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ABSTRACT: Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

The rise in Salmonella resistance to cephalosporins and fluoroquinolones has become a significant threat to public health. At issue, is whether agricultural use of antimicrobials is selecting antibiotic resistance in Salmonella and the degree to which large antimicrobial resistance gene reservoirs, present in animal manures, contribute to this resistance. Two in vivo studies were performed to address these questions. In the first study, chickens were administered Salmonella and commensals, including an Escherichia coli strain with a mobile, ceftiofur-resistance plasmid, in order to determine how antibiotic administration impacted resistance in E. coli and Salmonella. All antibiotics administered to chickens increased streptomycin resistance in E. coli. However, only ceftiofur administration increased resistance in Salmonella and specifically to extended-spectrum β-lactams and cephalosporins (ESBL). There was no significant increase in ESBL-resistant Salmonella in chickens administered a ceftiofur-resistance plasmid donor. In the second study, chickens were administered two different isolates of S. enterica Typhimurium and a chicken resistome to serve as a gene donor. Birds were subsequently administered chlortetracycline or streptomycin. Antimicrobial administration significantly altered aminoglycoside and tetracycline resistance in the Enterobacteriaceae population. However, there was no significant increase in antimicrobial resistant Salmonella. Administration of a chicken resistome had no significant impact on prevalence of resistance in Enterobacteriaceae populations, including Salmonella. Evident, from both studies, was that these treatments had minimal effect on increasing the prevalence of resistance in Salmonella, suggesting that other factors may be more important in dissemination of antimicrobial resistant Salmonella in chickens.

The objective of this study was to determine the effects of protected sodium butyrate (SB), and protected sodium butyrate plus essential oils (carvacrol and ginger; SBEO) on the cecal microbiota of broilers challenged with Eimeria maxima and Clostridium perfringens. Birds were assigned to 4 treatments (8 replicates pens of 58 birds/pen): unchallenged control; challenged control; challenged and supplemented with SB; challenged and supplemented with SBEO. On d 13, challenged birds were orally inoculated with ~5,000 Eimeria maxima oocysts. On d 18–19, the same birds were exposed to Clostridium perfringens via drinking water (~8 log CFU/ml). Cecal excreta was collected at d 12, 18, 21, and 28 for microbiota analysis through 16s rRNA sequencing using Illumina MiSeq platform and analyzed using QIIME v. 1.9.1 The cecal microbiota was analyzed over time within each experimental group. The inclusion of SB alone or in combination with EO contributed to larger variations in the cecal microbiota over time than the unsupplemented treatments, as shown by the diversity indices. The community structure and abundance of the cecal microbiota were significantly different across ages, especially in the groups supplemented with SB and SBEO. As shown in the PCoA analysis, the supplementation of SB led to a more stable microbial community and lower between-sample variability over time. In the unchallenged control birds, Ruminococcus decreased (p = 0.006), whereas Bacteroides and Clostridiales increased (p ≤ 0.02) as the birds aged. In the challenged control group, however, the frequency of Coprococcus and Blautia decreased as birds aged (p ≤ 0.01), and, Clostridiales did not increase. Supplementation of SB, but not SBEO, increased the frequency of Lactobacillus (p = 0.01) on d 12 compared to d 18 and d 28, and prevented the reduction in the frequency of Blautia as the birds aged. Nevertheless, supplementation of SB and SBEO contributed to unique changes in the predicted functions of the cecal microbiota over time, which was not observed in the unsupplemented birds. SB and SBEO modulated the diversity, composition, and predictive function of the cecal microbiota which may have lowered the negative impact of necrotic enteritis (NE).