Efficacy of a Novel, Native Microbial Feed Supplement on the Mitigation of Necrotic Enteritis in Cobb 500 Broiler Chickens

Andrew Izzo, Grant Gogul, Josh Lefler, Allison Wells, James Gaffney, Jennifer Cao, Norman Pitt, Brian Dirks and Mallory Embree

Copyright: © 2022. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Andrew Izzo, Grant Gogul, Josh Lefler, Allison Wells, James Gaffney, Jennifer Cao, Norman Pitt, Brian Dirks and Mallory Embree, 2022. Efficacy of a Novel, Native Microbial Feed Supplement on the Mitigation of Necrotic Enteritis in Cobb 500 Broiler Chickens. International Journal of Poultry Science, 21: 57-64.

DOI: 10.3923/ijps.2022.57.64

URL: https://scialert.net/abstract/?doi=ijps.2022.57.64

MATERIALS AND METHODS

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Experimental design: In Trial 1, a total of 540 Cobb 500 male broiler chickens were divided into three treatment groups (180 birds/treatment) and placed in concrete floor pens on the day of hatch. In Trial 1,each treatment group consisted of six pens, each containing 30 birds. In Trial 2, a total of 936 Cobb 500 male broiler chickens were divided into three treatment groups (312 birds/treatment) and placed in concrete floor pens on the day of hatch. Each Trial 2 treatment group consisted of 12 pens, each containing 26 birds. All other aspects of the experimental design were consistent between Trial 1 and Trial 2.

Chicks were placed in floor pens that had dimensions of 4’×4’ and contained fresh pine shavings top-dressed with used pine shavings. All broilers were fed the same standard, corn/soy-based diet. Additionally, the MFS treatment group (MFS) was supplemented with an MFS containing 1×10⁶ CFU of both H. xylanolytica ASCUSBR21 and C. beijerinckii ASCUSBR67 (Native Microbials, San Diego, CA). The treatment was mixed as a lyophilized powder with a base mash of corn and soy feed to create treatment feed at the test concentrations. The remaining groups received calcium carbonate without the MFS.

All birds were vaccinated for Marek’s disease at the hatchery. Additionally, all birds were vaccinated for Infectious Bronchitis and Newcastle disease upon receipt at the facility, as well as for coccidiosis via spray cabinet before placement into study pens. Feed was provided ad libitum via a single hanging tube feeder in each pen. Birds were fed a three-phase commercial-type corn/soy mash diet consisting of a starter (day 0-16), grower (day 17-35) and finisher (day 36-42). All feed was sampled and tested at each change in feed to ensure that the feed met or exceeded nutritional requirements as designated by the National Research Council²⁹. Water was provided ad libitum via a bell drinker. Pens were housed within an environmentally controlled building. The building temperature was regulated based on the breed recommendation for Cobb 500 broiler chickens³⁰. A commercial lighting program was used based on the breed recommendation for Cobb 500 broilers³⁰.

All procedures were approved by the Colorado Quality Research Institutional Animal Care and Use Committee and overseen by onsite veterinarians.

Clostridium perfringens challenge: The MFS treatment group (MFS) and positive control group (POS) received an oral challenge of Clostridium perfringens (CL-15, Type A, α and β2 toxins). The strain used, referred to as CL-15, was received from Microbial Research, Inc. and was isolated from a NE outbreak on a commercial broiler farm in Colorado, USA. This strain of C. perfringens is known to produce Net B, a toxin with direct links to causing severe avian NE³¹.

On day 17, each pen in the MFS and POS groups received an oral C. perfringens challenge. Approximately 25 g of feed mixed with 1.5 mL of inoculum was administered to the POS and MFS pens per bird. In Trial 1, the C. perfringens inoculum had a concentration of 7.7×10⁷ CFU mL^–1. In Trial 2, the C. perfringens inoculum had a concentration of 5.6×10⁸ CFU mL^–1. All other aspects of the Clostridium perfringens challenge were consistent between Trial 1 and Trial 2.

All feeder trays in the POS and MFS pens were removed after the inoculated feed was consumed. The negative control group (NEG) also had their feeder trays removed when the trays for the challenged groups were removed. The concentration of liquid inoculum of C. perfringens was determined pre-administration using spectrophotometry at a wavelength of 600 nm using a standard curve. Clostridium perfringens concentrations were subsequently confirmed post-administration by plating the inoculum onto tryptose-sulfite-cycloserine agar, incubating anaerobically for 18 h at 37°C. Black colonies were presumed to be C. perfringens and counted to confirm the colony forming unit (cfu)concentration of inoculum.

Data collection: Facility conditions and bird health were observed twice daily by trained facility staff for the duration of both studies. Throughout each study, any bird that was removed from the study was necropsied and had sex, weight and probable cause of death recorded. Birds were weighed by pen on days 0,17, 28, 35 and 42. The remaining feed in each pen was weighed and recorded prior to feed change on days 17, 28, 35 and 42. This data was used to calculate feed intakes for the following time periods: days 0-17, 0-28, 17-28, 0-35, 28-35, 0-42 and 35-42.

Necrotic enteritis lesion scoring: On study days 21 and 28, 5 birds were removed from each pen at random, weighed and euthanized using cervical dislocation. The number of birds removed per pen was decreased and replaced by the number of birds that died from NE prior to lesion scoring up to 5 birds per pen. Portions of their intestines were then scored for lesions resulting from NE. Lesions were scored using the following criteria: (0) Normal: no NE lesions, small intestine has normal elasticity (rolls back on itself after being opened); (1) Mild: small intestinal wall is thin and flaccid (remains flat when opened and does not roll back into normal positions after being opened), excess mucus covering mucus membrane, (2) Moderate: noticeable reddening and swelling of the intestinal wall, minor ulceration and necrosis of the intestine membrane, excess mucus, (3) Severe: extensive necrosis and ulceration of the small intestine membrane, significant hemorrhage, a layer of fibrin and necrotic debris on the mucous membrane, (4) Dead or moribund: dead bird (or bird that would likely die within 24 h) with NE gross lesions scored 2 or more. This was consistent between Trial 1 and Trial 2.

Statistical analysis: Data from each trial were analyzed using R software (version 3.5.1)³². One-way Analysis of Variance tests (ANOVA) were used to compare the performance data between treatment groups. A significant ANOVA result (p≤0.05) warranted further analysis via Fisher's Least Significant Difference (LSD) test (package agricolae version 1.3.3), in order to determine significance when comparing individual treatment groups. Pairwise comparisons generated via LSD were corrected using the Bonferroni procedure. p-values generated via both ANOVA and LSD were considered trending if p≤0.10 and significant if p≤0.05. ANOVA generated p-values are presented in tables and the LSD pairwise p-values are indicated by superscripts in tables.

RESULTS AND DISCUSSION

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Mortality and lesion scores: In Trial 1, there were no significant differences between any groups regarding mortality over the entire study period (day 0-42, p = 0.24, Table 1). Although not significant, the MFS group showed a reduction that was trending when compared to the POS (p = 0.10, Table 1) and numerically lower than the NEG control group (p = 0.26, Table 1). This result can be expected as the birds were vaccinated with only a 1× dose of coccidiosis vaccine and no other challenge conditions were applied to instigate a response until day 17 when C. perfringens was applied to the MFS and POS group. However, in the post-challenge period (day 17-28), MFS supplementation reduced necrotic mortality rate in Trial 1 (p = 0.05, Table 2).

There was also a trending reduction in NE specific mortality throughout the entire study (day 0-42) (p = 0.07, Table 1). There was a significant difference between the POS and NEG control groups (p≤0.05, Table 1). While not significantly different from the POS or NEG control groups, there was a numerical reduction between the MFS group and the POS control group suggesting that the MFS may be exerting a beneficial effect on the C. perfringens challenged birds (Table1). The reduction being non-significant may be related to the number of pens, as a sample size of six pens is small compared to similar studies^22,33,34. Additionally, the necrotic mortality data across the whole trial may be influenced by the lack of differences seen in either total mortality or NE associated mortality between groups after day 28. This pattern was expected, however, as most cases of NE in broilers are seen between 10 and 28 days of age^35,36. Finally, broilers supplemented with the MFS had significantly lower lesion scores compared to the POS group on Day 21 (p<0.05, Table 5). On Day 28, the MFS group displayed numerically reduced intestinal lesions when compared to the POS control (Table 5).

In Trial 2, there was a significant difference in mortality between groups, largely driven by the low mortality in the non-challenged NEG group (p< 0.01, Table 3). This difference in mortality was driven by NE, as the NE specific mortality rate was much higher in the MFS and POS group than the NEG group, accounting for approximately 80% of total mortality in the groups that received the C. perfringens challenge (p<0.01, Table 3).

MFS supplementation numerically reduced the necrotic mortality rate in the MFS group when compared to the POS group during the post-challenge period (day 17-28), despite the NE outbreak being much more intense than that in Trial 1 (Table 4). Additionally, lesion scores were higher in Trial 2 for challenged birds than in Trial 1 due to the intensity of the NE outbreak. There were no significant differences in lesion scoring between the POS and MFS groups in Trial 2 but the MFS lesion scores were numerically lower than the POS group on both sampling days (Table 5).

In both the POS and MFS groups of Trial 2, mortality due to NE was approximately 18%, suggesting that the outbreak of NE was much more intense in Trial 2 than in Trial 1 which had necrotic mortality rates of approximately 10 and 3% for POS and MFS, respectively (Table 1 and 3). While many factors may influence the severity of a NE outbreak, the likely source of this severity difference between trials is the C. perfringens inoculum used for the challenge^37,38. The inoculum used in Trial 1 was at a concentration of 7.7×10⁷ CFU mL^–1, while the concentration of the inoculum used for Trial 2 was approximately seven times higher at 5.6×10⁸ CFU mL^–1. As C. perfringens infection is a major factor in the development of NE, it is very likely that the increased concentration of the inoculum was a driving factor in the intensity of the outbreak in Trial 2^2,26.

The NE mortality reductions seen in this study may be due to a combination of several possible modes of action of the supplemented strains. Supplementation with live bacterial strains has been previously shown to affect both hosts and their microbiomes^39-41. The possible avenues the supplemented microorganisms took to ameliorate NE may include enhancing host immunity^42-45, improving intestinal barrier function^27,46,47 and promoting a better intestinal balance of beneficial microorganisms^39,48,49. In birds with a balanced microbiome, the microbiome contributes to protecting the bird from potentially pathogenic organisms^50,51. Bacterial supplementation to both healthy and challenged intestinal microbiomes has been associated with competitive exclusion, reducing the likelihood of a pathogenic bacteria attaching to the epithelial cells^52,53. These supplemented strains are also acting antagonistically with pathogens, in addition to competing for resources and space, by producing inhibitory metabolites, such as organic acids, to reduce the pathogen load in the gastrointestinal tract and prevent dysbiosis by promoting a more balanced gut microbiome^34,37,54. The microorganisms used in this trial (H. xylanolytica ASUCSBR21 and C. beijerinckii ASCUSBR67) were isolated from the gastrointestinal tracts of healthy broiler chickens that were more resilient to NE, so it is plausible that these organisms are contributing to these positive effects.

Based on these findings, it appears that the MFS had a rapid and impactful effect on NE immediately following the C. perfringens challenge. The reduced presence of lesions, in addition to the lower NE mortality rate, strengthens the support for the effectiveness of the bacterial strains in the MFS in reducing the severity of necrotic enteritis infection.

Performance: No significant effects of the MFS on body weight gain, feed intake per bird, or adjusted FCR were observed during Trial 1 (Table 1 and 2). However, adjusted FCR was numerically lower in every time period for the MFS group (Table 6). In Trial 2, the MFS supplemented group displayed an FCR decrease of 4 points when compared to the POS group across the whole trial period (p = 0.01, Table 3). In both trials, improvements in the performance of the MFS group began to increase toward the end of the trial. Additional studies utilizing a variety of challenge models, such as diet and nutrition-based challenges, may yield further insights into the efficacy and consistency of the results seen in the trials presented in this study. A future study with increased replicates and an extended trial period may elucidate these findings.