Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

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Abstract

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-γ RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as α toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-γ expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

Introduction

Necrotic enteritis (NE) is a small intestinal inflammatory disorder of economic significance (globally greater than 2 billion US dollars/year) that primarily affects neonatal chickens. The causative agent of NE is Clostridium perfringens, a low GC, Gram-positive, anaerobic, spore-forming bacterium that is found commonly in soil, sewage, and the gastrointestinal tract of animals and humans (McDonel, 1980, Shane et al., 1984, Lindsay, 1996). Risk factors for the onset of clinical disease include concurrent coccidial infection, the removal of antibiotic growth promoters, and the inclusion of diet components such as wheat and barley, which are high in water-soluble non-starch polysaccharides (Truscott and Al-Sheikhly, 1977, Shane et al., 1984, Riddell and Kong, 1992, Ficken and Wages, 1997, Langhout, 1998; Caplan and Jilling, 2001; Craven et al., 2001). Necrotic enteritis shares strikingly similar pathophysiological features with necrotizing enterocolitis (NEC), one of the most common and serious gastrointestinal disorders of newborn infants (Gibbs et al., 2007). NEC is responsible for the death of approximately 1000 infants per year, which is comparable to the number of children under 15 years of age who die of leukemia each year in the U.S (Gibbs et al., 2007). Preterm neonates, which are often fed parenterally, are at greatest risk for the development of NEC (Heird and Gomez, 1994, Heird and Gomez, 1996, Yeo, 2006). Although the etiology of NEC is unknown, intestinal inflammatory cascades associated with parenteral nutrition and the transition from parenteral to enteral support possibly contribute to the onset of the disorder (Heird and Gomez, 1996). In previous work with a piglet model of total parental nutrition (TPN), we observed an association between small intestinal inflammation and mucogenesis (Conour et al., 2002, Ganessunker et al., 1999). Enhanced mucus production in the TPN piglet model was associated with the selection of C. perfringens, which led to discovery of the mucolytic nature of this opportunistic pathogen (Deplancke et al., 2002).

The present study used a previously established chick model of NE (Hofacre et al., 1998) to test the hypothesis that the host inflammatory response to eliminate intestinal coccidia results in increased mucus production, and that this provides a growth advantage for C. perfringens due to its ability to utilize mucus as a substrate (Deplancke et al., 2002). In contrast, the direct suppression of coccidia with an ionophore would preclude a characteristic mucogenic response of the host, thereby eliminating conditions favorable for C. perfringens growth. Thus, we hypothesize that C. perfringens growth on mucus represents an initial and previously unrecognized stage of virulence, and that this mode of growth may explain, in part, why coccidiosis predisposes to NE.

Section snippets

Experimental animals and infection model

Cobb male chicks (Cobb-Vantress Hatchery, Cleveland, GA) were distributed over three tier battery cages in a conventional housing system. Five groups of birds (42 birds/group) were randomly allotted to five experimental treatments to test the main effect of coccidiosis (non-infected (NIF) vs. infection with field strains of Eimera vs. ionophore prophylaxis; Table 1). Birds were housed in separate batteries according to coccidia treatment status to avoid cross contamination. Birds were fed a

Infection (EAM/CP) compromised intestinal barrier function and increased intestinal lesions and bird mortality

Intestinal mucosa barrier function data are presented in Table 1 as the translocation of bacteria to the liver or spleen. At day 20, aerobic and anaerobic bacterial and C. perfringens CFUs were observed in the spleen for all birds independent of treatment. This outcome possibly reflects the immaturity of barrier function at this early age. Despite substantial bird-to-bird variation for all treatments, anaerobic bacterial and C. perfringens CFU/g were greater (p < 0.05) in EAM/CP-treated birds

Discussion

The present results support the hypothesis that coccidial infection predisposes to NE through the induction of local T cell-mediated inflammatory responses that enhance intestinal mucogenesis. This outcome was associated with alterations in the composition of the ileal microbiota and enhanced growth of the mucolytic bacterium C. perfringens, the causative agent of NE. While there is considerable evidence for intestinal goblet cell responsiveness to inflammatory mediators, the extent to which

Acknowledgements

We thank Madelyn Stumpf and Jennifer Croix for assistance with intestinal goblet cell histology and Shankar Chowdhury, John Conour, and Noriko Nakamura for review of the manuscript. This study was supported by Elanco Animal Health, Greenfield, Indiana.

References (53)

  • G.M. Morris et al.

    Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria

    Biotechnol. Adv.

    (2006)
  • A. Smirnov et al.

    Mucin dynamics in the chick small intestine are altered by starvation

    J. Nutr.

    (2004)
  • R.F. Wang et al.

    A 16S rDNA-based PCR method for rapid and specific detection of Clostridium perfringens in food

    Mol. Cell. Probes

    (1994)
  • K.L. Watkins et al.

    In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin

    Vet. Microbiol.

    (1997)
  • P.C. Allen et al.

    Recent advances in biology and immunobiology of Eimeria species and in diagnosis and control of infection with these coccidian parasites of poultry

    Clin. Microbiol. Rev.

    (2002)
  • C.D. Brown et al.

    Regulation of 15-lipoxygenase isozymes and mucin secretion by cytokines in cultured normal human bronchial epithelial cells

    Inflamm. Res.

    (2001)
  • M.S. Caplan et al.

    New concepts in necrotizing enterocolitis

    Curr. Opin. Pediatr.

    (2001)
  • L. Cohn et al.

    Cytokine regulation of mucus production in a model of allergic asthma

    Novartis Found Symp.

    (2002)
  • L. Cohn et al.

    Asthma: mechanisms of disease persistence and progression

    Annu. Rev. Immunol.

    (2004)
  • C.T. Collier et al.

    The effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis

    Antimicrob. Agents Chemother.

    (2003)
  • R. Collins

    Protozoan parasites of the intestinal tract: a review of Coccidia and Microsporida

    J. Am. Osteopath. Assoc.

    (1997)
  • J.E. Conour et al.

    Acidomucin goblet cell expansion induced by parenteral nutrition in the small intestine of piglets

    Am. J. Physiol. Gastrointest. Liver Physiol.

    (2002)
  • S.E. Craven et al.

    Incidence of Clostridium perfringens in broiler chickens and their environment during production and processing

    Avian Dis.

    (2001)
  • K. Dabbagh et al.

    IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

    J. Immunol.

    (1999)
  • M.L. Enss et al.

    Changes in colonic mucins of germfree rats in response to the introduction of a “normal” rat microbial flora: rat colonic mucin

    J. Exp. Anim. Sci.

    (1992)
  • M.D. Ficken et al.

    Necrotic enteritis

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