Avian Pathology
ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20
Necrotic enteritis predisposing factors in broiler chickens Robert J. Moore To cite this article: Robert J. Moore (2016): Necrotic enteritis predisposing factors in broiler chickens, Avian Pathology, DOI: 10.1080/03079457.2016.1150587 To link to this article: http://dx.doi.org/10.1080/03079457.2016.1150587
Accepted author version posted online: 29 Feb 2016.
Submit your article to this journal
Article views: 4
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Download by: [University of California, San Diego]
Date: 03 March 2016, At: 18:24
Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: 10.1080/03079457.2016.1150587
Cavp-2015-0258.R1
1
rip us c
Robert J. Moore1,2,3,*
School of Science, RMIT University, Bundoora West Campus, Bundoora , Victoria 3083,
M an
Australia. 2Poultry Cooperative Research Centre, University of New England, Armidale, New South Wales 2315, Australia. 3Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria
pt e
d
3800, Australia
ce
Short title: Necrotic enteritis predisposing factors
Corresponding author:
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
Necrotic enteritis predisposing factors in broiler chickens
*Robert J. Moore,
Email: [email protected]
Received 5 January 2016
Abstract
Necrotic enteritis in chickens develops as a result of infection with pathogenic strains of Clostridium perfringens and the presence of pre-disposing factors. Pre-disposing factors include elements that directly change the physical properties of the gut, either damaging the
t
epithelial surface, inducing mucus production, or changing gut transit times; factors that
rip
research into necrotic enteritis predisposing factors was directed by the simple hypothesis that
us c
low level colonization of C. perfringens commonly occurred within the gut of healthy
chickens and the predisposing factors lead to a proliferation of those bacteria to produce
M an
disease. More recently, with an increasing understanding of the major virulence factors of C. perfringens and the application of molecular techniques to define different clades of C. perfringens strains, it has become clear that the C. perfringens isolates commonly found in
d
healthy chickens are generally not strains that have the potential to cause disease. Therefore,
pt e
we need to re-evaluate hypotheses regarding the development of disease, the origin of disease causing isolates of C. perfringens, and the importance of interactions with other C.
ce
perfringens strains and with predisposing factors. Many predisposing factors that affect the physical and immunological characteristics of the gastrointestinal tract my also change the resident microbiota. Research directed towards defining the relative importance of each of
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
disrupt the gut microbiota; and factors that alter the immune status of birds. In the past
these different actions of predisposing factors will improve the understanding of disease pathogenesis and may allow refinement of experiment disease models.
Keywords: Necrotic enteritis, Clostridium perfringens, predisposing factors, virulence, pathogenicity, Eimeria, microbiota
Introduction
Necrotic enteritis (NE) has been estimated to cost the global poultry industry US$5-6 million per annum in production losses and current control measures (Wade and Keyburn, 2015). Clostridium perfringens strains expressing NetB toxin are the definitive cause of NE in
t
chickens (Keyburn et al., 2008, 2010; Smyth and Martin, 2010). However, a simple infection
rip
environment conducive to an increase in the abundance of C. perfringens. When pathogenic
us c
strains proliferate in the gastrointestinal tract (GIT) the increase in NetB toxin causes gross damage to the GIT epithelium. By understanding the steps that lead to C. perfringens
M an
colonization, proliferation and GIT damage, we will be better placed to design intervention strategies to control or avoid disease and could use the knowledge to guide further experimental investigation of the pathogenesis of disease. A more complete understanding of
d
the effects of predisposing factors and the mechanisms of action may also help to further
pt e
refine the experimental disease models used to study NE. There are many predisposing factors which have been experimentally demonstrated to
ce
increase the incidence or severity of NE. The evidence for predisposing factors is often mixed, with contradictory reports in the literature (e.g. feed particle size (Branton et al., 1987; Riddell and Kong, 1992), seasonal effects (Bains, 1968; Nairn and Bamford, 1967), feed
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
is not sufficient to precipitate disease; predisposing factors are necessary to produce an
restriction (Keyburn et al., 2008; Tsiouris et al., 2014)). This may reflect the complexity of disease development and the difficulties in controlling all possible influences when undertaking experimental analysis. Therefore caution is required when interpreting the literature and drawing conclusions. Often conclusions have been made on the basis of a single trial. In terms of identifying the most influential predisposing factors it is of value to
consider experimental disease induction models that have been repeatedly used. For example,
coccidial infection is commonly used in experimental disease models and generally makes the experimental induction of disease reasonably reliable; it is clearly a strong predisposing factor for NE. However, Eimeria infection is certainly not essential and a number of groups successfully produce disease without the use of Eimeria (Cooper and Songer, 2010; Feng et al., 2010; Keyburn et al., 2010; Smyth and Martin, 2010)
t
Many predisposing factors have more than one effect. For example, whole wheat can
rip
C. perfringens (Annett et al., 2002); Eimeria infection causes physical damage to the gut
us c
surface, induces immune changes in the bird and induces mucogenesis (Al-Sheikhly and AlSaieg, 1980; Collier et al., 2008); the addition of fish-meal in the diet can alter the gut
M an
microbiota profile (Stanley et al., 2014), provide high nutrient levels for C. perfringens growth (Drew et al., 2004), introduce gut damaging biogenic amines (Barnes et al., 2001), and act as a source of contaminating C. perfringens (Chakrabarty and Boro, 1981).
d
The physical, environmental and feed changes that can predispose birds to NE have
pt e
been documented and summarised elsewhere (Allaart et al., 2013; M’Sadeq et al., 2015; Williams, 2005). This minireview touches on those issues but emphasises the potential
ce
importance of perturbations in the GIT microbiota for development of NE and the predisposing factors that influence microbiota structure. A conceptual framework to help understand the predisposing factors that may play a role in NE disease development is
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
both change gut viscosity and provide complex carbohydrates that can cause proliferation of
presented below and outlined in Figure 1. In the figure four main areas of influence are shown; (i) physical changes to gut, (ii) changes to bird immune status, (iii) disruption of GIT microbiota, and (iv) proliferation of pathogenic C. perfringens.
Changes to the gut. Certain dietary components are predisposing factors for NE. Dietary components can alter the physical properties of the digesta, enhance the growth of C.
perfringens and more broadly modify the microbiota present in the GIT. Birds on wheat, rye, oats and barley-based diets are more likely to suffer severe NE than birds on corn-based diets (Branton et al., 1997; Riddell and Kong, 1992). Such grains have high levels of non-starch polysaccharides (NSPs) which can increase digesta viscosity and provide substrates for C. perfringens growth. In vitro analysis has shown that C. perfringens proliferates more in
t
digested wheat and barley diets compared to digested corn diets (Annett et al., 2002). NSPs
rip
sporulation of contaminating C. perfringens within the litter. Wet litter has been identified as
us c
a predisposing factor in a field survey of poultry producers (Hermans and Morgan, 2007). Coccidial infection has multiple effects on the GIT that predispose birds to NE.
M an
Physical damage to the GIT epithelium compromises gut integrity which (i) opens direct access to the intestinal basal layer, which may be an important site in the early stages of disease (Olkowski et al., 2008); (ii) may expose extracellular matrix molecules, such as
d
collagen, that have a role in C. perfringens adherence (Wade et al., 2015); (iii) causes serum
pt e
leakage into the gut which acts as a rich nutrient source for C. perfringens growth (Van Immerseel et al., 2004); and (iv) induces mucus production, providing another protein rich
2012).
ce
nutrient source to support C. perfringens proliferation (Collier et al., 2008; Forder et al.,
High levels of animal protein in the diet, particularly fish-meal, has been used as a
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
can also increase the water intake of birds resulting in wet litter, which can in turn induce
predisposing factor in experimental disease models (e.g. Cooper and Songer, 2010; Keyburn et al., 2006; Wu et al., 2010). The high protein levels in the GIT can induce C. perfringens growth and change microbiota composition; effects modulated by the supply of nutrients and possibly by the increase in the pH throughout the GIT.
Changes to immune status. Changes in the immune status of birds can increase the incidents of NE. The peak risk period for broiler birds to develop NE is at about 3 weeks of age. This is the time when maternal antibodies are disappearing from circulation and this waning of maternal antibodies may cause some susceptibility to infection or proliferation of C. perfringens. Maternal antibodies can protect chicks from disease (Keyburn et al., 2013;
rip
Infection with viruses such as Marek’s disease virus, infectious bursal disease virus
us c
and chicken anaemia virus can have immunosuppressive effects (Hoerr, 2010) which can increase the severity of NE and some studies have used these viruses as predisposing factors
M an
for NE disease modelling (McReynolds et al., 2004; Stringfellow et al., 2009). Other stresses on birds such as overcrowding, environmental ammonia and physiological stress also have the potential to cause immunosuppression (Hoerr, 2010) and hence may exacerbate NE and
d
indeed high stock density has been shown to be a predisposing factor (Tsiouris et al., 2015).
pt e
The genetics of birds appears to have some influence on susceptibility to NE as different lines of birds have different degrees of susceptibility (Jang et al., 2013) to NE and
ce
this may result from subtle difference in immune responses to C. perfringens (Hong et al., 2014; Kim et al., 2014).
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
(Heier et al., 2001), although this is yet to be clearly demonstrated.
t
Lovland et al., 2004) so their disappearance as the young birds develop could be an issue
Disruption of microbiota. Until recently the generally accepted model for NE development hypothesised that there were underlying low level populations of C. perfringens within the GIT of chickens that, under certain predisposing circumstances, proliferated to produce the disease (Cooper and Songer, 2009). With recent advances in the understanding of the main virulence factors of C. perfringens it has become obvious that most strains circulating at low levels in healthy birds are non-pathogenic (Lacey et al 2016; Timbermont et al., 2009). It is
therefore necessary to modify our hypothesis regarding the dynamics of the temporal and structural changes occurring within the intestinal populations of C. perfringens when disease develops. It now appears that a necessary step is the introgression of pathogenic isolates and their proliferation, at the expense of non-pathogenic strains, to dominate the C. perfringens population in diseased birds. It is likely that the microbial environment into which pathogenic
t
strains of C. perfringens infiltrate and proliferate to cause disease must play an important role
rip
It has been shown that within a C. perfringens challenged group of birds the birds that
us c
developed NE had differences in gut microbiota compared to birds that remained healthy (Feng et al., 2010; Stanley et al., 2012). The causal relationships between microbiota
M an
structure and NE development are yet to be elucidated; it has not been demonstrated whether the differences seen in the microbiota of diseased birds (Antonissen et al. 2016) results from the disease process or whether some underlying types of microbiota make birds more
d
susceptible to disease and hence it is within the birds with “susceptible microbiota” that
pt e
disease is more likely to develop.
Although a causal link between microbiota changes and disease progression is yet to
ce
be definitively established it is certainly true that many of the predisposing factors that physically alter the GIT and the immune status of the bird also affect the GIT microbiota. Many predisposing factors have been recognised to increase proliferation of C. perfringens
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
in NE disease susceptibility.
but parallel proliferation or inhibitory effects on other members of the GIT microbiota may also be of importance in predisposing birds to NE. Eimeria infection disrupts the microbiota
of the GIT (Stanley et al., 2014; Wu et al., 2014) and this effect, along with the physical changes to the GIT induced by coccidiosis, may play an important role in NE predisposition. It is unsurprising that the release of high protein nutritional sources following Eimeria infection, in the form of serum leakage from compromised GIT surfaces and increased
mucogenesis, will selectively favour certain members of the GIT microbiota as well as C. perfringens. Changes to feed, for example high protein diets that include fishmeal, also alter the composition of GIT microbiota (Stanley et al., 2014). Manipulation of feeding schedules, including sudden major changes to feed composition and withdrawal of feed for 8 to 12 hours prior to challenge are key parts of some experimental disease induction models (Feng et al.,
t
2010; Keyburn et al., 2006) and are also likely to significantly disrupt the GIT microbiota.
rip
al., 2015). One of the changes identified following mycotoxin treatment was a reduction in
us c
the segmented filamentous bacteria (SFB) and this may have significance for immune
development (Snel et al., 1995; Talham et al., 1999). A reduced representation of the SFB in
M an
NE diseased birds was also noted by Stanley et al. (Stanley et al., 2012).
There are a few bacteria, besides C. perfringens, that have been shown to affect the incidence of NE. Many studies have demonstrated that potentially probiotic strains of
d
bacteria and competitive exclusion cultures reduce the incidence of NE (e.g. Elwinger et al.,
pt e
1992; La Ragione et al., 2004; Layton et al., 2013; Tactacan et al., 2013). These microbial interactions are protective rather than predisposing but there are examples of bacterial
ce
infections that have been found to predispose to greater NE development; for example, early infection with a Salmonella enterica sv. Typhimurium isolate increased NE-related mortality and lesion scores, perhaps resulting from a change in host immune response (Shivaramaiah et
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Other predisposing factors such as mycotoxins also change GIT microbiota (Antonissen et
al., 2011). It is clear that bacteria in the GIT can have profound effects on C. perfringens induced disease development, either by directly affecting the way that C. perfringens can colonise and proliferate or by modulation of the chicken immune system. Further assessment of the changes to the GIT microbiota induced by predisposing factors should be carried out to determine if the extent, rate and nature of microbiota change is predictive of disease susceptibility. Is a disrupted and changing microbiota the key
predisposing element of the microbiota contribution to NE susceptibility or is the actual composition of the microbiota into which pathogenic strains of C. perfringens infiltrate more important? For use as a predisposing factor in disease model development, actively changing/disrupting the GIT microbiota is relatively easy to achieve by manipulation of the feed but producing a specific predetermined microbiota “type” in a trial setting is not
rip
Proliferation of pathogenic C. perfringens strains. The application of molecular genome
us c
typing techniques has shown that the GIT of NE diseased birds is usually dominated by a single C. perfringens type, unlike the case in healthy birds were there are generally a number
M an
of different isolates present (Lacey et al. 2016; Barbara et al., 2008; Nauerby et al., 2003). It therefore appears that an important element of the disease process is the selective proliferation of pathogenic C. perfringens strains.
d
A number of characteristics that may favour pathogenic strain proliferation have been
pt e
identified. Antimicrobial proteins such as perfrin may play a role. Timbermont et al. (Timbermont et al., 2014) found that the perfrin gene was only present in pathogenic strains
ce
that also carried the netB gene. The perfrin expressing strains inhibited the in vitro growth of commensal C. perfringens isolates that did not carry the perfrin gene. This could be an important mechanism driving the selective growth of pathogenic strains at the expense of the
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
currently possible.
commensal strains. The selective adhesive qualities of C. perfringens isolates may also be an important
factor that tips the balance in favour of pathogenic strains. Wade et al. (Wade et al., 2015) found that pathogenic strains adhered to extracellular matrix proteins much more strongly than commensal strains; this may lead to higher retention of pathogenic strains and hence their dominance of the C. perfringens population in disease birds. The plasmid that encodes
the NetB toxin also harbours a number of other genes that encode proteins that may potentially enhance the maintenance and proliferation of pathogenic strains of C. perfringens during disease progression. For example two glycoside hydrolases may be involved in mucus colonisation and degradation (Prescott et al., 2016). However, it is not yet clear whether strains expressing the hydrolases would be selectively advantaged or whether all C.
t
perfringens strains that are co-localised with the expressing strain would benefit from the
rip
colonisation and proliferation.
us c
The origin of pathogenic C. perfringens strains that go on to dominate the clostridial population in diseased birds is not clear. Non-pathogenic strains of C. perfringens are much
M an
more commonly isolated from healthy birds than are pathogenic strains (i.e. netB-positive strains). It is unclear whether pathogenic strains are present in healthy birds, but generally at much lower levels than non-pathogenic strains, or whether in many disease outbreaks the C.
d
perfringens that comes to dominate is newly introduced. Therefore, the importance of
pt e
contamination sources is not yet understood. The first published studies that investigated sources of contamination (Craven et al., 2001a, 2001b) did not differentiate between
ce
pathogenic and non-pathogenic strains as they were carried out before the distinction was clear but recent work by Engström et al. (2012) found a high prevalence of netB-positive strains in an empty broiler house, indicating that pathogenic strains may be prevalent in some
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
potential nutrient release. More work is required to understand the role of these proteins in
production environments. The finding that the gene encoding the main virulence factor, NetB, is carried on a conjugative plasmid (Bannam et al., 2011; Parreira et al., 2012) opens up the possibility of exchange of the plasmid between different C. perfringens strains. It is
possible that transfer of the NetB plasmid to a non-pathogenic strain could convert it to a pathogenic strain.
None of the elements that potentially enhance proliferation of pathogenic strains in preference to commensal strains can currently be directly manipulated by the commonly recognised and used predisposing factors. However, the growing understanding of the differences between pathogenic and non-pathogenic C. perfringens isolates highlights the need to carefully select appropriate strains to use in combination with predisposing factors in
rip us c
Conclusions
M an
Predisposing factors influence the incidence and severity of NE induced by C. perfringens. An understanding of what factors are predisposing has been important in both the production setting, where avoidance of those factors has helped poultry producers reduce the impact of
d
disease, and in research. Knowledge of predisposing factors has informed the development of
pt e
robust experimental infection models used to develop and test intervention strategies (e.g. vaccines, feed additives), has contributed to the study of NE pathogenesis and allowed the in
ce
vivo characterization of the pathogenic potential of C. perfringens wild-type strains and mutants. Many predisposing factors are likely to have multiple effects which contribute to the increase in NE. Direct physical effects on the GIT and digesta and host immune perturbation
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
experimental disease models.
have been previously identified as key properties of many predisposing factors but there is mounting evidence that effects on the GIT microbiota may also be important. For researchers, dissection of the direct and indirect effects of predisposing factors on the microbiota and how changes to the microbiota may influence the colonization and proliferation of pathogenic C.
perfringens may be a productive area of study.
Acknowledgments
Robert Moore gratefully acknowledges the ongoing support of the Poultry Cooperative Research Centre, established and supported under the Australian Government’s Cooperative
rip us c
References
M an
Allaart, J.G., van Asten, A.J.A.M., & Gröne, A. (2013). Predisposing factors and prevention of Clostridium perfringens-associated enteritis. Comparative Immunology, Microbiology and Infectious Diseases 36, 449–464.
d
Al-Sheikhly, F., & Al-Saieg, A. (1980). Role of Coccidia in the Occurrence of Necrotic
pt e
Enteritis of Chickens. Avian Diseases, 24, 324–333. Annett, C.B., Viste, J.R., Chirino-Trejo, M., Classen, H.L., Middleton, D.M., and Simko, E.
ce
(2002). Necrotic enteritis: effect of barley, wheat and corn diets on proliferation of Clostridium perfringens type A. Avian Patholology, 31, 598–601.
Antonissen, G., Croubels, S., Pasmans, F., Ducatelle, R., Eeckhaut, V., Devreese, M.,
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
Research Centre Program.
Verlinden, M., Haesebrouck, F., Eeckhout, M., Saeger, S.D., Antlinger, B., Novak, B., Martel, A., & Van Immerseel, F. (2015). Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Veterinary Research, 46, 98.
Antonissen, G., Eeckaut, V., Van Driessche, K., Onrust, L., Haesebrouck, F., Ducatelle, R., Moore, R.J., & Van Immerseel, F. (2016) Microbial shifts that predispose to necrotic enteritis. Avian Pathology, 45, 3. Bains, B.S. (1968). Necrotic enteritis of chickens. Australian Veterinary Journal, 44, 40. Bannam, T.L., Yan, X.-X., Harrison, P.F., Seemann, T., Keyburn, A.L., Stubenrauch, C.,
t
Weeramantri, L.H., Cheung, J.K., McClane, B.A., Boyce, J.D., Moore, R.J., & Rood,
rip
Plasmids. mBio, 2, e00190-11.
us c
Closely Related Independently Conjugative Toxin and Antibiotic Resistance
Barbara, A.J., Trinh, H.T., Glock, R.D., & Songer, J.G. (2008). Necrotic enteritis-producing
M an
strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks. Veterinary Microbiology, 126, 377–382.
Branton, S.L., Reece, F.N., & Hagler, W.M. (1987). Influence of a Wheat Diet on Mortality
d
of Broiler Chickens Associated with Necrotic Enteritis. Poultry Science, 66, 1326–
pt e
1330.
Branton, S.L., Lott, B.D., Deaton, J.W., Maslin, W.R., Austin, F.W., Pote, L.M., Keirs, R.W.,
ce
Latour, M.A., & Day, E.J. (1997). The effect of added complex carbohydrates or added dietary fiber on necrotic enteritis lesions in broiler chickens. Poultry Science,
76, 24–28.
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
J.I. (2011). Necrotic Enteritis-Derived Clostridium perfringens Strain with Three
Chakrabarty, A.K., & Boro, B.R. (1981). Prevalence of food-poisoning (enterotoxigenic) Clostridium perfringens type A in blood and fish meal. Zentralblatt Für Bakteriologie, Mikrobiologie und Hygiene. 1. Abt Originale B Hygiene, 172, 427– 433. Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I., & Gaskins, H.R. (2008). Coccidia-induced mucogenesis promotes the onset of necrotic
enteritis by supporting Clostridium perfringens growth. Veterinary Immunology and Immunopathology, 122, 104–115. Cooper, K.K., & Songer, J.G. (2009). Necrotic enteritis in chickens: A paradigm of enteric infection by Clostridium perfringens type A. Anaerobe, 15, 55–60. Cooper, K.K., & Songer, J.G. (2010). Virulence of Clostridium perfringens in an
t
experimental model of poultry necrotic enteritis. Veterinary Microbiology, 142, 323–
rip
Craven, S.E., Stern, N.J., Bailey, J.S., & Cox, N.A. (2001a). Incidence of Clostridium
processing. Avian Diseases, 45, 887–896.
us c
perfringens in broiler chickens and their environment during production and
M an
Craven, S.E., Cox, N.A., Stern, N.J., & Mauldin, J.M. (2001b). Prevalence of Clostridium perfringens in Commercial Broiler Hatcheries. Avian Diseases, 45, 1050–1053. Drew, M.D., Syed, N.A., Goldade, B.G., Laarveld, B., & Kessel, A.G.V. (2004). Effects of
d
dietary protein source and level on intestinal populations of Clostridium perfringens
pt e
in broiler chickens. Poultry Science, 83, 414–420. Elwinger, K., Schneitz, C., Berndtson, E., Fossum, O., Teglöf, B., & Engstöm, B. (1992).
ce
Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Veterinaria Scandinavica, 33, 369–378.
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
328.
Engström, B.E., Johansson, A., Aspan, A., & Kaldhusdal, M. (2012) Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis. Veterinary Microbiology, 159, 260264.
Feng, Y., Gong, J., Yu, H., Jin, Y., Zhu, J., & Han, Y. (2010). Identification of changes in the composition of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens. Veterinary Microbiology, 140, 116–121. Forder, R.E.A., Nattrass, G.S., Geier, M.S., Hughes, R.J., & Hynd, P.I. (2012). Quantitative analyses of genes associated with mucin synthesis of broiler chickens with induced
t
necrotic enteritis. Poultry Science, 91, 1335–1341.
rip
naturally occurring specific antibodies against Clostridium perfringens alpha toxin in
us c
Norwegian broiler flocks. Avian Diseases, 45, 724–732.
Hermans, P.G., & Morgan, K.L. (2007). Prevalence and associated risk factors of necrotic
Pathology, 36, 43–51.
M an
enteritis on broiler farms in the United Kingdom; a cross-sectional survey. Avian
Hoerr, F.J. (2010). Clinical aspects of immunosuppression in poultry. Avian Diseases, 54, 2–
d
15.
pt e
Hong, Y.H., Dinh, H., Lillehoj, H.S., Song, K.-D., & Oh, J.-D. (2014). Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic
ce
enteritis disease. Poultry Science, 93, 1383–1395. Jang, S.I., Lillehoj, H.S., Lee, S.-H., Lee, K.W., Lillehoj, E.P., Hong, Y.H., An, D.-J., Jeoung, H.-Y., & Chun, J.-E. (2013). Relative disease susceptibility and clostridial
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Heier, B.T., Lovland, A., Soleim, K.B., Kaldhusdal, M., & Jarp, J. (2001). A field study of
toxin antibody responses in three commercial broiler lines coinfected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis. Avian Diseases, 57, 684–687.
Keyburn, A.L., Sheedy, S.A., Ford, M.E., Williamson, M.M., Awad, M.M., Rood, J.I., & Moore, R.J. (2006). Alpha-toxin of Clostridium perfringens is not an essential
virulence factor in necrotic enteritis in chickens. Infection and Immunity, 74, 6496– 6500. Keyburn, A.L., Boyce, J.D., Vaz, P., Bannam, T.L., Ford, M.E., Parker, D., Di Rubbo, A., Rood, J.I., & Moore, R.J. (2008). NetB, a new toxin that is associated with avian ecrotic enteritis caused by Clostridium perfringens. PLoS Pathogens, 4, e26.
t
Keyburn, A.L., Yan, X.-X., Bannam, T.L., Van Immerseel, F., Rood, J.I., & Moore, R.J.
rip
strains expressing NetB toxin. Veterinary Research, 41, 21
us c
Keyburn, A.L., Portela, R.W., Ford, M.E., Bannam, T.L., Yan, X.X., Rood, J.I., & Moore, R.J. (2013). Maternal immunization with vaccines containing recombinant NetB toxin
M an
partially protects progeny chickens from necrotic enteritis. Veterinary Research, 44, 108.
Kim, D.K., Lillehoj, H.S., Jang, S.I., Lee, S.H., Hong, Y.H., & Cheng, H.H. (2014).
d
Transcriptional profiles of host-pathogen responses to necrotic enteritis and
pt e
differential regulation of immune genes in two inbreed chicken lines showing disparate disease susceptibility. PLoS ONE, 9, e114960.
ce
Lacey, J.A., Johanesen, P.A., Lyras, D., & Moore, R.J. (2016) Genomic diversity of necrotic enteritis associated strains of Clostridium perfringens. Avian Pathology, this issue.
La Ragione, R.M., Narbad, A., Gasson, M.J., & Woodward, M.J. (2004). In vivo
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
(2010). Association between avian necrotic enteritis and Clostridium perfringens
characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry. Letter in Applied Microbiology, 38, 197–205.
Layton, S.L., Hernandez-Velasco, X., Chaitanya, S., Xavier, J., Menconi, A., Latorre, J.D., Kallapura, G., Kuttappan, V.A., Wolfenden, R.E., Filho, R.L.A., Hargis, B.M., &
Téllez, G. (2013). The effect of a Lactobacillus-based probiotic for the control of necrotic enteritis in broilers. Food and Nutrition Sciences, 04, 1-7. Lee, K.W., Lillehoj, H.S., Jeong, W., Jeoung, H.Y., & An, D.J. (2011). Avian necrotic enteritis: Experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poultry Science, 90, 1381–1390.
t
Lovland, A., Kaldhusdal, M., Redhead, K., Skjerve, E., & Lillehaug, A. (2004). Maternal
rip
90.
us c
McReynolds, J.L., Byrd, J.A., Anderson, R.C., Moore, R.W., Edrington, T.S., Genovese, K.J., Poole, T.L., Kubena, L.F., & Nisbet, D.J. (2004). Evaluation of
M an
immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poultry Science, 83, 1948–1952.
M’Sadeq, S.A., Wu, S., Swick, R.A., & Choct, M. (2015). Towards the control of necrotic
pt e
Nutrition, 1, 1–11.
d
enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. Animal
Nairn, M.E., & Bamford, V.W. (1967). Necrotic enteritis of broiler chickens in western
ce
Australia. Australian Veterinary Journal, 43, 49–54. Nauerby, B., Pedersen, K., & Madsen, M. (2003). Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
vaccination against subclinical necrotic enteritis in broilers. Avian Pathology, 33, 81–
chickens. Veterinary Microbiology, 94, 257–266.
Olkowski, A.A., Wojnarowicz, C., Chirino-Trejo, M., Laarveld, B., & Sawicki, G. (2008). Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue. Research in Veterinary Science, 85, 543–553.
Parreira, V.R., Costa, M., Eikmeyer, F., Blom, J., & Prescott, J.F. (2012). Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative plasmids. PLoS ONE, 7, e49753. Prescott, J.F., Valeria, R.P., Gohari, I.M., Lepp, D., & Gong, J. (2016) The pathogenesis of necrotic enteritis in chickens: What we know and what we need to know. Avian
t
Pathology, this issue.
rip
chickens. Avian Diseases, 36, 499–503.
us c
Shivaramaiah, S., Wolfenden, R.E., Barta, J.R., Morgan, M.J., Wolfenden, A.D., Hargis, B.M., & Téllez, G. (2011). The role of an early Salmonella Typhimurium infection as
Diseases, 55, 319–323.
M an
a predisposing factor for necrotic enteritis in a laboratory challenge model. Avian
Smyth, J.A., & Martin, T.G. (2010). Disease producing capability of netB positive isolates of
d
C. perfringens recovered from normal chickens and a cow, and netB positive and
76–84.
pt e
negative isolates from chickens with necrotic enteritis. Veterinary Microbiology, 146,
ce
Snel, J., Heinen, P.P., Blok, H.J., Carman, R.J., Duncan, A.J., Allen, P.C., & Collins, M.D. (1995). Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of “Candidatus Arthromitus.”
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Riddell, C., & Kong, X.M. (1992). The influence of diet on necrotic enteritis in broiler
International Journal of Systematic Bacteriology, 45, 780–782.
Stanley, D., Keyburn, A.L., Denman, S.E., & Moore, R.J. (2012). Changes in the caecal microflora of chickens following Clostridium perfringens challenge to induce necrotic enteritis. Veterinary Microbiology, 159, 155–162.
Stanley, D., Wu, S.-B., Rodgers, N., Swick, R.A., & Moore, R.J. (2014). Differential responses of cecal microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis challenge model in chickens. PLoS ONE, 9, e104739. Stringfellow, K., McReynolds, J., Lee, J., Byrd, J., Nisbet, D., & Farnell, M. (2009). Effect of bismuth citrate, lactose, and organic acid on necrotic enteritis in broilers. Poultry
t
Science, 88, 2280–2284.
rip
(QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens.
us c
Journal of Applied Poultry Research, 22, 825–831.
Talham, G.L., Jiang, H.-Q., Bos, N.A., & Cebra, J.J. (1999). Segmented filamentous bacteria
M an
are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. Infection and Immunity, 67, 1992–2000. Timbermont, L., Lanckriet, A., Gholamiandehkordi, A.R., Pasmans, F., Martel, A.,
d
Haesebrouck, F., Ducatelle, R., & Van Immerseel, F. (2009). Origin of Clostridium
pt e
perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comparative Immunology, Microbiology and Infectious Diseases, 32, 503–512.
ce
Timbermont, L., De Smet, L., Van Nieuwerburgh, F., Parreira, V.R., Van Driessche, G., Haesebrouck, F., Ducatelle, R., Prescott, J., Deforce, D., Devreese, B., & Van Immerseel, F. (2014). Perfrin, a novel bacteriocin associated with netB positive
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Tactacan, G.B., Schmidt, J.K., Miille, M.J., & Jimenez, D.R. (2013). A Bacillus subtilis
Clostridium perfringens strains from broilers with necrotic enteritis. Veterinary Research, 45, 40.
Tsiouris, V., Georgopoulou, I., Batzios, C., Pappaioannou, N., Ducatelle, R., & Fortomaris, P. (2014). Temporary feed restriction partially protects broilers from necrotic enteritis. Avian Pathology, 43, 139–145.
Tsiouris, V., Georgopoulou, I., Batzios, C., Pappaioannou, N., Ducatelle, R., & Fortomaris, P. (2015). High stocking density as a predisposing factor for necrotic enteritis in broiler chicks. Avian Pathology, 44, 59–66. Van Immerseel, F., Buck, J.D., Pasmans, F., Huyghebaert, G., Haesebrouck, F., & Ducatelle, R. (2004). Clostridium perfringens in poultry: an emerging threat for animal and
t
public health. Avian Pathology, 33, 537–549.
rip
17.
us c
Wade, B., Keyburn, A.L., Seemann, T., Rood, J.I., & Moore, R.J. (2015). Binding of
Clostridium perfringens to collagen correlates with the ability to cause necrotic
M an
enteritis in chickens. Veterinary Microbiology, 180, 299–303.
Williams, R.B. (2005). Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34,
d
159–180.
pt e
Wu, S.-B., Rodgers, N., & Choct, M. (2010). Optimized Necrotic Enteritis Model Producing Clinical and Subclinical Infection of Clostridium perfringens in Broiler Chickens.
ce
Avian Diseases, 54, 1058–1065.
Wu, S.-B., Stanley, D., Rodgers, N., Swick, R.A., & Moore, R.J. (2014). Two necrotic enteritis predisposing factors, dietary fishmeal and Eimeria infection, induce large
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Wade, B., & Keyburn, A. (2015). The true cost of necrotic enteritis. World Poultry, 31, 16–
changes in the caecal microbiota of broiler chickens. Veterinary Microbiology, 169, 188–197.
Figure Legend Figure 1. Summary of predisposing factors for necrotic enteritis development in chickens. Predisposing factors are shown in circles and the major effects of these factors are shown in the ovals. Important factors that may drive the influence of the predisposing factors are
rip us c M an d pt e ce Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
shown in the small rectangular boxes.
ce
Ac d
pt e M an us c
rip
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20
Necrotic enteritis predisposing factors in broiler chickens Robert J. Moore To cite this article: Robert J. Moore (2016): Necrotic enteritis predisposing factors in broiler chickens, Avian Pathology, DOI: 10.1080/03079457.2016.1150587 To link to this article: http://dx.doi.org/10.1080/03079457.2016.1150587
Accepted author version posted online: 29 Feb 2016.
Submit your article to this journal
Article views: 4
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Download by: [University of California, San Diego]
Date: 03 March 2016, At: 18:24
Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: 10.1080/03079457.2016.1150587
Cavp-2015-0258.R1
1
rip us c
Robert J. Moore1,2,3,*
School of Science, RMIT University, Bundoora West Campus, Bundoora , Victoria 3083,
M an
Australia. 2Poultry Cooperative Research Centre, University of New England, Armidale, New South Wales 2315, Australia. 3Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria
pt e
d
3800, Australia
ce
Short title: Necrotic enteritis predisposing factors
Corresponding author:
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
Necrotic enteritis predisposing factors in broiler chickens
*Robert J. Moore,
Email: [email protected]
Received 5 January 2016
Abstract
Necrotic enteritis in chickens develops as a result of infection with pathogenic strains of Clostridium perfringens and the presence of pre-disposing factors. Pre-disposing factors include elements that directly change the physical properties of the gut, either damaging the
t
epithelial surface, inducing mucus production, or changing gut transit times; factors that
rip
research into necrotic enteritis predisposing factors was directed by the simple hypothesis that
us c
low level colonization of C. perfringens commonly occurred within the gut of healthy
chickens and the predisposing factors lead to a proliferation of those bacteria to produce
M an
disease. More recently, with an increasing understanding of the major virulence factors of C. perfringens and the application of molecular techniques to define different clades of C. perfringens strains, it has become clear that the C. perfringens isolates commonly found in
d
healthy chickens are generally not strains that have the potential to cause disease. Therefore,
pt e
we need to re-evaluate hypotheses regarding the development of disease, the origin of disease causing isolates of C. perfringens, and the importance of interactions with other C.
ce
perfringens strains and with predisposing factors. Many predisposing factors that affect the physical and immunological characteristics of the gastrointestinal tract my also change the resident microbiota. Research directed towards defining the relative importance of each of
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
disrupt the gut microbiota; and factors that alter the immune status of birds. In the past
these different actions of predisposing factors will improve the understanding of disease pathogenesis and may allow refinement of experiment disease models.
Keywords: Necrotic enteritis, Clostridium perfringens, predisposing factors, virulence, pathogenicity, Eimeria, microbiota
Introduction
Necrotic enteritis (NE) has been estimated to cost the global poultry industry US$5-6 million per annum in production losses and current control measures (Wade and Keyburn, 2015). Clostridium perfringens strains expressing NetB toxin are the definitive cause of NE in
t
chickens (Keyburn et al., 2008, 2010; Smyth and Martin, 2010). However, a simple infection
rip
environment conducive to an increase in the abundance of C. perfringens. When pathogenic
us c
strains proliferate in the gastrointestinal tract (GIT) the increase in NetB toxin causes gross damage to the GIT epithelium. By understanding the steps that lead to C. perfringens
M an
colonization, proliferation and GIT damage, we will be better placed to design intervention strategies to control or avoid disease and could use the knowledge to guide further experimental investigation of the pathogenesis of disease. A more complete understanding of
d
the effects of predisposing factors and the mechanisms of action may also help to further
pt e
refine the experimental disease models used to study NE. There are many predisposing factors which have been experimentally demonstrated to
ce
increase the incidence or severity of NE. The evidence for predisposing factors is often mixed, with contradictory reports in the literature (e.g. feed particle size (Branton et al., 1987; Riddell and Kong, 1992), seasonal effects (Bains, 1968; Nairn and Bamford, 1967), feed
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
is not sufficient to precipitate disease; predisposing factors are necessary to produce an
restriction (Keyburn et al., 2008; Tsiouris et al., 2014)). This may reflect the complexity of disease development and the difficulties in controlling all possible influences when undertaking experimental analysis. Therefore caution is required when interpreting the literature and drawing conclusions. Often conclusions have been made on the basis of a single trial. In terms of identifying the most influential predisposing factors it is of value to
consider experimental disease induction models that have been repeatedly used. For example,
coccidial infection is commonly used in experimental disease models and generally makes the experimental induction of disease reasonably reliable; it is clearly a strong predisposing factor for NE. However, Eimeria infection is certainly not essential and a number of groups successfully produce disease without the use of Eimeria (Cooper and Songer, 2010; Feng et al., 2010; Keyburn et al., 2010; Smyth and Martin, 2010)
t
Many predisposing factors have more than one effect. For example, whole wheat can
rip
C. perfringens (Annett et al., 2002); Eimeria infection causes physical damage to the gut
us c
surface, induces immune changes in the bird and induces mucogenesis (Al-Sheikhly and AlSaieg, 1980; Collier et al., 2008); the addition of fish-meal in the diet can alter the gut
M an
microbiota profile (Stanley et al., 2014), provide high nutrient levels for C. perfringens growth (Drew et al., 2004), introduce gut damaging biogenic amines (Barnes et al., 2001), and act as a source of contaminating C. perfringens (Chakrabarty and Boro, 1981).
d
The physical, environmental and feed changes that can predispose birds to NE have
pt e
been documented and summarised elsewhere (Allaart et al., 2013; M’Sadeq et al., 2015; Williams, 2005). This minireview touches on those issues but emphasises the potential
ce
importance of perturbations in the GIT microbiota for development of NE and the predisposing factors that influence microbiota structure. A conceptual framework to help understand the predisposing factors that may play a role in NE disease development is
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
both change gut viscosity and provide complex carbohydrates that can cause proliferation of
presented below and outlined in Figure 1. In the figure four main areas of influence are shown; (i) physical changes to gut, (ii) changes to bird immune status, (iii) disruption of GIT microbiota, and (iv) proliferation of pathogenic C. perfringens.
Changes to the gut. Certain dietary components are predisposing factors for NE. Dietary components can alter the physical properties of the digesta, enhance the growth of C.
perfringens and more broadly modify the microbiota present in the GIT. Birds on wheat, rye, oats and barley-based diets are more likely to suffer severe NE than birds on corn-based diets (Branton et al., 1997; Riddell and Kong, 1992). Such grains have high levels of non-starch polysaccharides (NSPs) which can increase digesta viscosity and provide substrates for C. perfringens growth. In vitro analysis has shown that C. perfringens proliferates more in
t
digested wheat and barley diets compared to digested corn diets (Annett et al., 2002). NSPs
rip
sporulation of contaminating C. perfringens within the litter. Wet litter has been identified as
us c
a predisposing factor in a field survey of poultry producers (Hermans and Morgan, 2007). Coccidial infection has multiple effects on the GIT that predispose birds to NE.
M an
Physical damage to the GIT epithelium compromises gut integrity which (i) opens direct access to the intestinal basal layer, which may be an important site in the early stages of disease (Olkowski et al., 2008); (ii) may expose extracellular matrix molecules, such as
d
collagen, that have a role in C. perfringens adherence (Wade et al., 2015); (iii) causes serum
pt e
leakage into the gut which acts as a rich nutrient source for C. perfringens growth (Van Immerseel et al., 2004); and (iv) induces mucus production, providing another protein rich
2012).
ce
nutrient source to support C. perfringens proliferation (Collier et al., 2008; Forder et al.,
High levels of animal protein in the diet, particularly fish-meal, has been used as a
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
can also increase the water intake of birds resulting in wet litter, which can in turn induce
predisposing factor in experimental disease models (e.g. Cooper and Songer, 2010; Keyburn et al., 2006; Wu et al., 2010). The high protein levels in the GIT can induce C. perfringens growth and change microbiota composition; effects modulated by the supply of nutrients and possibly by the increase in the pH throughout the GIT.
Changes to immune status. Changes in the immune status of birds can increase the incidents of NE. The peak risk period for broiler birds to develop NE is at about 3 weeks of age. This is the time when maternal antibodies are disappearing from circulation and this waning of maternal antibodies may cause some susceptibility to infection or proliferation of C. perfringens. Maternal antibodies can protect chicks from disease (Keyburn et al., 2013;
rip
Infection with viruses such as Marek’s disease virus, infectious bursal disease virus
us c
and chicken anaemia virus can have immunosuppressive effects (Hoerr, 2010) which can increase the severity of NE and some studies have used these viruses as predisposing factors
M an
for NE disease modelling (McReynolds et al., 2004; Stringfellow et al., 2009). Other stresses on birds such as overcrowding, environmental ammonia and physiological stress also have the potential to cause immunosuppression (Hoerr, 2010) and hence may exacerbate NE and
d
indeed high stock density has been shown to be a predisposing factor (Tsiouris et al., 2015).
pt e
The genetics of birds appears to have some influence on susceptibility to NE as different lines of birds have different degrees of susceptibility (Jang et al., 2013) to NE and
ce
this may result from subtle difference in immune responses to C. perfringens (Hong et al., 2014; Kim et al., 2014).
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
(Heier et al., 2001), although this is yet to be clearly demonstrated.
t
Lovland et al., 2004) so their disappearance as the young birds develop could be an issue
Disruption of microbiota. Until recently the generally accepted model for NE development hypothesised that there were underlying low level populations of C. perfringens within the GIT of chickens that, under certain predisposing circumstances, proliferated to produce the disease (Cooper and Songer, 2009). With recent advances in the understanding of the main virulence factors of C. perfringens it has become obvious that most strains circulating at low levels in healthy birds are non-pathogenic (Lacey et al 2016; Timbermont et al., 2009). It is
therefore necessary to modify our hypothesis regarding the dynamics of the temporal and structural changes occurring within the intestinal populations of C. perfringens when disease develops. It now appears that a necessary step is the introgression of pathogenic isolates and their proliferation, at the expense of non-pathogenic strains, to dominate the C. perfringens population in diseased birds. It is likely that the microbial environment into which pathogenic
t
strains of C. perfringens infiltrate and proliferate to cause disease must play an important role
rip
It has been shown that within a C. perfringens challenged group of birds the birds that
us c
developed NE had differences in gut microbiota compared to birds that remained healthy (Feng et al., 2010; Stanley et al., 2012). The causal relationships between microbiota
M an
structure and NE development are yet to be elucidated; it has not been demonstrated whether the differences seen in the microbiota of diseased birds (Antonissen et al. 2016) results from the disease process or whether some underlying types of microbiota make birds more
d
susceptible to disease and hence it is within the birds with “susceptible microbiota” that
pt e
disease is more likely to develop.
Although a causal link between microbiota changes and disease progression is yet to
ce
be definitively established it is certainly true that many of the predisposing factors that physically alter the GIT and the immune status of the bird also affect the GIT microbiota. Many predisposing factors have been recognised to increase proliferation of C. perfringens
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
in NE disease susceptibility.
but parallel proliferation or inhibitory effects on other members of the GIT microbiota may also be of importance in predisposing birds to NE. Eimeria infection disrupts the microbiota
of the GIT (Stanley et al., 2014; Wu et al., 2014) and this effect, along with the physical changes to the GIT induced by coccidiosis, may play an important role in NE predisposition. It is unsurprising that the release of high protein nutritional sources following Eimeria infection, in the form of serum leakage from compromised GIT surfaces and increased
mucogenesis, will selectively favour certain members of the GIT microbiota as well as C. perfringens. Changes to feed, for example high protein diets that include fishmeal, also alter the composition of GIT microbiota (Stanley et al., 2014). Manipulation of feeding schedules, including sudden major changes to feed composition and withdrawal of feed for 8 to 12 hours prior to challenge are key parts of some experimental disease induction models (Feng et al.,
t
2010; Keyburn et al., 2006) and are also likely to significantly disrupt the GIT microbiota.
rip
al., 2015). One of the changes identified following mycotoxin treatment was a reduction in
us c
the segmented filamentous bacteria (SFB) and this may have significance for immune
development (Snel et al., 1995; Talham et al., 1999). A reduced representation of the SFB in
M an
NE diseased birds was also noted by Stanley et al. (Stanley et al., 2012).
There are a few bacteria, besides C. perfringens, that have been shown to affect the incidence of NE. Many studies have demonstrated that potentially probiotic strains of
d
bacteria and competitive exclusion cultures reduce the incidence of NE (e.g. Elwinger et al.,
pt e
1992; La Ragione et al., 2004; Layton et al., 2013; Tactacan et al., 2013). These microbial interactions are protective rather than predisposing but there are examples of bacterial
ce
infections that have been found to predispose to greater NE development; for example, early infection with a Salmonella enterica sv. Typhimurium isolate increased NE-related mortality and lesion scores, perhaps resulting from a change in host immune response (Shivaramaiah et
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Other predisposing factors such as mycotoxins also change GIT microbiota (Antonissen et
al., 2011). It is clear that bacteria in the GIT can have profound effects on C. perfringens induced disease development, either by directly affecting the way that C. perfringens can colonise and proliferate or by modulation of the chicken immune system. Further assessment of the changes to the GIT microbiota induced by predisposing factors should be carried out to determine if the extent, rate and nature of microbiota change is predictive of disease susceptibility. Is a disrupted and changing microbiota the key
predisposing element of the microbiota contribution to NE susceptibility or is the actual composition of the microbiota into which pathogenic strains of C. perfringens infiltrate more important? For use as a predisposing factor in disease model development, actively changing/disrupting the GIT microbiota is relatively easy to achieve by manipulation of the feed but producing a specific predetermined microbiota “type” in a trial setting is not
rip
Proliferation of pathogenic C. perfringens strains. The application of molecular genome
us c
typing techniques has shown that the GIT of NE diseased birds is usually dominated by a single C. perfringens type, unlike the case in healthy birds were there are generally a number
M an
of different isolates present (Lacey et al. 2016; Barbara et al., 2008; Nauerby et al., 2003). It therefore appears that an important element of the disease process is the selective proliferation of pathogenic C. perfringens strains.
d
A number of characteristics that may favour pathogenic strain proliferation have been
pt e
identified. Antimicrobial proteins such as perfrin may play a role. Timbermont et al. (Timbermont et al., 2014) found that the perfrin gene was only present in pathogenic strains
ce
that also carried the netB gene. The perfrin expressing strains inhibited the in vitro growth of commensal C. perfringens isolates that did not carry the perfrin gene. This could be an important mechanism driving the selective growth of pathogenic strains at the expense of the
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
currently possible.
commensal strains. The selective adhesive qualities of C. perfringens isolates may also be an important
factor that tips the balance in favour of pathogenic strains. Wade et al. (Wade et al., 2015) found that pathogenic strains adhered to extracellular matrix proteins much more strongly than commensal strains; this may lead to higher retention of pathogenic strains and hence their dominance of the C. perfringens population in disease birds. The plasmid that encodes
the NetB toxin also harbours a number of other genes that encode proteins that may potentially enhance the maintenance and proliferation of pathogenic strains of C. perfringens during disease progression. For example two glycoside hydrolases may be involved in mucus colonisation and degradation (Prescott et al., 2016). However, it is not yet clear whether strains expressing the hydrolases would be selectively advantaged or whether all C.
t
perfringens strains that are co-localised with the expressing strain would benefit from the
rip
colonisation and proliferation.
us c
The origin of pathogenic C. perfringens strains that go on to dominate the clostridial population in diseased birds is not clear. Non-pathogenic strains of C. perfringens are much
M an
more commonly isolated from healthy birds than are pathogenic strains (i.e. netB-positive strains). It is unclear whether pathogenic strains are present in healthy birds, but generally at much lower levels than non-pathogenic strains, or whether in many disease outbreaks the C.
d
perfringens that comes to dominate is newly introduced. Therefore, the importance of
pt e
contamination sources is not yet understood. The first published studies that investigated sources of contamination (Craven et al., 2001a, 2001b) did not differentiate between
ce
pathogenic and non-pathogenic strains as they were carried out before the distinction was clear but recent work by Engström et al. (2012) found a high prevalence of netB-positive strains in an empty broiler house, indicating that pathogenic strains may be prevalent in some
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
potential nutrient release. More work is required to understand the role of these proteins in
production environments. The finding that the gene encoding the main virulence factor, NetB, is carried on a conjugative plasmid (Bannam et al., 2011; Parreira et al., 2012) opens up the possibility of exchange of the plasmid between different C. perfringens strains. It is
possible that transfer of the NetB plasmid to a non-pathogenic strain could convert it to a pathogenic strain.
None of the elements that potentially enhance proliferation of pathogenic strains in preference to commensal strains can currently be directly manipulated by the commonly recognised and used predisposing factors. However, the growing understanding of the differences between pathogenic and non-pathogenic C. perfringens isolates highlights the need to carefully select appropriate strains to use in combination with predisposing factors in
rip us c
Conclusions
M an
Predisposing factors influence the incidence and severity of NE induced by C. perfringens. An understanding of what factors are predisposing has been important in both the production setting, where avoidance of those factors has helped poultry producers reduce the impact of
d
disease, and in research. Knowledge of predisposing factors has informed the development of
pt e
robust experimental infection models used to develop and test intervention strategies (e.g. vaccines, feed additives), has contributed to the study of NE pathogenesis and allowed the in
ce
vivo characterization of the pathogenic potential of C. perfringens wild-type strains and mutants. Many predisposing factors are likely to have multiple effects which contribute to the increase in NE. Direct physical effects on the GIT and digesta and host immune perturbation
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
experimental disease models.
have been previously identified as key properties of many predisposing factors but there is mounting evidence that effects on the GIT microbiota may also be important. For researchers, dissection of the direct and indirect effects of predisposing factors on the microbiota and how changes to the microbiota may influence the colonization and proliferation of pathogenic C.
perfringens may be a productive area of study.
Acknowledgments
Robert Moore gratefully acknowledges the ongoing support of the Poultry Cooperative Research Centre, established and supported under the Australian Government’s Cooperative
rip us c
References
M an
Allaart, J.G., van Asten, A.J.A.M., & Gröne, A. (2013). Predisposing factors and prevention of Clostridium perfringens-associated enteritis. Comparative Immunology, Microbiology and Infectious Diseases 36, 449–464.
d
Al-Sheikhly, F., & Al-Saieg, A. (1980). Role of Coccidia in the Occurrence of Necrotic
pt e
Enteritis of Chickens. Avian Diseases, 24, 324–333. Annett, C.B., Viste, J.R., Chirino-Trejo, M., Classen, H.L., Middleton, D.M., and Simko, E.
ce
(2002). Necrotic enteritis: effect of barley, wheat and corn diets on proliferation of Clostridium perfringens type A. Avian Patholology, 31, 598–601.
Antonissen, G., Croubels, S., Pasmans, F., Ducatelle, R., Eeckhaut, V., Devreese, M.,
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
Research Centre Program.
Verlinden, M., Haesebrouck, F., Eeckhout, M., Saeger, S.D., Antlinger, B., Novak, B., Martel, A., & Van Immerseel, F. (2015). Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Veterinary Research, 46, 98.
Antonissen, G., Eeckaut, V., Van Driessche, K., Onrust, L., Haesebrouck, F., Ducatelle, R., Moore, R.J., & Van Immerseel, F. (2016) Microbial shifts that predispose to necrotic enteritis. Avian Pathology, 45, 3. Bains, B.S. (1968). Necrotic enteritis of chickens. Australian Veterinary Journal, 44, 40. Bannam, T.L., Yan, X.-X., Harrison, P.F., Seemann, T., Keyburn, A.L., Stubenrauch, C.,
t
Weeramantri, L.H., Cheung, J.K., McClane, B.A., Boyce, J.D., Moore, R.J., & Rood,
rip
Plasmids. mBio, 2, e00190-11.
us c
Closely Related Independently Conjugative Toxin and Antibiotic Resistance
Barbara, A.J., Trinh, H.T., Glock, R.D., & Songer, J.G. (2008). Necrotic enteritis-producing
M an
strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks. Veterinary Microbiology, 126, 377–382.
Branton, S.L., Reece, F.N., & Hagler, W.M. (1987). Influence of a Wheat Diet on Mortality
d
of Broiler Chickens Associated with Necrotic Enteritis. Poultry Science, 66, 1326–
pt e
1330.
Branton, S.L., Lott, B.D., Deaton, J.W., Maslin, W.R., Austin, F.W., Pote, L.M., Keirs, R.W.,
ce
Latour, M.A., & Day, E.J. (1997). The effect of added complex carbohydrates or added dietary fiber on necrotic enteritis lesions in broiler chickens. Poultry Science,
76, 24–28.
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
J.I. (2011). Necrotic Enteritis-Derived Clostridium perfringens Strain with Three
Chakrabarty, A.K., & Boro, B.R. (1981). Prevalence of food-poisoning (enterotoxigenic) Clostridium perfringens type A in blood and fish meal. Zentralblatt Für Bakteriologie, Mikrobiologie und Hygiene. 1. Abt Originale B Hygiene, 172, 427– 433. Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I., & Gaskins, H.R. (2008). Coccidia-induced mucogenesis promotes the onset of necrotic
enteritis by supporting Clostridium perfringens growth. Veterinary Immunology and Immunopathology, 122, 104–115. Cooper, K.K., & Songer, J.G. (2009). Necrotic enteritis in chickens: A paradigm of enteric infection by Clostridium perfringens type A. Anaerobe, 15, 55–60. Cooper, K.K., & Songer, J.G. (2010). Virulence of Clostridium perfringens in an
t
experimental model of poultry necrotic enteritis. Veterinary Microbiology, 142, 323–
rip
Craven, S.E., Stern, N.J., Bailey, J.S., & Cox, N.A. (2001a). Incidence of Clostridium
processing. Avian Diseases, 45, 887–896.
us c
perfringens in broiler chickens and their environment during production and
M an
Craven, S.E., Cox, N.A., Stern, N.J., & Mauldin, J.M. (2001b). Prevalence of Clostridium perfringens in Commercial Broiler Hatcheries. Avian Diseases, 45, 1050–1053. Drew, M.D., Syed, N.A., Goldade, B.G., Laarveld, B., & Kessel, A.G.V. (2004). Effects of
d
dietary protein source and level on intestinal populations of Clostridium perfringens
pt e
in broiler chickens. Poultry Science, 83, 414–420. Elwinger, K., Schneitz, C., Berndtson, E., Fossum, O., Teglöf, B., & Engstöm, B. (1992).
ce
Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Veterinaria Scandinavica, 33, 369–378.
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
328.
Engström, B.E., Johansson, A., Aspan, A., & Kaldhusdal, M. (2012) Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis. Veterinary Microbiology, 159, 260264.
Feng, Y., Gong, J., Yu, H., Jin, Y., Zhu, J., & Han, Y. (2010). Identification of changes in the composition of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens. Veterinary Microbiology, 140, 116–121. Forder, R.E.A., Nattrass, G.S., Geier, M.S., Hughes, R.J., & Hynd, P.I. (2012). Quantitative analyses of genes associated with mucin synthesis of broiler chickens with induced
t
necrotic enteritis. Poultry Science, 91, 1335–1341.
rip
naturally occurring specific antibodies against Clostridium perfringens alpha toxin in
us c
Norwegian broiler flocks. Avian Diseases, 45, 724–732.
Hermans, P.G., & Morgan, K.L. (2007). Prevalence and associated risk factors of necrotic
Pathology, 36, 43–51.
M an
enteritis on broiler farms in the United Kingdom; a cross-sectional survey. Avian
Hoerr, F.J. (2010). Clinical aspects of immunosuppression in poultry. Avian Diseases, 54, 2–
d
15.
pt e
Hong, Y.H., Dinh, H., Lillehoj, H.S., Song, K.-D., & Oh, J.-D. (2014). Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic
ce
enteritis disease. Poultry Science, 93, 1383–1395. Jang, S.I., Lillehoj, H.S., Lee, S.-H., Lee, K.W., Lillehoj, E.P., Hong, Y.H., An, D.-J., Jeoung, H.-Y., & Chun, J.-E. (2013). Relative disease susceptibility and clostridial
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Heier, B.T., Lovland, A., Soleim, K.B., Kaldhusdal, M., & Jarp, J. (2001). A field study of
toxin antibody responses in three commercial broiler lines coinfected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis. Avian Diseases, 57, 684–687.
Keyburn, A.L., Sheedy, S.A., Ford, M.E., Williamson, M.M., Awad, M.M., Rood, J.I., & Moore, R.J. (2006). Alpha-toxin of Clostridium perfringens is not an essential
virulence factor in necrotic enteritis in chickens. Infection and Immunity, 74, 6496– 6500. Keyburn, A.L., Boyce, J.D., Vaz, P., Bannam, T.L., Ford, M.E., Parker, D., Di Rubbo, A., Rood, J.I., & Moore, R.J. (2008). NetB, a new toxin that is associated with avian ecrotic enteritis caused by Clostridium perfringens. PLoS Pathogens, 4, e26.
t
Keyburn, A.L., Yan, X.-X., Bannam, T.L., Van Immerseel, F., Rood, J.I., & Moore, R.J.
rip
strains expressing NetB toxin. Veterinary Research, 41, 21
us c
Keyburn, A.L., Portela, R.W., Ford, M.E., Bannam, T.L., Yan, X.X., Rood, J.I., & Moore, R.J. (2013). Maternal immunization with vaccines containing recombinant NetB toxin
M an
partially protects progeny chickens from necrotic enteritis. Veterinary Research, 44, 108.
Kim, D.K., Lillehoj, H.S., Jang, S.I., Lee, S.H., Hong, Y.H., & Cheng, H.H. (2014).
d
Transcriptional profiles of host-pathogen responses to necrotic enteritis and
pt e
differential regulation of immune genes in two inbreed chicken lines showing disparate disease susceptibility. PLoS ONE, 9, e114960.
ce
Lacey, J.A., Johanesen, P.A., Lyras, D., & Moore, R.J. (2016) Genomic diversity of necrotic enteritis associated strains of Clostridium perfringens. Avian Pathology, this issue.
La Ragione, R.M., Narbad, A., Gasson, M.J., & Woodward, M.J. (2004). In vivo
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
(2010). Association between avian necrotic enteritis and Clostridium perfringens
characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry. Letter in Applied Microbiology, 38, 197–205.
Layton, S.L., Hernandez-Velasco, X., Chaitanya, S., Xavier, J., Menconi, A., Latorre, J.D., Kallapura, G., Kuttappan, V.A., Wolfenden, R.E., Filho, R.L.A., Hargis, B.M., &
Téllez, G. (2013). The effect of a Lactobacillus-based probiotic for the control of necrotic enteritis in broilers. Food and Nutrition Sciences, 04, 1-7. Lee, K.W., Lillehoj, H.S., Jeong, W., Jeoung, H.Y., & An, D.J. (2011). Avian necrotic enteritis: Experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poultry Science, 90, 1381–1390.
t
Lovland, A., Kaldhusdal, M., Redhead, K., Skjerve, E., & Lillehaug, A. (2004). Maternal
rip
90.
us c
McReynolds, J.L., Byrd, J.A., Anderson, R.C., Moore, R.W., Edrington, T.S., Genovese, K.J., Poole, T.L., Kubena, L.F., & Nisbet, D.J. (2004). Evaluation of
M an
immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poultry Science, 83, 1948–1952.
M’Sadeq, S.A., Wu, S., Swick, R.A., & Choct, M. (2015). Towards the control of necrotic
pt e
Nutrition, 1, 1–11.
d
enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. Animal
Nairn, M.E., & Bamford, V.W. (1967). Necrotic enteritis of broiler chickens in western
ce
Australia. Australian Veterinary Journal, 43, 49–54. Nauerby, B., Pedersen, K., & Madsen, M. (2003). Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
vaccination against subclinical necrotic enteritis in broilers. Avian Pathology, 33, 81–
chickens. Veterinary Microbiology, 94, 257–266.
Olkowski, A.A., Wojnarowicz, C., Chirino-Trejo, M., Laarveld, B., & Sawicki, G. (2008). Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue. Research in Veterinary Science, 85, 543–553.
Parreira, V.R., Costa, M., Eikmeyer, F., Blom, J., & Prescott, J.F. (2012). Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative plasmids. PLoS ONE, 7, e49753. Prescott, J.F., Valeria, R.P., Gohari, I.M., Lepp, D., & Gong, J. (2016) The pathogenesis of necrotic enteritis in chickens: What we know and what we need to know. Avian
t
Pathology, this issue.
rip
chickens. Avian Diseases, 36, 499–503.
us c
Shivaramaiah, S., Wolfenden, R.E., Barta, J.R., Morgan, M.J., Wolfenden, A.D., Hargis, B.M., & Téllez, G. (2011). The role of an early Salmonella Typhimurium infection as
Diseases, 55, 319–323.
M an
a predisposing factor for necrotic enteritis in a laboratory challenge model. Avian
Smyth, J.A., & Martin, T.G. (2010). Disease producing capability of netB positive isolates of
d
C. perfringens recovered from normal chickens and a cow, and netB positive and
76–84.
pt e
negative isolates from chickens with necrotic enteritis. Veterinary Microbiology, 146,
ce
Snel, J., Heinen, P.P., Blok, H.J., Carman, R.J., Duncan, A.J., Allen, P.C., & Collins, M.D. (1995). Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of “Candidatus Arthromitus.”
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Riddell, C., & Kong, X.M. (1992). The influence of diet on necrotic enteritis in broiler
International Journal of Systematic Bacteriology, 45, 780–782.
Stanley, D., Keyburn, A.L., Denman, S.E., & Moore, R.J. (2012). Changes in the caecal microflora of chickens following Clostridium perfringens challenge to induce necrotic enteritis. Veterinary Microbiology, 159, 155–162.
Stanley, D., Wu, S.-B., Rodgers, N., Swick, R.A., & Moore, R.J. (2014). Differential responses of cecal microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis challenge model in chickens. PLoS ONE, 9, e104739. Stringfellow, K., McReynolds, J., Lee, J., Byrd, J., Nisbet, D., & Farnell, M. (2009). Effect of bismuth citrate, lactose, and organic acid on necrotic enteritis in broilers. Poultry
t
Science, 88, 2280–2284.
rip
(QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens.
us c
Journal of Applied Poultry Research, 22, 825–831.
Talham, G.L., Jiang, H.-Q., Bos, N.A., & Cebra, J.J. (1999). Segmented filamentous bacteria
M an
are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. Infection and Immunity, 67, 1992–2000. Timbermont, L., Lanckriet, A., Gholamiandehkordi, A.R., Pasmans, F., Martel, A.,
d
Haesebrouck, F., Ducatelle, R., & Van Immerseel, F. (2009). Origin of Clostridium
pt e
perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comparative Immunology, Microbiology and Infectious Diseases, 32, 503–512.
ce
Timbermont, L., De Smet, L., Van Nieuwerburgh, F., Parreira, V.R., Van Driessche, G., Haesebrouck, F., Ducatelle, R., Prescott, J., Deforce, D., Devreese, B., & Van Immerseel, F. (2014). Perfrin, a novel bacteriocin associated with netB positive
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Tactacan, G.B., Schmidt, J.K., Miille, M.J., & Jimenez, D.R. (2013). A Bacillus subtilis
Clostridium perfringens strains from broilers with necrotic enteritis. Veterinary Research, 45, 40.
Tsiouris, V., Georgopoulou, I., Batzios, C., Pappaioannou, N., Ducatelle, R., & Fortomaris, P. (2014). Temporary feed restriction partially protects broilers from necrotic enteritis. Avian Pathology, 43, 139–145.
Tsiouris, V., Georgopoulou, I., Batzios, C., Pappaioannou, N., Ducatelle, R., & Fortomaris, P. (2015). High stocking density as a predisposing factor for necrotic enteritis in broiler chicks. Avian Pathology, 44, 59–66. Van Immerseel, F., Buck, J.D., Pasmans, F., Huyghebaert, G., Haesebrouck, F., & Ducatelle, R. (2004). Clostridium perfringens in poultry: an emerging threat for animal and
t
public health. Avian Pathology, 33, 537–549.
rip
17.
us c
Wade, B., Keyburn, A.L., Seemann, T., Rood, J.I., & Moore, R.J. (2015). Binding of
Clostridium perfringens to collagen correlates with the ability to cause necrotic
M an
enteritis in chickens. Veterinary Microbiology, 180, 299–303.
Williams, R.B. (2005). Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34,
d
159–180.
pt e
Wu, S.-B., Rodgers, N., & Choct, M. (2010). Optimized Necrotic Enteritis Model Producing Clinical and Subclinical Infection of Clostridium perfringens in Broiler Chickens.
ce
Avian Diseases, 54, 1058–1065.
Wu, S.-B., Stanley, D., Rodgers, N., Swick, R.A., & Moore, R.J. (2014). Two necrotic enteritis predisposing factors, dietary fishmeal and Eimeria infection, induce large
Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
Wade, B., & Keyburn, A. (2015). The true cost of necrotic enteritis. World Poultry, 31, 16–
changes in the caecal microbiota of broiler chickens. Veterinary Microbiology, 169, 188–197.
Figure Legend Figure 1. Summary of predisposing factors for necrotic enteritis development in chickens. Predisposing factors are shown in circles and the major effects of these factors are shown in the ovals. Important factors that may drive the influence of the predisposing factors are
rip us c M an d pt e ce Ac
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
shown in the small rectangular boxes.
ce
Ac d
pt e M an us c
rip
Downloaded by [University of California, San Diego] at 18:24 03 March 2016
t
