Date of Award

8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Food Technology

Committee Chair/Advisor

Xiuping Jiang

Committee Member

Annel K. Greene

Committee Member

E. Jeffery Rhodehamel

Committee Member

William C. Bridges Jr.

Abstract

Necrotic enteritis (NE), a severe enteric disease in poultry caused by the anaerobic bacterium Clostridium perfringens (Cp), leads to major economic losses worldwide due to reduced growth performance and increased mortality rates in broiler chickens. Damage to the intestinal epithelium caused by coccidia (Eimeria spp.), a protozoan parasite, is a major predisposing factor in NE, facilitating rapid proliferation and toxin production by Cp. Traditionally, antibiotics have been used to effectively control and manage coccidiosis and NE in poultry. However, due to factors like development of antibiotic resistance, government bans, and rise in consumer demand for antibiotic-free poultry products, there is a shift in the industry to reduce reliance on antibiotic growth promoters and investigate natural products as alternatives. The goal of this research was to explore the potential of natural feed additives such as Nigella sativa (black cumin), kefir, and bacteriophage alone and in combination for alleviating NE through both in vitro and in vivo studies in broiler chickens.

Initially, supplementation of black cumin seed oil (BCSO) in poultry feed to combat coccidiosis and reduce NE in broilers was investigated. A commercially available BCSO oil product, selected for its high anti-Cp activity from a preliminary study was used in the animal trials and GC-MS analysis of the product revealed the presence of bioactive compounds p-cymene, thymoquinone, carvacrol, and thymol. Conducted in two phases, trial 1 evaluated three concentrations (1, 2, and 5 mL/kg) and second trial focused on two concentrations (2 and 5 mL/kg) of BCSO. Each in vivo study consisted of Cobb 500 male broiler chicks, with 384 in the first trial and 320 in the second, distributed in a randomized complete block experimental design. The birds were challenged with E. maxima and Cp strain Cp#6 in trial 1 and Cp#4 in trial 2. During the trials, live performance, disease outcomes, and Cp populations were measured. In the first trial using the Cp#6 strain, BCSO concentrations of 2 and 5 mL/kg lowered NE lesion score and mortality rate to 1.6%, compared to 7.8% in the positive control, without any detrimental effect on growth performance. In the second trial with the Cp#4 strain, BCSO decreased NE lesion score and mortality rate to 35.9% from 51.6% in the positive control and also enhanced weight gain during Cp infection in broilers. Additionally, the population of vegetative cells and spores of Cp in cecal samples decreased for all treatments in trial 2. This study underscores the potential of BCSO at 2 and 5 mL/kg in enhancing broiler live performance and reducing NE symptoms caused by two different Cp strains with varying severity, positioning BCSO as a promising natural alternative to traditional antimicrobial feed additives.

Next, to understand the genomic characteristics that contribute to the virulence and antibiotic resistance of Cp strains, whole genome sequencing of Cp#4 and Cp#6 was performed. The sequencing results revealed that Cp#4 genome was 3,771,271 base pairs in length with a GC content of 27.82%, while the Cp#6 genome was 3,469,973 base pairs in length with a GC content of 28.01%, containing 252 and 253 subsystems, respectively. Comparative genomic analysis indicated that Cp#4 is both alpha-toxin and NetB toxin positive, suggesting a more robust virulence factor profile compared to Cp#6, which lacks the NetB toxin i.e. crucial for NE development. Both strains were found to carry additional virulence genes such as alpha-clostripain, collagenase, and perfringolysin O. Furthermore, each strain harbored 8 common antibiotic resistance genes, including those for tetracycline resistance, and 8 phage elements. The genomic exploration of Cp strains Cp#4 and Cp#6 offered valuable insights into the genetic determination of virulence, pathogenicity, and antibiotic resistance, underling the importance to develop a more targeted therapeutic or nontherapeutic approach to combat NE. Due to the more virulent nature of Cp#4, the subsequent animal trials used this strain for inducing NE in chickens.

Due to potential problems such as oxidation and cost effectiveness in using BCSO, the efficacy of black cumin seeds (BCS) and a natural probiotic kefir were assessed as antibiotic alternatives in broiler chickens. An in vivo study was conducted with 384 Cobb 500 male broilers administered with 5% BCS in feed and 20% kefir in drinking water, separately and in combination, as treatments. NE was induced in broilers using E. maxima and Cp (Cp#4) challenges. The results from this study showed that treatments of BCS, kefir, and their combination did not negatively affect broiler performance. BCS treated and the combination treatment had lower NE scores (P>0.05) in comparison to the positive control and exhibited no significant difference (P>0.05) from antibiotic control. Furthermore, there was no significant difference (P>0.05) in mortality rates between the treatment groups and the antibiotic control, with the combination of BCS and kefir lowering mortality to 14.1%, compared to 31.3% in the positive control. In addition, both spores and vegetative cells of Cp decreased for all treatments. The findings from this study suggest that both BCS and kefir proved to be successful in reducing Cp infection and mortality with the combination treatment being most efficacious.

The discovery of some intact BCS seeds in the gizzards and feces of chickens, indicating their incomplete digestion, prompted further evaluation of crushed Nigella sativa (black cumin) seeds (CBCS) and bacteriophage, both individually and in combination, as dietary supplements. Clostridium phage CU1 from the Myoviridae family, with a genome of 51,700 bp and 34.3% GC content and lacking virulence and antibiotic resistance genes, was isolated and characterized. The challenge study involved feeding 384 Cobb 500 male broilers with 5% CBCS in feed, 1:15 bacteriophage (109 PFU/mL) in drinking water from days 19 to 26, both individually and in combination, with E. maximaand Cp#4 challenges. The treatments with CBCS, bacteriophage, and their combination did not negatively impact the broilers' growth performance. Both the CBCS and the combination treatments significantly lowered NE scores (P

Furthermore, the impact of 5% CBCS, 1:15 phage CU1, and their combination on the cecal microbiota of chickens afflicted with NE was analyzed using 16S rRNA sequencing. The predominant bacterial phyla identified included Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and Bacteroidetes. While alpha diversity showed no significant changes on day 22 after Cp challenge across treatments, significant alterations in Shannon indices (P=0.05) were observed by day 28, although Chao richness remained unchanged (P>0.05). Beta diversity analysis revealed significant community variation in cecal microbiota profiles (PEubacterium on day 22, and Colidextribacter and DTU089 on day 28 in CBCS treatment group. For bacteriophage treatment, significant genera included Lactobacillus, Anaerostipes, DTU089, [Eubacterium]_nodatum_group, and [Clostridium]_innocuum_group on day 22, while Monoglobus was prominent on day 28. Whereas the combination treatment of CBCS and phage did not have any significant characterizing genera. These results highlight the potential of incorporation of black cumin seeds and bacteriophage alongside nutritional interventions in modifying gut microflora of cecum in chickens, thereby improving gastrointestinal health and alleviating NE.

In summary, natural products of Nigella sativa seeds and oil, kefir, and bacteriophage proved to be effective in mitigating necrotic enteritis in broiler chickens without adversely affecting growth performance. Also, CBCS proved to be more effective in alleviating disease outcomes compared to BCS. Moreover, the combined treatments of black cumin seeds and kefir and crushed back cumin seeds with bacteriophage showed the most promising outcome in reducing lesion scores and morality rates. Therefore, Nigella sativa, kefir, and bacteriophage present viable alternatives to antibiotics for disease prevention in the poultry industry, helping to combat antibiotic resistance.

Author ORCID Identifier

https://orcid.org/0000-0003-2549-3043

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