Brief Summary of Minutes of Annual Meeting

Objective 1. Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals.

 A. Campylobacter jejuni

Iowa

Pathogenesis of Campylobacter jejuni clone SA. We identified a highly virulent, tetracycline-resistant C. jejuni clone (named clone SA) that has recently emerged in ruminant reservoirs and become the predominant cause of sheep abortion in the United States. To understand the virulent mechanisms of clone SA, we took advantage of natural competence of C. jejuni, positive selection in animal model, and next generation sequencing to directly identify the genetic determinant causing abortion. We proved that sequence polymorphisms in the outer membrane protein are both necessary and sufficient for inducing abortion in pregnant animals. These findings identify a key virulence determinant responsible for abortion induction by Campylobacter and illustrate the powerfulness and necessity of combining experimental evolution with genomic approaches for understanding bacterial pathogenesis.

 B. Salmonella

Washington State

Antimicrobial resistance profiling of > 600 isolates belonging to twelve most prevalent poultry associated Salmonella serotypes (MPPSTs) recovered from the US poultry sources. Most of the isolates were pan-susceptible (52%) or displayed resistance to 1 (24.5%) or 2 (20.5%) antibiotic classes, whereas few isolates (3%) were resistant to ≥3 antibiotic classes. Multi-drug resistance (≥3 antibiotic classes) including resistance to third generation cephalosporin (2.4%) was primarily limited to Heidelberg and Kentucky. All MPPSTs except Kentucky were recently identified by CDC among the top 30 clinically relevant serotypes isolated from human illnesses in the US. The prevalence of Enteritidis (R2 = 0.650) and Heidelberg (R2 = 0.899) significantly (P < 0.01) correlated with the annual incidence of human illness due to these serotypes. 

We assessed the antimicrobial activity of chlorine against twelve MPPSTs using a chicken-meat-based model to simulate the processing environment within the immersion chilling tank. We observed that the level of chicken meat extract contamination is an important contributing factor for survival of Salmonella against chlorine treatment. 

Genetic characterization of S. Kentucky isolates recovered from the US poultry (n=140) and human (n=29). Based on PFGE patterns, two major clusters (A and B) were identified at a similarity level of 50%. Our data suggests that the clinical isolates within cluster B are likely of domestic origin and clinical isolates within cluster A are likely of international origin and indicate a possible international spread of multi-drug resistant S. Kentucky.

C. Shiga toxin-producing E. coli (STEC)

Nebraska

Comparison of enrichment broths for supporting growth of Shiga toxin-producing Escherichia coli. We observed that STEC grew significantly better when enriched in EC (E. coli broth) compared to TSB. Modification of TSB by the addition of bile salts improved the growth and detection of STEC compared to TSB alone.

Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and pre-intervention carcass surfaces of feedlot cattle at harvest. This study indicates that “top 6” and O157:H7 EHEC are present on hides, and to a lesser extent, pre-intervention carcasses of feedlot cattle at harvest. However, continued improvement in non-O157 detection methods is needed for accurate estimation of prevalence given the discordant results across protocols.

Prevalence and concentration of enterohemorrhagic Escherichia coli in culled dairy cows at harvest.   Matched samples (n = 300) were collected from 100 cows at harvest and tested by a culture-based method and 2 molecular-based methods, NeoSEEK STEC (NS), and Atlas STEC EG2 Combo (Atlas). This study provides evidence that non-O157 EHEC are ubiquitous on hides of culled dairy cattle, and feces are an important source of non-O157 EHEC hide contamination.

Hemorrhagic colitis associated with enterohemorrhagic Escherichia coli O165:H25 infection in a yearling feedlot heifer. A 1-year-old feedlot beef heifer was moribund with neurological signs and bloody diarrhea.   An E. coli O165:H25 strain was isolated from the colonic mucosal tissue, and by microarray analysis contained diverse typical virulence genes. To our knowledge: this is the first report of disease in cattle associated with EHEC O165:H25 infection; the oldest bovine EHEC disease case with isolation of the pathogen; and the first bovine case to demonstrate grossly-evident, hemorrhagic, colonic mucosal erosions associated with EHEC infection.

Kansas

Seasonal aspects of prevalence of non-O157 STEC in feces of feedlot cattle. We conducted a study to determine the prevalence of seven STEC-7 and their virulence genes in feces of commercial feedlot cattle during summer and winter months. We did identify seasonal differences and fecal shedding was highly variable between pens of cattle, which needs to be explored further.

Regional, feedlot, and pen-level variability in prevalence of STEC. We conducted a study looking at regional, feedlot, and pen-level variability in prevalence of non-O157 STEC in feces of feedlot cattle. The pre-harvest fecal prevalence estimates, and their distribution across pens, feedlots, and states obtained in this study provide necessary data to integrate into the quantitative microbial risk assessment.

Quantification of Microbial Transfer from Hides to Carcasses during Commercial Beef Slaughter Operations. We are collecting hide and carcass samples from each of 4 large commercial slaughter plants in the north and south regions. We are evaluating matched samples through the beef slaughter process at five sampling points: 1) hides (hide-on) on the kill floor; 2) pre-wash carcass; 3) post-wash/pre-evisceration carcass; 4) post-evisceration carcass; 5) post-evisceration/post-final intervention carcass.

Prevalence and concentration of STEC-7 on hides of fed and culled cattle harvested within the same plant and time. Our group and others have generated limited data on STEC prevalence and concentration for each of these cattle types. However, there is a challenge when synthesizing data/results from multiple independent studies that may differ in several important factors (e.g., time/season, geography, and diagnostic methods) other than simply cattle type. We have established a collaboration to address this issue.

Basic Reproduction Number and Transmission Dynamics of Shiga toxin-producing E. coli. We developed a Markov chain model to simulate the dynamics of 7 serogroups of Shiga toxin-producing Escherichia coli (O26, O45, O103, O111, O121, O145, and O157) in cattle production environments based on a set of cross-sectional data in two years in two states. We demonstrated that different detection sensitivity levels of the pathogen change the dynamics of prevalence, thus better detection techniques and estimates of their performance are critical for risk assessment and future studies.

Role of stable flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) in the confined cattle environment. Stable flies were collected over three summer months and processed individually for STEC. Our data indicated that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.

Role of house flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) in the confined cattle environment. The results from this study indicate that house flies likely play a role as vectors and/or reservoirs of non-O157 STEC in confined cattle environments.

D. Brachyspira hampsonii

Minnesota

Identify the molecular epidemiology of pathogenic Brachyspira species circulating in U.S. swine herds. We characterized isolates from diverse epidemiological sources using MLST and compared the genotypes from multiple sites and systems within the U.S. Further, we compared genotypes from the U.S. with those identified globally. Though the species is genetically diverse, with 5 genetic groups identified to date, it represents a single species, genetically closest to the commensal Brachyspira species.

 E. Coronavirus

Ohio

Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets versus 26-day-old weaned pigs. Our study confirmed that the US PEDV PC21A is highly enteropathogenic in conventional, seronegative nursing piglets. Our study was the first to explore differences in the the innate immune responses of nursing and weaned pigs to PEDV infection.

Isolation and characterization of porcine deltacoronavirus (PDCoV) from pigs with diarrhea in the United States. PDCoV OH-FD22 strain collected from intestinal contents of a diarrheic pig from Ohio was successfully isolated in ST and LLC-PK cell cultures. The results of this study are critical for the development of new serologic tests for PDCoV and expansion of our knowledge of the biology and epidemiology of PDCoV in swine.

Illinois

In our study, the role of IFN responses for PEDV infections was examined. Our data suggest that type I IFNs seem to restrict PEDV, and in turn PEDV seems to code for IFN antagonizing proteins. There is a need to conduct such studies to understand the pathogenesis of PEDV, which will provides a new insights into the design of a new vaccine candidate for control of the disease.

Minnesota

Genomic and evolutionary inferences between American and global strains of PEDV. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n=126) to determine the most variable genomic areas. Our findings suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV.

Characterization and evolution of porcine deltacoronavirus in the United States. We investigated the presence of PDCoV in diagnostic samples, which were further categorized by case identification (ID), and the association between occurrence, age, specimen and location between March and September 2014. Approximately, 7% of the case IDs submitted from the US were positive for PDCoV. Our results indicate that oral fluids continue to be a valuable specimen to monitor swine herd health, and PDCoV has been circulating in the US prior to 2014.

Identification and complete genome of Seneca Valley Virus in vesicular fluid and sera of pigs affected with ediopathic vesicular cisease, Brazil. Numerous ongoing outbreaks in Brazilian swine herds have been characterized by vesicular lesions in sows and acute losses of neonatal piglets. The complete genome of Seneca Valley virus was identified in vesicular fluid and sera of sows, providing evidence of association between SVV and vesicular disease and viremia in affected animals.

South Dakota

We developed and validated new method for whole genome sequencing of PEDV directly from fecal samples. A total of 56 samples were tested using our method.

We also developed an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of PEDV. All assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.

F. Calicivirus

Ohio

The genetic mechanism of cell culture adaptation of an enteric calicivirus, porcine sapovirus (PoSaV) Cowden strain was investigated. By using the reverse genetics system, we identified that four (178, 289, 324, and 328) amino acid substitutions in VP1, but not the substitutions in the RdRp region, were critical for the cell culture adaptation of PoSaV Cowden strain. Our findings have revealed the molecular basis for PoSaV adaptation to cell culture.

Elucidation of the mechanisms of NoV (and infectious SaV) entry through the roots, their interaction with components of the vascular system, and their internal transport inside lettuce plants. Our preliminary results showed that following RNase treatment of the samples, HuNoV particles could not be detected in lettuce or spinach leaves when the virus was introduced through the petiole or the roots. In contrast, porcine SaV particles were detected under both settings.

Identification of the lettuce cell wall carbohydrate(s) involved in NoV binding and the inhibition of such binding by plant-derived lectins. Our results suggest that H type HBGA-like carbohydrates exist in lettuce tissues, and GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall materials.

G. Rotavirus

Minnesota

Widespread rotavirus H (RVH) in commercially raised pigs. In the United States, 30 positive RVH samples were detected form 10 different states. Sequencing analysis suggested RVH has been circulating in the United States swine population for at least one decade but probably longer.

Detection and high occurrence of porcine rotavirus A, B, and C by real time RT-PCR in diagnostic samples. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). Although RVA was detected at the highest overall percentage (62%), 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1–20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs.

 H. Antimicrobial Resistance

Iowa

Mutation and antibiotic resistance. Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. We performed experiments to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance. The mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni. The mutY point mutation also led to ∼ 700-fold increase in the emergence of ampicillin-resistant mutants. The G595 → T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter.

Function and regulation of multidrug transporters. The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. Among the 64 C. jejuni isolates examined in this study, We observed that overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. Differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

Rising fluoroquinolone resistance in Campylobacter of bovine origin. Here, we analyzed the antimicrobial susceptibilities of C. jejuni and C. coli obtained from 65 different feedlot cattle in different states with total 320 C. jejuni and 115 C. coli isolates. Our findings reveal the drastic increase in the prevalence of fluoroquinolone-resistant Campylobacter in feedlot cattle in the United States and highlight the need for enhanced effort to understand the ecology of antibiotic resistant Campylobacter in the ruminant reservoir.

Tennessee

Molecular basis of conjugation in Campylobacter jejuni. Conjugation is an important mechanism for horizontal gene transfer. Using a unique co-transformation strategy in conjunction with comparative genomics analysis, Cj1501c was identified as a key restriction-modification enzyme involved in high frequency conjugation in C. jejuni.

Development of bile salt hydrolase (BSH) inhibitors, novel alternative to antibiotic growth promoters.   Our previous studies suggested that BSH inhibitors are promising alternatives to AGP for enhanced animal growth performance and food safety. The in vivo efficacy of riboflavin, one BSH inhibitor identified from high-throughput screening, was evaluated in a chicken study. At 21 days of age, average body weight per bird in riboflavin-treated group is significantly higher than that in control group, supporting our hypothesis. The BSH from L. salivarius was successfully crystalized (1.8 Å resolution) and the motifs and amino acids critical for BSH activity were identified.

Ohio

Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania: Campylobacter occurred in 9.5% of the beef carcasses and 13.4% of the raw milk samples, respectively. Resistance to various antibiotics was observed. In addition, 46.3% isolates were classified as multi drug resistant Campylobacter.

Antimicrobial Resistance and Genotypic Diversity of Campylobacter Isolated from Pigs, Dairy, and Beef Cattle in Tanzania: Campylobacter occurred in 32.5, 35.4, and 19.6% of the pig, dairy, and beef cattle samples, respectively. Resistance to important antimicrobials and multi-drug resistance was also observed; with the highest resistance noted for ampicillin (75.7%) and the lowest for chloramphenicol (4.5%), MLST typing revealed seven novel sequence types (six C. jejuni and one C. coli).

Wyoming

We have developed MALDI-TOF MS protocols for identification and typing of antimicrobial resistant bacteria. Approximately 3,000 presumptive cephalosporin and fluoroquinolone resistant E. coli, macrolide resistant Enterococcus spp., and methicillin resistant Staphylococcus spp. isolates collected from 40 cattle facilities and associated wildlife were initially identified by MALDI-TOF MS.

Objective 2. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.

Campylobacter jejuni

Arizona

jejuni vaccine development. Recombinant attenuated Salmonella vaccines (RASV) are being utilized in industry compatible conditions for assessment of utility in production and refined to produce greater and more consistent reductions. Results indicate the potential for a reduced dose to maintain efficacy with regard to reduction of colonization by C. jejuni NCTC11168 following challenge. Studies investigating the efficacy of non-gavage-based, industry compatible delivery methods were also performed. A commercial spray cabinet was used to administer vaccine, as was addition of the vaccine to drinking water. Results of these studies indicate that these dosing methods may be viable options for effective delivery of this vaccine to poultry, though further development is required.

Tennessee

Development and evaluation of Campylobacter vaccine. Two large chicken trials were performed to immune response and protective efficacy of intranasal vaccination of prepared subunit vaccines.   Intranasal vaccination with encapsulated protein significantly elicited serum IgG and mucosal IgA response. However, the encapsulated DNA vaccine did not trigger significant immune response

Ohio

Control of broiler litter contamination with C. jejuni: Our approach evaluated the impact of different chemical treatment of litter with [aluminum sulfate (Alu), sodium bisulfate (Sob), and magnesium sulfate (Mgs)] and the combination of three chemicals (Alu+Sob+Mgs) on the C. jejuni survival. The litter treatment with combination of three compounds can significantly reduce the number of C. jejuni in ceca and in litter under specific experimental conditions. Metagenomics analysis also confirmed this finding.

The role of chemotaxis in C. jejuni’s pathobiology: Our findings highlighted important roles for chemotaxis proteins in the pathobiology of C. jejuni. Our study also revealed that certain chemotaxis proteins (Tlps) can serve as possible targets for controlling C. jejuni in hosts.

Transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants:   Our study highlighted a novel regulatory role for the Ppk enzymes, which advanced our understanding of the biology of this organism. Our findings further highlighted the importance of these enzymes as potential targets for anti-C. jejuni therapeutics

 B. STEC and ETEC

Nebraska

Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. Molecular and biochemical studies demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

Kansas

ETEC vaccinology. We used quantitative proteomics to identify 24 proteins that differed in abundance in membrane protein preparations derived from wild-type vs. a type II secretion system mutant of ETEC. A subset of these proteins and identified antigens were generated and evaluated in mice in terms of immunogenicity and protective efficacy. Immunization with either Skp or MipA provided an intermediate degree of protection, 68 and 64 %, respectively.

Role of lipid rafts in bacterial infection. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity.

Washington

We conducted an oral immunization trial of cattle with E. coli O157:H7, using a pool of three spontaneously Stx-negative E. coli from the EHEC1 lineage (beta-glucuronidase negative, sorbitol non-fermenting, eae positive, O157 antigen positive, fliC-H7 positive), administered in repeated frequent (2/wk for six weeks), high (10e9/dose) oral gavage challenges. Taking into account both O157 vaccine strains and challenge strains, the O157 immunized group did not exhibit reduced RAJ colonization compared to the sham-immunized group.

We are continuing to test the hypothesis that high summertime seasonal shedding of O157 results from introduction of new season forage crops. We are sampling pens of dairy and feedlot cattle just prior to and just after introduction of the new forage crops as well as transition feeding. Most of the data to date have focused on adult dairy cattle and shown relatively minor impact on STEC shedding including O157.

By comparing STEC shedding in adjacent pens with shared water troughs with shedding in adjacent pens lacking shared water troughs, the specific role of common water sources in STEC epidemiology was evaluated. The preliminary analysis, a general linear model of absolute STEC prevalence difference conditional on shared water through or shared fence line, found no effect of shared water through on absolute prevalence difference.

C. Salmonella

Washington

We completed virulence testing and metabolic profiling of a Salmonella pathogenicity island 13 (SPI-13) mutant of S. Enteritidis and demonstrated that SPI-13 contributes to (i) pathogenesis in streptomycin pre-treated mice but not in day-old chickens and (ii) the metabolic fitness of Salmonella Enteritidis through glucuronic acid and tyramine metabolism.

We tested the virulence of 90 wild-type Salmonella isolates belonging to twelve most prevalent poultry-associated Salmonella serotypes (MPPSTs) using avian macrophages as a surrogate model for intracellular survival. We found that there are significant inter- and intra-serotype differences in the invasion and survival of MPPSTs in avian macrophages and induction of nitric oxide. Most notably S. Schwarzengrund, a serotype associated with invasive Salmonella infection in human, showed higher invasion (3.35%) and higher intracellular survival (8.1%) whereas Kentucky, a serotype rarely associated with human disease, showed low invasion (1.7%) and low intracellular survival (1.15%) in avian macrophages.

D. Brachyspira

Minnesota

Identify the epidemiological associations of antibiograms of Brachyspira hyodysenteriae and “Brachyspira hampsonii” circulating in U.S. swine herds. General antimicrobial susceptibility trends for U.S. Brachyspira species showed high susceptibility to tiamulin, valnemulin, and carbadox and low susceptibility to tylosin and lincomycin. Very few U.S. B. hyodysenteriae strains showed low susceptibility to multiple drugs (little tiamulin resistance). The general patterns of antimicrobial susceptibility profiles varied by the species of Brachyspira, by type of site, and by system of origin (suggesting system-specific susceptibility).

Identify candidate virulence-associated genes of Brachyspira hyodysenteriae and “Brachyspira hampsonii”. To date, we have sequenced the genomes of 14 B. hyodysenteriae and 2 “B. hamsponii” isolates. Genomes of reference high and low virulence strains, one each, are available publically. After genome assembly (either de novo sequencing or by reference mapping), comparisons of high and low virulence B. hyodysenteriae strains have been performed using four different methods.

 E.  Calicivirus and Norovirus

Kansas

Entry of caliciviruses. We reported that bile acids facilitate virus escape from the endosomes into the cytoplasm for successful replication of porcine enteric calicivirus (PEC). This is through the ceramide formation by the activation of sphingomyelinase (ASM). Inhibition of ASM resulted in the retention of PEC, feline calicivirus, or murine norovirus in the endosomes in correlation with reduced viral replication, suggesting the importance of viral escape from the endosomes for the replication of various caliciviruses.

Norovirus antivirals. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of anti-norovirus therapeutics. We have reported the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrated in vivo efficacy of an inhibitor using the murine model of norovirus infection.

F. Rotavirus

Ohio

Broad-spectrum antibiotic and probiotic treatments: Effects on VirHRV disease severity and immune responses in Gn pigs colonized with/without the defined commensal microflora (DMF). We have demonstrated that in Gn pigs colonized with defined microflora, broad-spectrum antibiotic treatment resulted in increased HRV infection and disease severity, likely, altering innate and adaptive immune responses and bacterial composition. Additionally, we confirmed that EcN probiotic treatment moderated the above parameters, thus, ameliorating HRV disease.

Comparative in vivo and in vitro studies of porcine rotavirus G9P and human rotavirus Wa (G1P) in gnotobiotic pigs: The changing epidemiology of porcine and human group A rotaviruses (RV), including emerging G9 strains, may compromise the efficacy of current vaccines. Understanding the genetic features of the new emerging RV strains will contribute to development of new surveillance and safe and efficacious vaccines.

Wyoming

Develop improved methods to capture and detect viruses from bioaerosols. Modification of SKC BioSamplers for capture of viruses from bioaerosols improved detection sensitivity by 8.5x and 2x for the virus surrogates MS2 and Φ6 bacteriophages, respectively. The modifications to the SKC BioSampler are straight forward and add minimum cost, allowing for simplification of sampling procedures and addition of Sample buffer incorporated protectants.

G. Cryptosporidium parvum

Illinois

We have developed a battery of complementary in vitro and in vivo assays that allow us to quantitfy Cryptosporidium, Plasmoddium, and Toxoplasma microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity or growth of these parasites is inhibited by CpV and CDPK inhibitors.

H. Microbiome-Host Interactions

Ohio

Establishment of a human microbiota transplanted neonatal pig model for future research to assess interactions among enteric pathogens, diet and host factors (commensal bacteria and immunity). We have recently established a human microbiota transplanted neonatal Gn pig model. This model will allow increased knowledge of the interactions among the commensal microbiota, enteric pathogens, immunity and diet and to test future potential interventions to restore the affected immune function and microbial structure.

Minnesota

Characterizing the functional microbiome of the swine gut in response to treatment with growth promotor tylosin. The microbiome of animals treated with tylosin, an antimicrobial growth promoter, were analyzed with PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) to predict their metagenome and allow for metabolic analyses. The swine used in this study were housed in two independent commercial farms (10 swine per group) and were fed normal diet (Control) or diet containing tylosin (40 ppm). Large scale changes in function of gut microbiome were not observed between the two farms or tylosin treatment. The major functions found in the microbiome of animals were carbohydrate metabolism (10.6%), amino acid metabolism (9.4%), membrane transport (13.1%) and DNA replication and repair (9.4%). Other studies have also found that differences in gene function of a microbiome remain largely the same despite perturbation of microbial populations within the gut.

I. Clostridium difficile and Clostridium sordellii.

South Dakota

Genomic and phenomic characterization of Clostridium difficile and Clostridium sordellii. Despite strong similarities between the two Clostridial species, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues. To improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we performed a comparative genomic and phenomic analysis of the two. The proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies.

Objective 3. Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures.

The PIs and Graduate Students involved in the project have been continuing to give presentations and updates enteric diseases and food safety at various scientific, veterinary, and diagnostic meetings in the previous year. They have effectively disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians. They also have generated many high impact peer-reviewed journal articles. The members have actively organized various outreach/education activities, such as 1) Dr. Armstrong, as chair of the University of Arizona Food Safety Consortium, organized the 6th annual University of Arizona Food Safety Conference on September 18, 2015. The keynote address was provided by Bonnie Fernandez-Fenaroli, Executive Director for the Center for Produce Safety and outcomes from University of Arizona food safety research programs were presented. In attendance were members of the academic community, regulatory agencies and the food production and processing industries; and 2) Through several venue Kansas State have provided educational opportunities to professional and graduate students as well as continuing education to veterinarians on enteric pathogens in livestock production systems. The majority of the shared information has focused on the impacts of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter, and antimicrobial use/resistance in the beef industry, the conclusions that can be reached based on recent research, and the potential opportunities to reduce these pathogens in beef production systems.

Objective 4. Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings.

• NC-1202 members and their students presented their work in numerous national and international meetings. We held annual NC1202 meetings in Dec of 2015, and also sponsored 3 student awards for best oral and poster presentations at the Conference of Research Workers in Animal Disease meeting. 
• NC 1202 organized a Mini-Symposium focused on Microbiome and Animal Health on Dec 7, 2015. Seven keynote speakers were invited to present their research work. NC 1202 members discussed the theme for 2016 symposium and unanimously selected the topic of antimicrobial resistance.
• NC-1202 members have established active collaborations, partly reflected by joint grant/publications. Following are three active integrated food safety projects in which our NC1202 members are project directors and involve several NC1202 members at the participating institutions as co-project directors: 

1. Zhang, Q. (PD, Iowa State Univ.) and other Co-PDs who are NC1202 members: Jun Lin (Univ. of Tennessee), Gireesh Rajashekara (Ohio State Univ). USDA NIFA Food Safety Challenge Grant, Novel Approaches for Mitigation of Campylobacter in Poultry. $2,500,000. Award period: 07/01/2012 – 06/30/2017 
2. Law, B. (PD, Univ. of Arizona) and other Co-PIs who are NC1202 members: Roy Curtiss III is a scientific advisor, and Ken Roland from the Curtiss lab is the Co-PI. USDA NIFA Food Safety Challenge Grant, The Development of an Efficacious Vaccine to Reduce Campylobacter in Chickens. $2,500,000. Award period: 8/1/2012 – 7/31/2017.
3. Moxley, R. (PD, Univ. of Nebraska) and T.G. Nagaraja and David Renter (Co-PDs, Kansas State Univ.) USDA NIFA Coordinated Agricultural Program Award, Shiga-toxigenic Escherichia coli (STEC) in the Beef Chain: Assessing and Mitigating the Risk by Translational Science, Education and Outreach.  Total funding: $25,000,000 for 16 institutions, 51 collaborators. Award period: 1/1/2012 – 12/31/2016  

• Dr. Rajashekara led NC-1202 team for submission of a CAP project to NIFA Food Security Program in 2015, which was not awarded but received “High Priority” score.

Peer-reviewed journal articles

Wang, Yang; Dong, Yanni; Deng, Fengru; Liu, Dejun; Yao, Hong; Zhang, Qijing; Shen, Jianzhong; Liu, Zhihai; Gao, Yanan; Wu, Congming; Shen, Zhangqi. 2015. Species shift and multidrug resistance of Campylobacter from chicken and swine, China, 2008-2014. Journal of Antimicrobial Chemotherapy. In press.

Deng, Fengru, Jianzhong Shen, Maojun Zhang, Congming Wu, Qijing Zhang, and Yang Wang. 2015. Constitutive and inducible expression of the ribosomal RNA methylase gene erm(B) in Campylobacter. Antimicrob. Agents Chemother. 59(10):6661-4.

Qin, Shangshang, Hui Qi, Qijing Zhang, Di Zhao, Zhen-Zhen Liu, Hao Tian, Lijuan Xu, Hui Xu, Mengmeng Zhou, Xianju Feng, Hong-Min Liu. 2015. Emergence of extensively drug-resistant Proteus mirabilis harboring a conjugative NDM-1 plasmid and a novel Salmonella genomic island 1 variant (SGI1-Z). Antimicrob. Agents Chemother. 59(10): 6601–6604.

Dai, Lei, Wayne T Muraoka, Zuowei Wu, Orhan Sahin, and Qijing Zhang. 2015. A single nucleotide change in mutY increases the emergence of antibiotic-resistant Campylobacter jejuni mutants. Journal of Antimicrobial Chemotherapy 07/2015; DOI:10.1093/jac/dkv190

Grinnage-Pulley, Tara and Qijing Zhang. 2015. Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates. PLoS ONE 10(7):e0131534.

Sahin, Orhan, Issmat I. Kassem, Zhangqi Shen, Jun Lin, Gireesh Rajashekara, and Qijing Zhang. 2015. Campylobacter in Poultry: Ecology and Potential Interventions. Avian Diseases 59(2):185-200.

Kovač, Jasna, Katarina Šimunović, Wu Zuowei, Anja Klančnik, Franz Bucar, Qijing Zhang, Sonja Smole Možina. 2015. Antibiotic resistance modulation and modes of action of (-)-[alpha]-pinene in Campylobacter jejuni. PLoS ONE 10(4): e0122871.

Kandasamy, S., Vlasova, A.N., Chattha, K.S., Fisher, D., Shao, L., Rajashekara, G., Saif, L.J. 2015. Escherichia coli Nissle colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model. JVI (Accepted).

Shao, L., Fischer, D.D., Kandasamy, S., Rauf, A., Langel, S.N., Wentworth, D.E., Stucker, K.M., Halpin, R.A., Lam, H.C., Marthaler, D., Saif, L.J., Vlasova, A.N. 2015. Comparative in vitro and in vivo studies of porcine rotavirus G9P[13] and human rotavirus Wa G1P[8]. J Virol. 2015 Oct 14. pii: JVI.02401-15. [Epub ahead of print].

Vlasova, A.N., Kandasamy, S., Chattha, K.S., Rajashekara, G., Saif, L.J. 2015. Comparison of probiotic lactobacilli and bifidobacteria effects, immune responses and rotavirus vaccines and infection in different host species. Veterinary Immunology and Immunopathology. (In press).

Jung, K., Annamalai. T., Lu, Z., Saif, L.J. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing versus 26-day-old weaned pigs. Veterinary Microbiology. July 9;178(1-2): 31-40. 2015.

Jung, K. B. Eyerly, T. Annamalai, Z. Lu and L.J. Saif. 2015. Structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus. Vet. Microbiol.177(3-4):373-378.

Annamalai, T., L.J. Saif Z. Lu, and K. Jung. 2015. Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs. Vet Immunol and Immunopath doi:10.1016/j.vetimm.2015.09.006.

Hu, H., Jung, K., Vlasova, A.N., Chepngeno, J., Lu, Z., Wang, Q., Saif, L.J. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. Journal of Clinical Microbiology 53, 1537-1548. 2015.

Esseili MA, Chin A, Saif L, Miller SA, Qu F, Lewis Ivey ML, Wang Q. 2015. Postharvest Survival of Porcine Sapovirus, a Human Norovirus Surrogate, on Phytopathogen-Infected Leafy Greens. J Food Prot 78:1472-1480.

Esseili MA, Saif LJ, Farkas T, Wang Q. 2015. Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce. Appl Environ Microbiol 81:5085-5092.

Esseili MA, Gao X, Tegtmeier S, Saif LJ, Wang Q. 2015. Abiotic stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and its surrogates on Preharvest Leafy Greens. Appl Environ Microbiol doi:10.1128/AEM.02763-15.

Zhongyan Lu, Masaru Yokoyama, Ning Chen, Oka Tomoichiro, Kwonil Jung, Kyeong-Ok Chang, Thavamathi Annamalai, Qiuhong Wang*, and Linda J. Saif*. Molecular mechanism of cell culture adaptation of porcine sapovirus. (J. Virol, Accepted).

Kashoma IP, Kassem II, Kumar A, Kessy BM, Gebreyes W, Kazwala RR and Rajashekara G (2015) Antimicrobial Resistance and Genotypic Diversity ofCampylobacter Isolated from Pigs, Dairy, and Beef Cattle in Tanzania. Front. Microbiol. 6:1240. doi: 10.3389/fmicb.2015.01240

Chandrashekhar K, Kassem II, Nislow C, Gangaiah D, Candelero-Rueda RA, Rajashekara G. Transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants. Virulence. 2015 Nov 5:1-5. [Epub ahead of print] PubMed PMID: 26537695.

Kashoma IP, Kassem II, John J, Kessy BM, Gebreyes W, Kazwala RR, Rajashekara G. Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania. Microb Drug Resist. 2015 Jul 8. [Epub ahead of print] PubMed PMID: 26153978.

Chandrashekhar K, Gangaiah D, Pina-Mimbela R, Kassem II, Jeon BH, Rajashekara G. Transducer like proteins of Campylobacter jejuni 81-176: role in chemotaxis and colonization of the chicken gastrointestinal tract. Front Cell Infect Microbiol. 2015 May 27;5:46. doi: 10.3389/fcimb.2015.00046. eCollection 2015. PubMed PMID: 26075188; PubMed Central PMCID: PMC4444964.

Zeng, X., B. Gillespie, and J. Lin. 2015. Important role of a putative lytic transglycosylase Cj0843 in β-lactam resistance in Campylobacter jejuni. Frontiers in Microbiology (section Antimicrobials, Resistance and Chemotherapy) 6:1292. DOI:10.3389/fmicb.2015.01292

Zhang, Y., J. Lin, Q. Zhong. 2015. S/O/W emulsions prepared with sugar beet pectin to enhance the viability of probiotic Lactobacillus salivarius NRRL B-30514. Food Hydrocolloids (In Press).

Xu, F., F. Guo, C. Wu, G. Cui, X. Zeng, B.Yang, H. Zhou, J. Lin. 2015. Transcriptomic analysis of Campylobacter jejuni NCTC 11168 in response to epinephrine and norepinephrine. Frontiers in Antimicrobials, Resistance and Chemotherapy. 6:452. doi: 10.3389/fmicb.2015.00452.

Zeng, X., D. Ardeshna, and J. Lin. 2015. Heat shock enhanced conjugation efficiency in standard Campylobacter jejuni strains. Applied and Environmental Microbiology. 81:4546-4552.

Zhang, Y., J. Lin, Q. Zhong. 2015. The increased viability of probiotic Lactobacillus salivarius NRRL B-30514 encapsulated in emulsions with multiple lipid-protein-pectin layers. Food Research International. 71:9-15.

Lin, J., K. Nishino, M.C. Roberts, M. Tolmasky, R.I. Aminov, and L. Zhang. 2015. Mechanisms of antibiotic resistance. Frontiers in Microbiology (section Antimicrobials, Resistance and Chemotherapy). 6:34. Doi:10.3389/fmicb.2015.00034

Mirajkar NS and Gebhart CG. Comparison of agar dilution and Etest with broth microdilution for susceptibility testing of swine Brachyspira species. J. Vet. Diagn. Invest. 2015 (In press).

Mirajkar, NS, AZ Bekele, YY Chandler, and CJ Gebhart, 2015. Molecular epidemiology of novel pathogen “Brachyspira hampsonii” reveals relationships between diverse genetic groups, regions, host species, and other pathogenic and commensal Brachyspira. J. Clin. Microbiol. PMID:26135863

Mirajkar N.S. and Gebhart C.J. Understanding the molecular epidemiology and global relationships of Brachyspira hyodysenteriae from swine herds in the United States: A Multi-Locus Sequence Typing Approach. PLoS ONE 9(9): e107176. doi: 10.1371/journal.pone.0107176

Vannucci FA, Linhares DC, Barcellos DE, Lam HC, Collins J, Marthaler D. Identification and Complete Genome of Seneca Valley Virus in Vesicular Fluid and Sera of Pigs Affected with Idiopathic Vesicular Disease, Brazil. Transbound Emerg Dis. 2015 Dec;62(6):589-93. doi: 10.1111/tbed.12410. Epub 2015 Sep 7. PMID: 26347296

Borewicz KA, Kim HB, Singer RS, Gebhart CJ, Sreevatsan S, Johnson T, Isaacson RE. Changes in the porcine intestinal microbiome in response to infection with Salmonella enterica and Lawsonia intracellularis. Plos One 2015 Oct 13;10(10):e0139106. doi: 10.1371/journal.pone.0139106. eCollection 2015.

Homwong N, Jarvis MC, Lam HC, Diaz A, Rovira A, Nelson MI, Marthaler D. Characterization and evolution of porcine deltacoronavirus in the United States. Prev Vet Med. 2015 Nov 10. pii: S0167-5877(15)30045-3. doi: 10.1016/j.prevetmed.2015.11.001. [Epub ahead of print] PMID: 26611652.

Jarvis MC, Lam HC, Zhang Y, Wang W, Hesse RA, Hause BM, Vlasova A, Wang Q, Zhang J, Nelson MI, Murtaugh MP, Marthaler D. Genomic and evolutionary inferences between American and global strains of porcine epidemic diarrhea virus. Prev Vet Med. 2015 Nov 10. pii: S0167-5877(15)30041-6. doi: 10.1016/j.prevetmed.2015.10.020. [Epub ahead of print] PMID: 26611651.

Johnson, T., Singer, R., Isaacson, R., Danzeisen, J., Lang, K., Kobluk, K., Rivet, B., Borewicz, K., Frye, J., Englen, M., Anderson, J., and Davies, P. In vivo transmission of an IncA/C plasmid in Escherichia coli depends on tetracycline concentration, and acquisition results in variable cost of fitness. Appl. Environ. Microbiol. 10, 2015.

Pragman, A., Isaacson, R., Wendt, C., and Reilly, C. A method for determining taxonomical contributions to group differences in microbiomic investigations. Journal of Computational Biology, Accepted March 14, 2015.

Kim, H. B. and Isaacson, R. E. The pig gut microbial diversity: understanding the pig gut microbial ecology through the next generation high throughput sequencing. Vet Microbiol. 177:242-251 (2015).

Pérez-Méndez A, Chandler JC, Paar J, Doolittle M, Bisha B, Goodridge LD. 2015. Field-based evaluation of an integrated F-RNA coliphage concentration and detection method. Applied and Environmental Microbiology (submitted).

Chandler JC, Manley WA, Pérez-Méndez A, Bisha B, Adkins JA, Henry CS, Prenni JE, Goodridge LD. 2015. Molecular and phenotypic properties of cantaloupe associated Listeria monocytogenes. Food Microbiology (submitted).

Johnson DC, Bzdek JP, Fahrenbruck CR, Chandler JC, Bisha B, Goodridge LD, Hybertson BM. 2015. An innovative non-thermal plasma reactor to eliminate microorganisms in water. Desalination and Water Treatment. DOI: 10.1080/19443994.2015.1024752.

Bisha B, Brehm-Stecher BF. 2014. Flow cytometry for rapid detection of Salmonella spp. in seed sprouts. ScienceOpen Research. DOI: 10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1.

Wijemanne, P., J. Xing, E. M. Berberov, D. B. Marx, D. H. Francis, and R. A. Moxley. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains. PLoS One 10(3):e0117663.

Stromberg, Z. R., G. L. Lewis, D. B. Marx, and R. A. Moxley. 2015. Comparison of enrichment broths for supporting growth of Shiga toxin-producing Escherichia coli. Curr. Microbiol. 71:214-219.

Stromberg, Z. R., N. Baumann, G. L. Lewis, N. Sevart, N. Cernicchiaro, D. G. Renter, D. B. Marx, R. K. Phebus, and R. A. Moxley. 2015. Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and carcass surfaces of beef feedlot cattle at harvest. Foodborne Pathog. Dis. 12:631-638.

Stromberg, L. R., Z. R. Stromberg, A. Banisadr, S. W. Graves, R. A. Moxley, and H. Mukundan. 2015. Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli. J. Microbiol. Methods 116:1-7.

Moxley, R. A., Z. R. Stromberg, G. L. Lewis, J. D. Loy, B. W. Brodersen, I. R. Patel, and J. Gangiredla. 2015. Hemorrhagic colitis associated with enterohemorrhagic Escherichia coli O165:H25 infection in a yearling feedlot heifer. JMM Case Reports 2:1-6. doi:10.1099/jmmcr.0.005004.

Stromberg, Z. R., G. L. Lewis, S. S. Aly, T. W. Lehenbauer, N. Cernicchiaro, N. and R. A. Moxley. Prevalence and concentration of enterohemorrhagic Escherichia coli in culled dairy cattle at harvest. J. Food Prot. Accepted and in press.

Chopyk, J., R. Moore, Z. R. Stromberg, G. L. Lewis, D. G. Renter, N. Cernicchiaro, R. A. Moxley, and K. E. Wommack. Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides. Microbiome. Submitted.

Stromberg, L. R., N. W. Hengartner, K. L. Swingle, R. A. Moxley, S. W. Graves, G. A. Montaño, H. Mukundan. Membrane insertion for the detection of lipopolysaccharides: exploring the dynamics of amphiphile-in-lipid assays. PLoS Pathog. Submitted.

Stromberg, Z. R., G. L. Lewis, and R. A. Moxley. Comparison of agar media for the detection and quantification of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O121, O145, and O157 in cattle feces. J. Food Prot. Submitted.

Scaria J, Suzuki H, Ptak CP, Chen JW, Zhu Y, Guo XK, Chang YF. Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii, two related pathogens with differing host tissue preference. BMC Genomics. 2015 Jun 10;16:448.

Okda F, Liu X, Singrey A, Clement T, Nelson J, Christopher-Hennings J, Nelson EA, Lawson S. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus. BMC Vet Res. 2015 Aug 1;11:180.

Zhou M, Duan Q, Li Y, Yang Y, Hardwidge PR, Zhu, G. Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling, Archives of Microbiology, 2015, May 3.

Kumar A, Hays M, Lim F, Foster LJ, Zhou M, Zhu G, Miesner T, Hardwidge PR. Protective enterotoxigenic Escherichia coli antigens in a murine intranasal challenge model, PLoS Neglected Tropical Diseases, 2015, Aug 5;9(8):e0003924.

Zhou M, Yang Y, Chen P, Hu H, Hardwidge PR, Zhu G. More than a locomotive organelle: flagella in Escherichia coli, Applied Microbiology and Biotechnology, 2015.

Zhang Y, Vadlani, PV, Kumar A, Hardwidge PR, Govind R, Tanaka T, Kondo A. Enhanced D-lactic acid production from renewable resources using engineered Lactobacillus plantarum, Applied Microbiology and Biotechnology, 2015.

Cull CA, Renter DG, Bello N.B., Ives S.E., Babcock A.H. Performance and carcass characteristics of commercial feedlot cattle from a study of vaccine and direct-fed microbial effects on E. coli O157:H7 fecal shedding. J An Sci. 2015; 93(6): 3144-3151.

Dewsbury DM, Renter DG, Shridhar PB, Noll LW, Shi X, Nagaraja TG, Cernicchiaro N. Summer and winter prevalence of Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121, O145, and O157 in feces of feedlot cattle. Foodborne Pathog Dis. 2015; (8):726-732.

Noll LW, Baumgartner WC, Shridhar PB, Cull CA, Dewsbury D, Shi,X, Cernicchiaro N, Renter DG, Nagaraja TG. Pooling of immunomagnetic separation beads does not affect sensitivity of detection of six serogroups of Shiga toxin-producing Escherichia coli in cattle feces. Journal of Food Protection (accepted for publication).

Ison S, Delannoy S, Bugarel , Nagaraja TG, Renter DG, den Bakker H, Nightingale K, Fach P, Loneragan G. Targeted amplicon sequencing for SNP genotyping of attaching and effacing Escherichia coli O26:H11 cattle strains using a high-throughput library preparation technique. Appl Enviorn Microbiol (accepted for publication).

Noll LW, Shridhar PB, Dewsbury DM, Shi X, Cernicchiaro N, Renter DG, Nagaraja TG. A comparison of culture- and PCR-based methods to detect six major non-O157 serogroups of Shiga toxin-producing Escherichia coli in cattle feces. PLoS One. 2015; 10(8):e0135446.

Noll LW, Shridhar PB, Shi X, An B, Cernicchiaro N, Renter DG, Nagaraja TG, Bai J. A four-plex real-time PCR assay, based on rfbE, eae, stx1, and stx2 genes, for the detection and quantification of Escherichia coli O157 in cattle feces. Foodborne Pathogens and Disease 12(9):787-794.

Shivanna V, Kim Y, Chang KO. Bile acid-mediated Activation of the Acid Sphingomyelinase Facilitates Endosomal Escape of Porcine Enteric Calicivirus. 2015. Virology. 483:218-28.

Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Kyeong-Ok Chang, William C. Groutas, Recent Advances in the Discovery of Norovirus Therapeutics. J Med Chem. 2015 Aug 17.

Kim Y, Shivanna V, Hua DH, Groutas WC, and Chang KO. Broad-spectrum protease inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.2015. J Virol. 89(9):4942-50.

Kankanamalage AM. Uy WP, Mandadapu SR, Alliston KR, Chang KO, Kim Y, Lovell S, Groutas WC. Structure-Based Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic and Cell-Based Studies. 2015. J Med Chem. 58(7):3144-55.

Toro M, Cao G, Rump L, Nagaraja TG, Meng J, Gonzalez-Escalona N. Genome Sequences of 64 Non-O157:H7 Shiga Toxin-Producing Escherichia coli Strains. Genome Announc. 2015 Oct 1;3(5). pii: e01067-15. doi: 10.1128/genomeA.01067-15.

Amachawadi RG, Scott HM, Vinasco J, Tokach MD, Dritz SS, Nelssen JL, Nagaraja TG. Effects of In-Feed Copper, Chlortetracycline, and Tylosin on the Prevalence of Transferable Copper Resistance Gene, tcrB, Among Fecal Enterococci of Weaned Piglets. Foodborne Pathog Dis. 2015 Aug;12(8):670-8. doi: 10.1089/fpd.2015.1961.

Amachawadi RG, Nagaraja TG. First Report of Anaerobic Isolation of Salmonella enterica from Liver Abscesses of Feedlot Cattle. J Clin Microbiol. 2015 Sep;53(9):3100-1. doi: 10.1128/JCM.01111-15.

Ison SA, Delannoy S, Bugarel M, Nightingale KK, Webb HE, Renter DG, Nagaraja TG, Loneragan GH, Fach P. Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States. Appl Environ Microbiol. 2015 Jun;81(11):3671-8. doi: 10.1128/AEM.00397-15.

Agga GE, Scott HM, Vinasco J, Nagaraja TG, Amachawadi RG, Bai J, Norby B, Renter DG, Dritz SS, Nelssen JL, Tokach MD. Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs. Prev Vet Med. 2015 May 1;119(3-4):179-89.doi: 10.1016/j.prevetmed.2015.02.008.

Amachawadi RG, Scott HM, Aperce C, Vinasco J, Drouillard JS, Nagaraja TG. Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle. J Appl Microbiol. 2015 Jun;118(6):1287-97. doi: 10.1111/jam.12790.

Kumar A, Menon S, Nagaraja TG, Narayanan S. Identification of an outer membrane protein of Fusobacterium necrophorum subsp. necrophorum that binds with high affinity to bovine endothelial cells. Vet Microbiol. 2015 Mar 23;176(1-2):196-201. doi: 10.1016/j.vetmic.2014.12.015.

Amachawadi RG, Scott HM, Nitikanchana S, Vinasco J, Tokach MD, Dritz SS, Nelssen JL, Goodband RD, Nagaraja TG. Nasal carriage of mecA-positive methicillin-resistant Staphylococcus aureus in pigs exhibits dose-response to zinc supplementation. Foodborne Pathog Dis. 2015 Feb;12(2):159-63. doi: 10.1089/fpd.2014.1851.

Guard J, Sanchez-Ingunza R, Shah DH, Rothrock MJ, Gast RK, Jones DR. Recovery of Salmonella enterica serovar Enteritidis from hens initially infected with serovar Kentucky. Food Chem. 189:86-92, 2015. PubMed PMID: 26190605.

Sheng H, Shringi S, Baker KN, Minnich SA, Hovde CJ, Besser TE. Standardized E. coli O157:H7 exposure studies in cattle: Evidence that bovine factors do not drive increased summertime colonization. Appl Environ Microbiol. 2015 Nov 25. pii: AEM.02839-15. [Epub ahead of print] PubMed PMID: 26607594.

Spencer SE, Besser TE, Cobbold RN, French NP. 'Super' or just 'above average'? Supershedders and the transmission of Escherichia coli O157:H7 among feedlot cattle. J R Soc Interface. 2015 Sep 6;12(110):0446. doi: 10.1098/rsif.2015.0446. PubMed PMID: 26269231; PubMed Central PMCID: PMC4614454.

Zhao Z, Eberhart LJ, Orfe LH, Lu SY, Besser TE, Call DR. Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI. Appl Environ Microbiol. 2015 Oct;81(20):6953-63. doi: 10.1128/AEM.01704-15. Epub 2015 Jul 24. PubMed PMID: 26209678; PubMed Central PMCID: PMC4579430.

Davis MA, Sischo WM, Jones LP, Moore DA, Ahmed S, Short DM, Besser TE. Recent Emergence of Escherichia coli with Cephalosporin Resistance Conferred by blaCTX-M on Washington State Dairy Farms. Appl Environ Microbiol. 2015 Jul;81(13):4403-10. doi: 10.1128/AEM.00463-15. Epub 2015 Apr 24. PubMed PMID: 25911480; PubMed Central PMCID: PMC4475894.

Jaros P, Cookson AL, Campbell DM, Duncan GE, Prattley D, Carter P, Besser TE, Shringi S, Hathaway S, Marshall JC, French NP. Geographic divergence of bovine and human Shiga toxin–producing Escherichia coli O157:H7 genotypes, New Zealand. Emerg Infect Dis. 2014 Dec;20(12):1980-9. PubMed PMID: 25568924; PubMed Central PMCID: PMC4257794.

Mellor GE, Fegan N, Gobius KS, Smith HV, Jennison AV, D'Astek BA, Rivas M, Shringi S, Baker KN, Besser TE. Geographically distinct Escherichia coli O157 isolates differ by lineage, Shiga toxin genotype, and total shiga toxin production. J Clin Microbiol. 2015 Feb;53(2):579-86. doi: 10.1128/JCM.01532-14. Epub 2014 Dec 10. PubMed PMID: 25502531; PubMed Central PMCID: PMC4298522.

Pereira RV, Siler JD, Ng JC, Davis MA, Warnick LD. Effect of preweaned dairy calf housing system on antimicrobial resistance in commensal Escherichia coli. J Dairy Sci. 2014 Dec;97(12):7633-43.

Book chapters

Marthaler D, Bohac A, Becker A, Peterson N. Next Generation Sequencing for Porcine Coronaviruses. Leyi Wang (Eds): Animal Coronaviruses, 978-1-4939-3412-6

Moxley, R. A., and G. R. Acuff. Chapter 22, Peri- and post-harvest factors in the control of Shiga toxin-producing Escherichia coli in beef. Enterohemorrhagic Escherichia coli, 2^nd Ed., American Society for Microbiology Press, Washington, D.C., pp. 437-456.