Comparison of the pathogenicity of five clostridium perfringens isolates using an eimeria maxima coinfection necrotic enteritis disease model in commercial broiler chickens

Authors: LIU, LIHENG - US Department Of Agriculture (USDA); YAN, XIANGHE - US Department Of Agriculture (USDA); Lillehoj, Hyun; SUN, ZHIFENG - US Department Of Agriculture (USDA); ZHAO, HONGYAN - US Department Of Agriculture (USDA); XIANYU, ZHEZI - US Department Of Agriculture (USDA); LEE, YOUNGSUB - US Department Of Agriculture (USDA); MELVILLE, STEPHEN - Virginia Polytechnic Institution & State University; GU, CHANGQIN - US Department Of Agriculture (USDA); Li, Charles

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2021
Publication Date: 4/15/2021
Citation: Liu, L., Yan, X., Lillehoj, H.S., Sun, Z., Zhao, H., Xianyu, Z., Lee, Y., Melville, S., Gu, C., Li, C.Z. 2021. Comparison of the pathogenicity of five clostridium perfringens isolates using an eimeria maxima coinfection necrotic enteritis disease model in commercial broiler chickens. Avian Diseases. https://doi.org/10.1637/aviandiseases-D-19-00098.
DOI: https://doi.org/10.1637/aviandiseases-D-19-00098

Interpretive Summary: According to the World Organization for Animal Health (OIE), necrotic enteritis (NE) and coccidiosis are two top priority enteric diseases in poultry, and negatively impacts the poultry industry with estimated annual economic losses of $6 and 2 billion worldwide, respectively. NE is caused by a bacterium called Clostridium perfringens (CP), while coccidiosis is caused by several parasites called Eimeria species. CP can produce toxins in the bird to damage the gut and results in body weight loss and underperformance of poultry growth. Eimeria spp is ubiquitous present in the chicken farms, can also cause gut damage, and therefore is a major predisposing factor for NE disease. It is not clear what kind of CP strains can induce severe NE disease in the presence of Eimeria species. Incidence of NE disease has increased in broiler chicken during a recent decade in parallel with reduction or withdrawal of growth promoting antibiotics from poultry feed, in responses to strict government regulations to address public concerns over the emergence and spread of multi-drug resistant bacteria. For elucidation of the NE disease infection mechanism of pathogens, 5 CP strains from different sources and backgrounds were selected to determine whether they could induce the NE disease in the presence of Eimerial parasites. These five strains were either originally isolated from healthy chicken intestine (LLY_N11), soil (S13), food poisoning (SM101) or from NE outbreak farm (Del1 and LLY_Tpel17). The capabilities of these bacterial strains were also tested in forming a thin, slimy bacterial film that adheres to a plastic surface (called biofilm). In the presence of Eimerial coinfection, nearly all the CP strains tested were able to reproduce NE lesions, nor matter the source and bacterial background. There was no direct correlation between biofilm formation capability and infection capability of CP strains in chickens. One virulent CP strain called LLY_Tpel17 has a unique tpeL toxin gene which contributed to NE disease severity: reducing the growth rate and inducing severe intestine gross lesions. Availability of the virulent netB+tpel+ CP strain will facilitate the development of practical and stable Eimeria / CP dual infection NE challenge model that will benefit future investigation of the CP pathogenesis and vaccine development.

Technical Abstract: This study tested the capability of several Clostridium perfringens strains from different backgrounds on forming biofilm and influencing pathogenesis of necrotic enteritis in co-infection with Eimeria spp. The virulence and pathogenicity of CP strains are sure important in inducing NE lesion, but less elucidated in CP strains in dual infections. In this study, five strains of CP from different sources and genetic backgrounds, LLY_N11 (isolated from healthy chicken intestine), Strain 13 (from soil), SM101 (from food poisoning), Del1 (netB+ from NE-afflicted chickens) and LLY_Tpel17 (netB+tpeL+ from NE-afflicted chickens) were evaluated for their pathogenicity abilities to induce necrotic enteritis lesions when birds pre-infected with 5x103 sporulated oocysts of Eimeria maxima (EM) per chicken were administrated with 1x109 colony forming unites of CP by oral gavage in 2-wk-old broiler chickens. In the birds infected with EM followed by one time dosing of CP (EM/CP), only the group of EM/LLY_Tpel17 had a mortality of 25%, and had broad and severe lesions. No mortality was observed in EM alone or CP alone groups. In the all groups of EM/CP dual infections, lesions were all observed, no matter the source of these strains. The relative body weight gain% showed statistically significant decrease in all EM/CP dual infection groups except EM / SM101 group. Our studies confirmed that the Eimerial pre-infection destroys the intestine integrity, and provides necessary conditions for C. perfringens outgrowth. Strain LLY_Tpel17 with NetB and Tpel toxin genes is very virulent with least relative body weight gain and causes very severe intestine lesions with Eimeria maxima dual infection, and should be considered as a good challenge strain when co-infected with EM in studies of pathogenesis or alternatives to antibiotics. Key words: Clostridium perfringens, Eimeria maxima, necrotic enteritis, Biofilm, pathogenicity.