Protein Flow to the Hindgut as a Trigger to Clostridium-Related Dysbacteriosis in Broilers

Poor gut health in broiler chickens is a generalized term that means different things to different people. Problems associated with shifts of intestinal bacterial populations (i.e. dysbacteriosis) gained notoriety as a key production issue with the removal of antibiotic growth promoters in the European Union (Wilson et al, 2005). However, it remains a common challenge in any production environment where chickens are produced on litter. The most common external symptoms of this malady include diarrhea; wet, watery or poorly formed feces that under more severe circumstances may be yellow in color with a foamy appearance. When combined with undigested feed particles some describe this as 'feed passage' or 'flushing'. All of these presentations will typically lead to wet litter and increased mortality along with a wide variety of other secondary problems including necrotic enteritis (NE) and gangrenous dermatitis; pododermatitis and synovitis, reduced flock and carcass uniformity and higher rates of field and plant condemnations.

Systematic study of poor gut health associated with dysbacteriosis is complicated by the fact that a variety of interacting factors contribute to its etiology. In many cases the initiating factors may be mild parasitic infection, exogenous pathogens or toxins. The host response to these insults contributes significantly to the course of the problem and the ultimate impact on flock performance; however, it is quite common that no clear clinical manifestations are associated with it. Therefore, the term that has been used to describe this form of poor gut health is subclinical enteritis (Hoerr, 1998) which, because it is not obvious, goes untreated and continues to reduce production efficiency. The purpose of the current report is to describe a reproducible experimental model that provides a means of creating subclinical enteritis such that the interacting factors that contribute to and mitigate this syndrome can be systematically studied.

Necrotic enteritis (NE) is only one representation of intestinal bacterial overgrowth in chickens however; it serves as a good example of an enteric disease that originates from a multi-factorial problem that typically begins with subclinical enteritis. Experimental models for producing NE (Prescott et al, 1978; Truscott and Al-Shaikhly, 1977; Williams et al, 2002) have drawn from field experience linking its occurrence to coccidiosis challenge, an association of diets containing fishmeal and animal protein, as well as grains with high content of poorly digestible non-starch polysaccharides (NSP). All published models for creation of NE in broilers include a substantial multi-day challenge with actively replicating C. perfringens (Cp) either orally or cloacally administered. We have chosen to eliminate this aspect of NE models from our studies as we are particularly interested in the factors that trigger the initiation of Cp growth. In addition, Cp are typically already present in the hindgut of day old chicks (Lee, 2002) where they reside in low concentrations and typically do not actively grow in percentage of the microbiota present. Furthermore, we are interested in studying subclinical enteritis since the degree of severity is such that it may allow us to more readily elucidate the factors that can tip the balance of the relationship between microbiota and host to either positively or negatively impact overall gut health.

Considering the elements of NE challenge models, the key triggers include some form of digestive failure along with gastrointestinal tract (GIT) inflammation and oxidative stress. Both factors have been reported to occur in the presence of NSP containing diets (Tierlynck et al, 2009). Feeding cereals like wheat, barley or rye creates highly viscous digesta and increases intestinal bacterial populations (Bedford and Classen, 1992). It also creates morphological changes to the intestine consistent with inflammation and this is mitigated by feeding antibiotics (Tierlynck et al, 2009). The host response to GIT inflammation includes hypersecretion of fluid and goblet cell hypertrophy with excess mucous secretion (Hoerr, 1998). The physical consequences of these responses include reduced digestibility and flushing of the intestinal digesta into the hindgut. Our hypothesis for development of this experimental model is that the effects of inflammation, in combination with the relative inability of the chicken to adequately digest NSP, increase the nutrient flow into the hindgut. Consequently, microbial populations in the ceca - which are established based on their relative ability to compete for nutrients - become imbalanced, resulting in the ability of certain organisms that typically cannot compete in the nutrient restricted environment of the ceca, to begin to actively grow (Collier et al, 2003; Choct et al, 2006).

Figure 1. Each of these four factors can

Intestinal barrier function is also challenged in the face of inflammation and oxidative stress. The action of phagocytic cells employs significant oxidative potential to destroy engulfed bacteria. While this is a necessary function, excessive inflammation creates oxidative challenges that can also result in damage to intestinal epithelial cell tight junctions. This contributes to a loss of gut barrier function (McQuaid and Keenan, 1998; Dibner et al, 2009). The addition of cycling Eimeria contributes to local inflammation and provides an additional challenge to barrier function associated with migrating sporozoites and merozoites. A loss of barrier function inevitably results in a systemic innate immune response that triggers the synthesis and secretion of a number of acute phase proteins such as alpha-1- acid glycoprotein (AGP). We have used AGP as an indicator of intestinal barrier function.

Therefore, the combination of NSP containing diets and cycling Eimeria provides an environment that can promote Cp growth in the hindgut and along with the normal anti-peristalsis on the hindgut, results in translocation of Cp into a mucous rich environment in the small intestine in which it can proceed to grow and ultimately produce the toxins associated with NE (Collier et al, 2003). Thus, digestive failure, excess nutrient flow to the hindgut, intestinal inflammation with oxidative stress, and barrier function failure with a resulting systemic acute phase response can each serve individually as a trigger to subclinical enteritis; however, ultimately, all 4 will likely be involved in an episode of poor gut health (Fig 1).

 

Using this theoretical framework, a series of 3 experiments was conducted to validate this model as a means of studying factors that can contribute to or mitigate the effects of subclinical enteritis.

Trial 1. In this first proof of principle experiment a 28-day broiler trial was conducted in which a 22% CP, 1.21%/1.07% total/digestible lysine mash diet was fed that contained 33% rye, 25% wheat, and 31% soybean meal. Treatments were in a 2 X 3 factorial arrangement that included two factors of cycling Eimeria as a 3X overdose of a 3-species live oocyst vaccine (1ADVENT® coccidiosis control) or nothing, and 3 feed additive factors: a negative control, an antibiotic (bacitracin methylene disalicyclate, 60 ppm:BMD) or an NSP enzyme mixture containing xylanase, glucanase and glycosidase (2CIBENZATM CSM).

Trial 2. In the second trial the diet was modified to assess the impact of an animal protein source that was less well digested than soybean meal when fed from 1 to 21 days of age. Previous studies have reported that the level of fishmeal but not soy protein concentrate significantly increased the growth of Cp in the ileum and cecum of the chicken (Drew et al, 2004). For this trial we added 7% feather meal in a complete diet formulated to 23.5% CP and 1.38%/1.21% total/digestible lysine. All birds were challenged with a 3X overdose of the same live oocyst coccidiosis vaccine at day of age and feed additive treatments included diets with and without betaine, NSP enzymes (CIBENZA™ CSM), and a protease enzyme (3CIBENZATM DP100)and a combination of the two types of enzymes. Betaine has been reported to improve osmotic balance and performance in the face of coccidial challenge (Allen and Fetterer, 2007) and to improve the efficiency of the innate immune response (Klasing et al, 2002).

Trial 3. To further examine the impact of dietary protein on ileal Cp and intestinal barrier function we used two diets, one with no animal protein and formulated to 22% CP and 1.38%/1.21% total/digestible lysine, the second diet was nearly identical to the first with the exception that it contained 14% poultry by-product meal (PBM) and was formulated to provide an excess of CP (30%) and total/digestible lysine (1.65%/1.38). We were concerned that the high level of rye (38%) in Trial 2 may have overridden any potential effect of the protease as a result of the very high viscosity of the diet. Therefore, in Trial 3 the level of rye in the diet was reduced from 38% to 20% with corn added to make up the energy difference. Again all birds were subjected to an overdose of the same live oocyst coccidiosis vaccine with the exception that this occurred on day 7 of the study instead of day 1.

 

The overdose of coccidiosis vaccine in this high viscosity diet resulted in a 4-5% reduction (P<.01) in the efficiency of gain, however, there were no interactions with any of the feed additive effects (Trial 1). Therefore, results are presented as main effects averaged across coccidiosis challenge. Addition of the feed additives improved 28-day Performance Index ((period gain*period ivability)/period feed efficiency), PI) from 145 for control to 188 and 284 for antibiotic and NSP enzymes, respectively (Fig 2; P<.01). The PI improvement was more consistent for NSP enzymes than that of the antibiotic and was also associated with a significant reduction in digesta viscosity (P<.01) throughout the trial. NSP enzymes were also associated with a 1.5 to 2.5 log reduction in cultured Cp from the hindgut and lower ileum (Fig 3; P<.01), consistent with a previous report (Choct et al, 2006), however, the antibiotic did not have a significant effect on Cp number. Overall livability for the trial was in excess of 95%, there were no treatment related differences and no Clostridiumrelated deaths.

Intestinal morphometry at day 15 and 22 of the study was used to assess intestinal health with respect to the various treatments employed in the trial (data not shown). Overdose of the coccidiosis vaccine affected intestinal morphometry negatively, showing significantly reduced mucosa development, villus height and higher (poorer) crypt/villus ratios in the duodenum and mid-small intestine at both 15 and 22 days compared to non-challenged controls. These negative effects, which are consistent with local inflammatory responses, were more pronounced in the mid-small intestine for challenged birds in the absence of NSP enzymes on day 15; however, ileal morphometry was not significantly affected at either day 15 or 22 by any additives or coccidial challenge. Addition of the NSP enzymes improved intestinal morphometry (duodenum and mid-small intestine) as represented by reduced crypt/villus ratio, indicating that at least a portion of the improved performance was related to improved gut health and reduced demands on the crypt stem cell proliferation.

Figure 2. Trial 1-NSP enzymes (CIBENZA™ CSM) and to a lesser extent antibiotic (BMD) improved performance index in high NSP diets, (P<.05).

Figure 3. Trial 1- Addition of NSP enzymes reduced C. perfringens in lower intestinal tract (Ileum + Ceca).

Figure 4. Trial 2 - Performance Index was improved primarily by NSP enzymes (CSM) and the effect was enhanced in the presence of betaine. (a, b,c,d; P<.01).

These results indicate that subjecting broilers to this challenge of dietary NSP-containing ingredients created intestinal inflammation and stimulated Cp growth in the lower GIT. Furthermore, addition of NSP enzymes and to a lesser extent an antibiotic improved performance while the NSP enzymes alone mitigated intestinal inflammation and reduced Cp overgrowth.

Results for Trial 2 demonstrated that Performance Index (PI) was improved by addition of NSP enzymes (Fig 4; P<.01) which was modestly more effective in the presence of the osmolyte, betaine (Bet X CSM = .037). There was only a small PI improvement with addition of the protease by itself that was not observed in the presence of betaine (Bet X DP100 = 0.049), primarily due to a PI improvement of the negative control in the presence of betaine. The addition of protease to the NSP enzymes did not result in a further PI improvement.

Digestive viscosity was reduced the most by NSP enzymes (Fig 5; P<.01), however, the protease reduced viscosity in the presence of betaine (Bet X DP100 < .05). Again, addition of NSP enzymes reduced Cp across other treatments by 1.5 to 2 logs (Fig 6; P<.01) from samples obtained only from the lower ileum. The acute phase protein, AGP (Fig. 7) was measured in serum as an indicator of intestinal barrier function. This protein is indicative of a systemic immune response, consequently elevated AGP would suggest some level of barrier function failure. The results indicated that both protease and NSP enzymes reduced AGP levels similarly in the presence of betaine and that the combination of the two types of enzymes was additive (Fig 7; P<.05). This suggests that in the presence of an osmolyte, both types of enzymes have some positive impact on intestinal barrier function in this subclinical enteritis model. However, while the addition of NSP enzymes to this diet reduced ileal concentrations of Cp, there was no evidence that addition of the protease in this high feather meal diet had any similar inhibitory impact on Cp growth. It is possible that the high viscosity associated with the NSP diet prevented appropriate interaction of the protease with its substrate.

Figure 6. Trial 2- Ileal C. perfringens were reduced by NSP enzymes (CSM) irrespective of protease (DP 100) or betaine treatments (a,b,c; P<.01)

Figure 7. Trial 2 - Protease (DP100), NSP enzyme (CSM) and betaine all contributed to a reduction in acute phase protein response (AGP) indicating all contributed to maintaining intestinal gut barrier function (a,b,c;P<.05)

Figure 8. Trial 3 – The high protein diet (30% CP) containing 14% poultry by-product meal (PBM) tended to reduce performance index while addition of protease (DP100) tended to maintain it at levels comparable to normal protein (22% CP).

The purpose of Trial 3 was to further examine the impact of animal protein on performance and subclinical enteritis. In this case the protein from PBM was in excess of requirement and also more highly fermentable than feather meal. The 28-day results indicated a reduction in PI with the 30% protein PBM diet that was increased to that of the normal protein diets with the addition of protease (Fig 7). While these results were not significant, they are directionally consistent with the potential for excess protein in the hindgut to have a promoting influence on bacterial overgrowth and consequently a negative influence on performance. Ileal concentrations of Cp were substantially lower in the normal protein diet than was observed for Trial 2 (~2.4 logs vs. ~5.0 logs) which is likely due to the reduction in rye content of the diet in this trial. The ileal Cp levels for the 30% CP PBM diet were increased approximately 2 log units compared to the normal protein diet (Fig 9; P<.01). Addition of the protease to the normal protein diet had no impact on ileal Cp levels however, protease addition to the high protein diet resulted in a 2 log reduction in Cp to levels that were similar to the normal protein diet. Similar to results observed in Trial 2, addition of protease in this trial resulted in reductions of serum AGP levels (Fig 9; P<.10) regardless of dietary protein level. These results indicate that minimizing flow of digestible animal protein into the hindgut in the face of cycling Eimeria will reduce Cp levels whether this is done by with lower dietary protein or the addition of a protease to increase digestibility in the upper GIT thereby minimizing protein flow to the hindgut. Furthermore, addition of the protease in this gut health challenge model improved intestinal barrier function as measured by acute phase protein response, irrespective of dietary protein level.

Figure 9. Trial 3 - Addition of protease (DP 100) reduced the elevated ileal Cp levels resulting from feeding the 30% CP diet (a,b; P<.05) and reduced systemic acute phase response (AGP) irrespective of dietary protein level (a,b; P<.10).

 

The purpose of these three trials was to evaluate the validity of a gut health challenge model that was designed to produce subclinical enteritis in young broiler chickens. If successful, this would provide a system that would facilitate the study of the many interacting factors that contribute to this widespread and often unpredictable problem. The theoretical framework for this model is predicated on the concept that 1) intestinal inflammation and oxidative stress, 2) digestive failure, with 3) increased nutrient flow to the hindgut, and 4) intestinal barrier failure are all involved to some degree in a gut health challenge. Results have shown that each of these 4 factors is influenced by this model and the lack of significant mortality is consistent with the subclinical gut health challenge model we intended to create.

These studies focused primarily on the influence of increased nutrient flow to the hindgut on the stimulation of Cp growth and demonstrated the role that substrate specific digestive enzymes play in minimizing Cp growth and supporting maintenance of a healthy gut. We have also observed that while Cp is a reproducible marker of intestinal overgrowth of a potential pathogen, there is no need to challenge the birds with exogenous Cp in order to produce these effects. In fact, it's possible that this approach more closely mimics what occurs in the normal commercial environment. This model system will be used in the future to evaluate additional factors that can support the maintenance of intestinal barrier function, modulate the intestinal inflammatory response and resulting oxidative stress, and promote the maintenance of the appropriate microbiota in the gastrointestinal tract of the chicken.

 

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