Veterinary Microbiology 169 (2014) 188–197
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Two necrotic enteritis predisposing factors, dietary fishmeal and Eimeria infection, induce large changes in the caecal microbiota of broiler chickens Shu-Biao Wu a,c,*,1, Dragana Stanley b,c,d,1, Nicholas Rodgers a,c, Robert A. Swick a,c, Robert J. Moore b,c a
School of Environmental and Rural Science, University of New England, Armidale, New South Wales 2351, Australia Australian Animal Health Laboratory, CSIRO Animal, Food and Health Sciences, Geelong, Victoria 3220, Australia Poultry Cooperative Research Centre, University of New England, Armidale, New South Wales 2351, Australia d Central Queensland University, School of Medical and Applied Sciences, Bruce Highway, Rockhampton, Queensland 4702, Australia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 July 2013 Received in revised form 8 January 2014 Accepted 9 January 2014
It is widely established that a high-protein fishmeal supplemented starter diet and Eimeria infection can predispose birds to the development of clinical necrotic enteritis symptoms following Clostridium perfringens infection. However, it has not been clearly established what changes these treatments cause to predispose birds to succumb to necrotic enteritis. We analysed caecal microbiota of 4 groups of broilers (n = 12) using deep pyrosequencing of 16S rDNA amplicons: (1) control chicks fed a control diet, (2) Eimeria infected chicks fed control diet, (3) chicks fed fishmeal supplemented diet and lastly (4) both fishmeal fed and Eimeria infected chicks. We found that the high-protein fishmeal diet had a strong effect on the intestinal microbiota similar to the previously reported effect of C. perfringens infection. We noted major changes in the prevalence of various lactobacilli while the total culturable Lactobacillus counts remained stable. The Ruminococcaceae, Lachnospiraceae, unknown Clostridiales and Lactobacillaceae families were most affected by fishmeal with increases in a number of operational taxonomic units (OTUs) that had previously been linked to Crohn’s disease and reductions in OTUs known to be butyrate producers. Eimeria induced very different changes in microbiota; Ruminococcaceae groups were reduced in number and three unknown Clostridium species were increased in abundance. Additionally, Eimeria did not significantly influence changes in pH, formic, propionic or isobutyric acid while fishmeal induced dramatic changes in all these measures. Both fishmeal feeding and Eimeria infection induced significant changes in the gut microbiota; these changes may play an important role in predisposing birds to necrotic enteritis. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Necrotic enteritis Chicken Microbiota Eimeria Fishmeal
1. Introduction Necrotic enteritis (NE) is an intestinal disease of chickens caused by Clostridium perfringens. It is estimated * Corresponding author at: School of Environmental and Rural Science, University of New England, Armidale, New South Wales 2351, Australia. Tel.: +61 267732238; fax: +61 67733922. E-mail address: [email protected] (S.-B. Wu). 1 These authors contributed equally to the work. 0378-1135/$ – see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2014.01.007
to cost the global poultry industry over $2 billion per year in control, treatment and lost production costs (Van der Sluis, 2000). The extent of the economic losses has been a catalyst for growth in the international research effort directed at understanding the pathogenesis and treatment of the disease. C. perfringens infection alone is not sufficient to experimentally produce disease; other predisposing factors need to be incorporated to reproducibly induce disease (Shojadoost et al., 2012). The use of high protein
S.-B. Wu et al. / Veterinary Microbiology 169 (2014) 188–197
diet containing fishmeal and Eimeria infection are two predisposing factors that have been widely used (Fernandes da Costa et al., 2013; Williams et al., 2003; Wu et al., 2010). The reasons these predisposing factors promote clostridial disease are not fully understood, however, some explanations have been proposed. There is evidence that Eimeria-assisted induction of necrotic enteritis may be related to damage caused to the epithelium releasing serum and other nutrients (Van Immerseel et al., 2009) or the induction of mucogenesis (Collier et al., 2008) providing a niche for C. perfringens colonisation and proliferation. Fishmeal-based diets have been reported to increase the abundance of C. perfringens (Drew et al., 2004) and this may be due to the high available nutrient content in a fishmeal diet causing proliferation of C. perfringens and hence more disease (Kocher, 2003). It has previously been reported that caecal microbiota is altered in necrotic enteritis affected birds compared to healthy birds (Stanley et al., 2012). We hypothesised that one of the primary effects of predisposing factors may be to change the gastrointestinal microbiota and thus provide a disrupted environment in which C. perfringens can proliferate and cause disease. It has previously been shown that Eimeria can cause large shifts in microbiota composition (Hume et al., 2006; Oviedo-Rondon et al., 2006; Perez et al., 2011). However, it is not yet established what the nature of those shifts are as the previous studies utilised gel-based microbiota profiling to observe differences between infected and uninfected animals without identifying any of the differential phylotypes. To our knowledge the influence of fishmeal on total microbiota composition has not yet been investigated. The caecum is the main site of C. perfringens colonisation in healthy birds. The caecum carries a diverse and dynamic microflora, sampling both microflora descending via normal peristalsis and microflora ascending via retrograde gut movement. Therefore, if the necrotic enteritis predisposing factors (fish meal and Eimeria) influence the GIT microbiota it is likely that these affects would be seen in the cecum. Hence, in this study we have phylogenetically profiled the complex bacterial populations in the caeca of treated chickens, to identify the microbiota community shifts that could contribute to C. perfringens infection advancing to clinical necrotic enteritis. In the present study pyrosequencing of 16S rRNA genes was used to provide deep analysis of the microbial populations to identify bacterial phylotypes that are most affected by the necrotic enteritis predisposing treatments. 2. Material and methods 2.1. Animal trial and sample collection Day old Cobb 500 male broiler chickens (Baiada Country Road Hatchery, Tamworth, NSW) were raised in floor pens in a temperature-controlled room. Chicks were vaccinated against Marek’s disease, infectious bronchitis, and Newcastle disease and subjected to artificial fluorescent illumination of 16 h from days 0 to 35. The shed temperature was initially maintained at 33–34 8C and gradually decreased by 3 8C per week until 22–24 8C was
189
reached by the third week. Water and feed were provided ad libitum. The research facility was thoroughly cleaned and disinfected prior to bird placement. Forty-eight pens were established, each with 28 birds, and 12 pens were used per treatment, giving a total of 336 birds per treatment and 1344 birds for the whole trial. Pens were assigned to one of the 4 groups: control, high fishmeal feed, Eimeria challenge group and a final group with both fishmeal and Eimeria. The birds were given a typical Australian starter diet (215 g/kg crude protein, 12.24 MJ/kg) until day 7; on day 8, 25% fishmeal (Skretting, Tasmania; 686 g/kg crude protein, 86 g/kg ether extract,
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Two necrotic enteritis predisposing factors, dietary fishmeal and Eimeria infection, induce large changes in the caecal microbiota of broiler chickens Shu-Biao Wu a,c,*,1, Dragana Stanley b,c,d,1, Nicholas Rodgers a,c, Robert A. Swick a,c, Robert J. Moore b,c a
School of Environmental and Rural Science, University of New England, Armidale, New South Wales 2351, Australia Australian Animal Health Laboratory, CSIRO Animal, Food and Health Sciences, Geelong, Victoria 3220, Australia Poultry Cooperative Research Centre, University of New England, Armidale, New South Wales 2351, Australia d Central Queensland University, School of Medical and Applied Sciences, Bruce Highway, Rockhampton, Queensland 4702, Australia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 July 2013 Received in revised form 8 January 2014 Accepted 9 January 2014
It is widely established that a high-protein fishmeal supplemented starter diet and Eimeria infection can predispose birds to the development of clinical necrotic enteritis symptoms following Clostridium perfringens infection. However, it has not been clearly established what changes these treatments cause to predispose birds to succumb to necrotic enteritis. We analysed caecal microbiota of 4 groups of broilers (n = 12) using deep pyrosequencing of 16S rDNA amplicons: (1) control chicks fed a control diet, (2) Eimeria infected chicks fed control diet, (3) chicks fed fishmeal supplemented diet and lastly (4) both fishmeal fed and Eimeria infected chicks. We found that the high-protein fishmeal diet had a strong effect on the intestinal microbiota similar to the previously reported effect of C. perfringens infection. We noted major changes in the prevalence of various lactobacilli while the total culturable Lactobacillus counts remained stable. The Ruminococcaceae, Lachnospiraceae, unknown Clostridiales and Lactobacillaceae families were most affected by fishmeal with increases in a number of operational taxonomic units (OTUs) that had previously been linked to Crohn’s disease and reductions in OTUs known to be butyrate producers. Eimeria induced very different changes in microbiota; Ruminococcaceae groups were reduced in number and three unknown Clostridium species were increased in abundance. Additionally, Eimeria did not significantly influence changes in pH, formic, propionic or isobutyric acid while fishmeal induced dramatic changes in all these measures. Both fishmeal feeding and Eimeria infection induced significant changes in the gut microbiota; these changes may play an important role in predisposing birds to necrotic enteritis. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Necrotic enteritis Chicken Microbiota Eimeria Fishmeal
1. Introduction Necrotic enteritis (NE) is an intestinal disease of chickens caused by Clostridium perfringens. It is estimated * Corresponding author at: School of Environmental and Rural Science, University of New England, Armidale, New South Wales 2351, Australia. Tel.: +61 267732238; fax: +61 67733922. E-mail address: [email protected] (S.-B. Wu). 1 These authors contributed equally to the work. 0378-1135/$ – see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2014.01.007
to cost the global poultry industry over $2 billion per year in control, treatment and lost production costs (Van der Sluis, 2000). The extent of the economic losses has been a catalyst for growth in the international research effort directed at understanding the pathogenesis and treatment of the disease. C. perfringens infection alone is not sufficient to experimentally produce disease; other predisposing factors need to be incorporated to reproducibly induce disease (Shojadoost et al., 2012). The use of high protein
S.-B. Wu et al. / Veterinary Microbiology 169 (2014) 188–197
diet containing fishmeal and Eimeria infection are two predisposing factors that have been widely used (Fernandes da Costa et al., 2013; Williams et al., 2003; Wu et al., 2010). The reasons these predisposing factors promote clostridial disease are not fully understood, however, some explanations have been proposed. There is evidence that Eimeria-assisted induction of necrotic enteritis may be related to damage caused to the epithelium releasing serum and other nutrients (Van Immerseel et al., 2009) or the induction of mucogenesis (Collier et al., 2008) providing a niche for C. perfringens colonisation and proliferation. Fishmeal-based diets have been reported to increase the abundance of C. perfringens (Drew et al., 2004) and this may be due to the high available nutrient content in a fishmeal diet causing proliferation of C. perfringens and hence more disease (Kocher, 2003). It has previously been reported that caecal microbiota is altered in necrotic enteritis affected birds compared to healthy birds (Stanley et al., 2012). We hypothesised that one of the primary effects of predisposing factors may be to change the gastrointestinal microbiota and thus provide a disrupted environment in which C. perfringens can proliferate and cause disease. It has previously been shown that Eimeria can cause large shifts in microbiota composition (Hume et al., 2006; Oviedo-Rondon et al., 2006; Perez et al., 2011). However, it is not yet established what the nature of those shifts are as the previous studies utilised gel-based microbiota profiling to observe differences between infected and uninfected animals without identifying any of the differential phylotypes. To our knowledge the influence of fishmeal on total microbiota composition has not yet been investigated. The caecum is the main site of C. perfringens colonisation in healthy birds. The caecum carries a diverse and dynamic microflora, sampling both microflora descending via normal peristalsis and microflora ascending via retrograde gut movement. Therefore, if the necrotic enteritis predisposing factors (fish meal and Eimeria) influence the GIT microbiota it is likely that these affects would be seen in the cecum. Hence, in this study we have phylogenetically profiled the complex bacterial populations in the caeca of treated chickens, to identify the microbiota community shifts that could contribute to C. perfringens infection advancing to clinical necrotic enteritis. In the present study pyrosequencing of 16S rRNA genes was used to provide deep analysis of the microbial populations to identify bacterial phylotypes that are most affected by the necrotic enteritis predisposing treatments. 2. Material and methods 2.1. Animal trial and sample collection Day old Cobb 500 male broiler chickens (Baiada Country Road Hatchery, Tamworth, NSW) were raised in floor pens in a temperature-controlled room. Chicks were vaccinated against Marek’s disease, infectious bronchitis, and Newcastle disease and subjected to artificial fluorescent illumination of 16 h from days 0 to 35. The shed temperature was initially maintained at 33–34 8C and gradually decreased by 3 8C per week until 22–24 8C was
189
reached by the third week. Water and feed were provided ad libitum. The research facility was thoroughly cleaned and disinfected prior to bird placement. Forty-eight pens were established, each with 28 birds, and 12 pens were used per treatment, giving a total of 336 birds per treatment and 1344 birds for the whole trial. Pens were assigned to one of the 4 groups: control, high fishmeal feed, Eimeria challenge group and a final group with both fishmeal and Eimeria. The birds were given a typical Australian starter diet (215 g/kg crude protein, 12.24 MJ/kg) until day 7; on day 8, 25% fishmeal (Skretting, Tasmania; 686 g/kg crude protein, 86 g/kg ether extract,
