Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
The avian pathogenic E. coli (APEC) is one of the major bacterial pathogens of significant concerns to the US and global poultry industry. APEC causes wide range of localized and systemic infections in poultry, including yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis and salphingitis; collectively referred as colibacillosis. Colibacillosis culminates in multi-million dollars annual losses to all the facets of poultry industry and remains as a serious impediment to the sustainable poultry production worldwide. Current control methods using antibiotic medication and vaccination have limitation as APEC is resistance to multiple antibiotics and the available vaccines fail to confer protection against diverse and heterologous APEC serotypes. Therefore, developing new and potent anti-APEC therapeutics as an alternative to current control measures are urgently needed. We previously identified probiotic-derived antimicrobial peptides (P-1 and P-2) that in small pilot studies reduced the colonization of APEC in cecum and internal organs of chickens. These peptides are heat and proteolysis resistant, effective against diverse APEC serotypes, antibiotic-resistant APEC strains and against biofilm-protected APEC. Further, these peptides function by disrupting outer membrane of APEC, a mechanism to which resistance is less likely to occur. Here, we plan to evaluate the efficacy, safety and applicability of identified anti-APEC peptides (P-1 and P-2) in drinking water of chickens and to elucidate their mechanism(s) of action. Our overarching goal is to develop these peptides as new anti-APEC therapeutics as an alternative to antibiotics. We expect that these peptides will provide a novel therapeutic solution to enhance the control of APEC infections in poultry, which will consequently promote the sustainable poultry production and benefit the public health.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Here, we plan to determine the therapeutic doses of peptides (P-1 and P-2) for drinking water delivery in chickens followed by testing the efficacy and safety of peptides in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. We will further elucidate and/or validate the mechanism(s) of action (MOA) of peptides by measuring direct drug-target binding using biophysical methods as well as employing affinity-based pulldown and thermal proteome profiling (TPP) approaches. Our overall aim is to evaluate the efficacy, safety and applicability of identified anti-APEC peptides (P-1 and P-2) in drinking water of chickens and to elucidate/validate their mechanism(s) of action. This will be accomplished via conducting the studies described in the following objectives:Objective 1:Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water of chickens and evaluate efficacy, safety and applicability in large number of chickens under conditions mimicking the field settings and/or natural APEC infections.?Objective 2: Elucidate/validate the mechanism(s) of action (MOAs) of peptides by measuring direct drug-target binding, affinity-based pulldown assay, and thermal proteome profiling.Our overarching goal is to develop these peptides as new anti-APEC therapeutics as an alternative to antibiotics. We expect that these peptides will provide a novel therapeutic solution to enhance the control of APEC infections in poultry, which will consequently promote the sustainable poultry production and benefit the public health.We will collaborate with Technology Commercialization Office (TCO),UIUCand to advance these peptides into commercial products and seek FDA approval.
Project Methods
Objective 1.1: Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water.In this objective, the therapeutic dose of peptides (P-1 and P-2) will be determined in drinking water of chickens. Chickens (N=80) will be divided into 8 groups (n=10/group) Briefly, on day 2, chickens will be challenged orally with rifampicin-resistant (Rifr) APEC O78 (1-2×109CFU/chicken). Peptides will be administered at three different doses (50 mg/L, 100 mg/L and 200 mg/L) in drinking water for 5 days starting one day after APEC challenge (day 3 to day 7),. The clinical signs and mortality will be monitored until 7 days post-infection (dpi; from day 3 to day 9). Chickens that die during this period will be necropsied on the same day, lesions in internal organs (liver, heart, lung, and air-sacs) will be scored, and APEC load will be quantified in cecum and internal organs (liver, heart, lung, and kidney) by plating on MacConkey agar plates supplemented with 50 μg/mL rifampicin. At day 9 (7 dpi), all live birds will be necropsied, lesions will be scored, and the APEC load will be quantified in cecum and internal organs.Objective 1.2: Evaluate efficacy, safety and applicability of peptides.A) Efficacy assessment: The efficacy of peptides (P-1 and P-2) will be evaluated under field simulated conditions using the most effective dose identified from obj 1.1. A total of 360 one-day-old broiler chickens will be raised on built-up floor litter in six different groups (n=60/group). The chickens will be infected, and peptides will be administered in drinking water. To compare the efficacy with currently used antibiotic, sulfadimethoxine will be administered for 5 days following manufacturer's recommendation (0.05%). The efficacy of the combination of peptides (1:1) will be also assessed. Clinical signs and mortality will be monitored until day 42, chickens will be necropsied at day 9 (7 dpi; n=30 chickens/group; short-term efficacy), 12 (5 days post-treatment of peptides for residue analysis; n=5 chickens/group), and 42 (40 dpi; slaughter age; n=25 chickens/group; long- term efficacy) and internal organs will be collected for quantification of APEC load. The APEC lesions will be scored.B) Safety assessment:I) Impact on performance parameters: The body weight gain (BWG) and feed conversion ratio (FCR) are the key indices of safety of drugs in chickens. The body weight will be measured weekly [day 1, 7, 14, 21, 28, 35, and 42] and feed intake will be recorded daily in order to determine weekly and total BWG and FCR.II) Evaluation of resistance to peptides: APEC colonies isolated from different organs of peptides treated chickens at day 9 or day 42 (n=20 isolates/group, 5 isolates from each organ) will be used for testing whether APEC acquire resistance to peptides.III) Impact on gut microbiome: The effect of peptides on the gut microbiome will be analyzed using 16S rRNA V4-V5 sequencing using illumina platform. Briefly, DNA will be extracted from the collected cecal samples (10 samples/group) and sequenced at the MCIC, OSU.IV) Quantitation of peptides residues in chicken's tissues: We will quantify the peptidesresidues in muscle, liver, and kidney at day 9 (2 days post-treatment) and day 12 (5 days post-treatment) to determine the drug withdrawal period and day 42 to confirm the absence of peptide's residues. Liver (n=5/group), muscle (n=5/group), and kidney (n=5/group) will be collected from peptides treated groups. All the tissues will be snap-frozen, and shipped to MS&P facility at OSU to quantify the peptide's residues in the tissues using HPLC-MS triple quadrupole (QQQ) Quantiva. Code of Federal Regulations (CFR) Title 21 #21CFR556) will provide the basis for the peptide residue limits.C) Pharmacokinetics (PK) and stability assessment of peptides:For PK study, blood (2 mL, n=5 chickens each time point) will be collected at 0, 1, 2, 4, 8, 12, 16 and 24 h post administration of peptides. Peptides' concentrations in plasma will be quantitated using HPLC-MS triple QQQ Quantiva. For the stability study, samples (2 mL, n=3) of drinking water will be collected at 0, 4, 8, 12, 16, 20 and 24 h post administration of peptides. The concentrations of the peptides in drinking water will be quantitated using HPLC-MS triple QQQ Quantiva.Objective 2: Elucidate the mechanism(s) of action (MOAs) of peptides.A) Drug-target binding validation using biophysical methods: The mlaA and ompC genes will be cloned into pET-21b vector and expression of MlaAHisand OmpCHiswill be achieved by addition of IPTG in PlysS. The OM proteins will be purified using Ni-affinity chromatography. and further purified by gel filtration using Superdex 200, and confirmed by immunoblot using anti-MlaA and -OmpC antibodies. The binding affinities of peptides with MlaAHisand OmpCHiswill be determined by surface plasmon resonance (SPR) using NiHC1000 sensor chip with a BIAcore T100 instrument.B) Additional Target identification:I) Affinity-based pulldown assay:Biotin probes will be synthesized linking non-essential amino acid residues of these peptides. Biotinylated peptides will be loaded onto streptavidin column and incubated to allow binding with streptavidin. The APEC O78 whole-cell lysate, will be added to the columns and incubated to allow binding with biotinylated peptides. The binding lysate will be eluted using elution buffer. The eluted protein(s) will be then examined using SDS-PAGE and quantified using HPLC-MS/MS. The proteins will be identified using Mascot in Proteome Discoverer 2.2 and E. coli Uniprot database will be used with false discovery rate of below 1%. The protein(s) highly abundant in the eluted buffer compared to the control will be considered as potential targets.II) Thermal proteome profiling (TPP):Logarithmic phase APEC O78 will be treated with two different concentrations (high: 4X, low: 1X of MBC) of peptides for 20 minutes followed by thermal treatment of bacterial cultures at 11 different temperatures (42°C to 72°C with 3°C successive increments). APEC unexposed to peptide and non-heat treated (room temperature, 25°C) will be used as controls. The soluble/non-denatured proteins will be extracted and the quantitative proteomics will be performed using Bruker maXis electrospray ionization (ESI) quadrupole LC-MS/MS coupled with 3000 RSLCnano system. The proteins will be identified using Mascot in Proteome Discoverer 2.2 and E. coli Uniprot database will be used with false discovery rate of below 1%.Data analysis:The statistical significance of reduction in mortality, APEC lesions and APEC load in the peptide treated groups will be determined by one-way ANOVA followed by post-hoc Tukey's test (P<0.05). The comparison of mortality, APEC lesions, APEC load, BWG and FCR between peptide and antibiotic treated groups will be made by Kruskal-Wallis test (P<0.05).For the microbiota analysis, QIIME 2 bioinformatics platform will be used. The taxonomic analysis will be performed using Naive Bayes classifiers trained on Silva 132 99% OTUs (silva-132-99-nb-classifier.qza) database. The phylogenetic diversity will be analyzed using align-to-tree-mafft-fasttree pipeline and alpha (Shannon's diversity index) and beta diversity (Bray-Curtis distance) will be analyzed using core-metrics-phylogenetic pipeline. The statistical comparison (P<0.05) of the taxonomic composition between untreated and peptide and antibiotic treated groups will be made using Kruskal-Wallis test. The alpha and beta diversity will be analyzed using Kruskal-Wallis and PERMANOVA tests (P<0.05), respectively.For proteomic analysis from TPP studies, TPP package using R pipeline in Bioconductor software will be used. The proteins significantly (P<0.05) altered in their abundance across the thermal treatments will be identified through built-in statistical analysis in the TPP package.