping gene marker. The in vivo NE challenge test using a multiple oral CP inoculations combined with high protein diet showed that the Tpel 17 strain was the most virulent in inducing NE lesion, followed by the Tpel 19 as the next virulent strain in broiler chickens. Furthermore, Tpel 17-infected group showed the slowest growth rate in body weight at Day 5 post-infection. Availability of the virulent netB+tpel+ CP strains will facilitate the development of CP-alone NE disease challenge model that will provide increased insights on the CP pathogenesis and vaccine development. Key words: Clostridium perfringens, necrotic enteritis, netB, tpel, virulence