Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (Gallus gallus domesticus) during Downstream Poultry Processing

Antibiotic growth promoters that have been historically employed to control pathogens and increase the rate of animal development for human consumption are currently banned in many countries. Probiotics have been proposed as an alternative to control pathogenic bacteria. Traditional culture methods typically used to monitor probiotic effects on pathogens possess significant limitations such as a lack in sensitivity to detect fastidious and non-culturable bacteria, and are both time consuming and costly. Here, we tested next generation pyrosequencing technology as a streamline and economical method to monitor the effects of a probiotic on microbial communities in juvenile poultry (Gallus gallus domesticus) after exposure to several microbiological challenges and litter conditions. Seven days and repeated again at 39 days following hatching, chicks were challenged with either Salmonella enterica serovar Enteritidis, Campylobacter jejuni, or no bacteria in the presence of, or without a probiotic (i.e., Bacillus subtilis) added to the feed. Three days following each of two challenges (i.e., days 10 and 42, respectively) the microbiome distributions of the poultry caecum were characterized based on 16S rDNA analysis. Generated PCR products were analyzed by automated identification of the samples after pooling, multiplexing and sequencing. A bioinformatics pipeline was then employed to identify microbial distributions at the phylum and genus level for the treatments. In conclusion, our results demonstrated that pyrosequencing technology is a rapid, efficient and cost-effective method to monitor the effects of probiotics on the microbiome of poultry propagated in an agricultural setting

Genetic Diversity in Camptotheca Decaisne

RAPD markers were used to reveal the genetic diversity of the genus Camptotheca. Three primers (OPA02, OPA03, and OPA04), generating 44 polymorphic bands using randomly amplified polymorphic DNA (RAPD) markers, were able to discriminate among 25 Camptotheca populations. The band size varied from 268-4,411bp, with an average of 15 bands/primer. Of these populations, cultivars ‘Katie’ (KT) and ‘Ang’ (AG) of C. lowreyana and cultivated A1a (HJ) of C. acuminata can be easily distinguished by their unique bands. Population differentiation of Camptotheca was found to be higher than in other species with similar breeding systems. Cluster analysis of the genetic distance values and dendrogram from RAPD markers were consistent with the phenotypic data and both support the current taxonomic treatment of Camptotheca. Camptotheca acuminata var. acuminata appeared as the closest relative of C. yunnanensis, followed at some distance by C. lowreyana and, further away, C. acuminata var. tenuifolia

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme

Effects of Storage Conditions on the Morphology and Titer of Lentiviral Vectors

Lentiviral vectors are commonly used in laboratory experiments to stably integrate transgenes into host genomes. It has long been observed that storage of virus stocks leads to a decrease in viral titer, but the mechanisms driving this decrease have yet to be identified. To that end, lentiviral vector stocks were generated and stored as follows: room temperature for less than one hour, -80ºC for 24 hours, 4ºC for three days and 4ºC for 7 days. These stocks were subsequently evaluated with regard to their transducing ability and their morphology, specifically particle diameter. The vector that was stored at room temperature served as the control with viral morphology similar to other VSV-G pseudotyped viruses. These stocks were able to transduce ~100% of HEK 293T cells. Particles were unstable under the storage conditions tested, as evidenced by the fact that all stocks stored at -80°C and 4°C required concentration with an ultracentrifuge to generate a preparation suitable for visualization with TEM. The vector stored at -80°C for 24 hours exhibited some morphological changes, but only a slight decrease in titer. The morphology of vectors stored at 4°C for 3 and 7 days was not significantly different from the room temperature control, although titer was reduced to 60% and 30-40%, respectively. Thus, the decrease in titer observed in the lentiviral stocks generated and stored during this investigation appears to be the result of viral particle instability rather than morphological changes to individual particles

Enzymes as Feed Additive to Aid in Responses Against Eimeria Species in Coccidia-Vaccinated Broilers Fed Corn-Soybean Meal Diets with Different Protein Levels

This research aimed to evaluate the effects of adding a combination of exogenous enzymes to starter diets varying in protein content and fed to broilers vaccinated at day of hatch with live oocysts and then challenged with mixed Eimeria spp. Five hundred four 1-d-old male Cobb-500 chickens were distributed in 72 cages. The design consisted of 12 treatments. Three anticoccidial control programs [ionophore (IO), coccidian vaccine (COV), and coccidia-vaccine + enzymes (COV + EC)] were evaluated under 3 CP levels (19, 21, and 23%), and 3 unmedicated-uninfected (UU) negative controls were included for each one of the protein levels. All chickens except those in unmedicated-uninfected negative controls were infected at 17 d of age with a mixed oral inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Live performance, lesion scores, oocyst counts, and samples for gut microflora profiles were evaluated 7 d postinfection. Ileal digestibility of amino acids (IDAA) was determined 8 d postinfection. Microbial communities (MC) were analyzed by G + C%, microbial numbers were counted by flow cytometry, and IgA concentrations were measured by ELISA. The lowest CP diets had poorer (P ≤ 0.001) BW gain and feed conversion ratio in the preinfection period. Coccidia-vaccinated broilers had lower performance than the ones fed ionophore diets during pre- and postchallenge periods. Intestinal lesion scores were affected (P ≤ 0.05) by anticoccidial control programs, but responses changed according to gut section. Feed additives or vaccination had no effect (P ≥ 0.05) on IDAA, and diets with 23% CP had the lowest (P ≤ 0.001) IDAA. Coccidial infection had no effect on MC numbers in the ileum but reduced MC numbers in ceca and suppressed ileal IgA production. The COV + EC treatment modulated MC during mixed coccidiosis infection but did not significantly improve chicken performance. Results indicated that feed enzymes may be used to modulate the gut microflora of cocci-vaccinated broiler chickens

The Addition of Arachidin 1 or Arachidin 3 to Human Rotavirus-infected Cells Inhibits Viral Replication and Alters the Apoptotic Cell Death Pathway

Rotavirus (RV) infections are a leading cause of severe gastroenteritis in infants and children under the age of five. There are two vaccines available in the United States and one in India that can be administered early in childhood, however they only protect against specific strains1. From our previous work, both arachidin-1 (A1) and arachidin-3 (A3) from peanut (Arachis hypogaea) hairy root cultures significantly inhibit simian RV replication2,3,4. The purpose of this study was to determine if a human intestinal cell line, HT29.f8, infected with a human RV, Wa, was affected by A1 and A3. Cell viability assays were utilized to determine if A1 and A3 affect the HT29.f8 cells with/without RV infections. At eighteen hours post infection (hpi), supernatants from the RV-infected HT29.f8 cells with/without the arachidins were used in plaque forming assays to quantify and compare the amount of infectious RV particles that are produced during an infection. Transmission electron microscopy (TEM) was used to visualize cell ultrastructure and individual RV particles. Additionally, tunable resistive pulse sensing technology (TRPS) using the qNano system by IZON was employed to quantify and measure virus particle sizes, and display the size distribution of RV particles. Likewise, quantitative real time polymerase chain reactions (qRT-PCR) were performed to determine if A1 and A3 regulated cell death pathways in the HT29.f8 cell line. This data will guide our future studies to determine the antiviral mechanism(s) of action of A1 and A3

Arachidin-1 and Arachidin-3 Modulation of Rotavirus-infected MA104 Cells

Rotavirus (RV) causes severe life-threatening diarrhea in young children and immunocompromised individuals. There are several licensed attenuated vaccines for young children, but there are no vaccines or antiviral therapeutics for immunocompromised patients of any age. Previously, our laboratory demonstrated that arachidin 1 (A1) and arachidin 3 (A3) decreases the number of infectious simian RV particles and RV non-structural protein 4 (NSP4) in a human intestinal cell line which suggests effects on RV replication. This study examined the effects of the arachidins on the human RV (Wa)-infected African green monkey kidney cell line, MA104. The addition of either A1 or A3 did not decrease the viability of MA104 cells, however plaque forming assays measured significant decreases in the number of infectious RV particles with the addition of the arachidins. Correspondingly, western blot analyses revealed a change in the presence of VP6 and NSP4 (structural and nonstructural RV proteins, respectively). This implies that like the simian RV, Wa replication is also affected by both A1 and A3. Additionally, tunable resistive pulse sensing technology (TRPS) measured changes in the population distribution of released nanoparticles between 60-140 nanometers. Additionally, TEM morphometric analyses showed ultrastructural changed in RV-infected cells treated with A1 or A3. This included nucleus to cytoplasm ratios that were determined by TEM and whole cell fluorescent assays that disclosed significant nuclear size alterations with the addition of RV which implied modifications of the apoptosis and autophagy pathways. Moreover, the increased presence of autophagic vesicles seen with RV+A1 reinforced the model of a switch from the apoptosis to the autophagy pathway. In addition, immunoblot assays reveal the presence of cannabinoid 1 and 2 receptors on MA104 cells. These receptors bind A1 and A3 and are important in signaling in the endocannabinoid system. This implies a role for Witcher et al.: Arachidin-1 and Arachidin-3 Modulation of Rotavirus-infected MA10 2 A1 and A3 in modulating cannabinoid receptor cell signaling in RV-infected cells which indicates a mechanism of action of A1 and A3 with potential RV therapeutic activity

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme

A Time Course Study of Rotavirus-Infected Intestinal Cells Treated with Stilbenoids and the Regulation of Apoptosis

This is a time course study of virus –host interactions that are modified with the addition of two small natural products. They appear to effect virus replication and the host response to the infection