FUTIs: an In-Person or Online Graphing, Bioinformatics, and Scientific Literacy Exercise That Explores the Presence of Antibiotic Resistance in Foodborne Urinary Tract Infections.

We developed a course-based undergraduate research experience (CURE) that gives students an opportunity to practice the process of science in a context that intersects with their everyday lives: purchasing grocery store chicken. Student mastery of concepts was assessed by pre- and postassessment questions and lab report worksheets that guided them through the process of writing a scientific paper. Learning to produce graphs from large data sets and comparing the results with published data emphasized quantitative reasoning, while working as a group and writing helped students practice scientific communication. Most students (>90%) met the learning objectives, and students in both groups reported feeling more confident producing graphs and figures; they also showed large gains in confidence and interest in bioinformatics. Lab protocols require biosafety level 2 safety guidelines; however, students in an online or dry lab setting can use the compiled data sets and whole-genome sequences to complete the objectives. Group discussions and essay prompts at the end encourage students to use evidence-based arguments to make decisions that impact the global issue of antimicrobial resistance.

Isolation of Porcine Reproductive and Respiratory Syndrome Virus GP5-Specific, Neutralizing Monoclonal Antibodies From Hyperimmune Sows.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease which impacts the pig industry worldwide. The disease is caused by PRRS viruses (PRRSV-1 and -2) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in growing pigs. Current PRRSV vaccines provide limited protection; only providing complete protection against closely related strains. The development of improved PRRSV vaccines would benefit from an increased understanding of epitopes relevant to protection, including those recognized by antibodies which possess the ability to neutralize distantly related strains. In this work, a reverse vaccinology approach was taken; starting first with pigs known to have a broadly neutralizing antibody response and then investigating the responsible B cells/antibodies through the isolation of PRRSV neutralizing monoclonal antibodies (mAbs). PBMCs were harvested from pigs sequentially exposed to a modified-live PRRSV-2 vaccine as well as divergent PRRSV-2 field isolates. Memory B cells were immortalized and a total of 5 PRRSV-specific B-cell populations were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1 isolates. Antibodies against GP5 protein, commonly thought to possess a dominant PRRSV neutralizing epitope, were found to be highly abundant, as four out of five B cells populations were GP5 specific. One of the GP5-specific mAbs was shown to be neutralizing but this was only observed against homologous and not heterologous PRRSV strains. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of broadly neutralizing antibodies against other porcine pathogens.

Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a Model.

Emerging viral infectious diseases present a major threat to the global swine industry. Since 2015, Senecavirus A (SVA) has been identified as a cause of vesicular disease in different countries and is considered an emerging disease. Despite the growing concern about SVA, there is a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious diseases. Using SVA as a model, we demonstrated that Oxford Nanopore MinION sequencing could be used as a robust tool for the investigation and surveillance of emerging viral diseases. Our results identified that MinION sequencing allowed for rapid, unbiased pathogen detection at the species and strain level for clinical cases. SVA whole genome sequences were generated using both direct RNA sequencing and PCR-cDNA sequencing methods, with an optimized consensus accuracy of 94% and 99%, respectively. The advantages of direct RNA sequencing lie in its shorter turnaround time, higher analytical sensitivity and its quantitative relationship between input RNA and output sequencing reads, while PCR-cDNA sequencing excelled at creating highly accurate sequences. This study developed whole genome sequencing methods to facilitate the control of SVA and provide a reference for the timely detection and prevention of other emerging infectious diseases.

Leveraging a Validated in silico Approach to Elucidate Genotype-Specific VP7 Epitopes and Antigenic Relationships of Porcine Rotavirus A.

Rotavirus A (RVA) remains one of the most widespread causes of diarrheal disease and mortality in piglets despite decades of research and efforts to boost lactogenic immunity for passive protection. Genetic changes at B cell epitopes (BCEs) may be driving failure of lactogenic immunity, which relies on production of IgA antibodies to passively neutralize RVA within the piglet gut, yet little research has mapped epitopes to swine-specific strains of RVA. Here we describe a bioinformatic approach to predict BCEs on the VP7 outer capsid protein using sequence data alone. We first validated the approach using a previously published dataset of VP7-specific cross-neutralization titers, and found that amino acid changes at predicted BCEs on the VP7 protein allowed for accurate recapitulation of antigenic relationships among the strains. Applying the approach to a dataset of swine RVA sequences identified 9 of the 11 known BCEs previously mapped to swine strains, indicating that epitope prediction can identify sites that are known to drive neutralization escape in vitro. Additional genotype-specific BCEs were also predicted that may be the cause of antigenic differences among strains of RVA on farms and should be targeted for further confirmatory work. The results of this work lay the groundwork for high throughput, immunologically-relevant analysis of swine RVA sequence data, and provide potential sites that can be targeted with vaccines to reduce piglet mortality and support farm health.

Establishment and characterization of a porcine B cell lymphoma cell line.

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3- and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

The PRRSV-Specific Memory B Cell Response Is Long-Lived in Blood and Is Boosted During Live Virus Re-exposure.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine health and well-being worldwide largely due to an insufficient understanding of the adaptive immune response to infection leading to ineffective PRRSV control. The memory and anamnestic response to infection are critical gaps in knowledge in PRRSV immunity. The lack of effective tools for the evaluation of the memory response previously hindered the ability to effectively characterize the porcine memory response to infection. However, the creation and validation of a PRRSV nsp7-specific B cell tetramer now facilitates the ability to detect very rare memory B cells and thus define the memory response of the pig. Here, we describe the PRRSV nsp7-specific B cell response following vaccination and challenge in six key secondary lymphoid organs including the identification of PBMCs as the tissue of interest for the memory immune response in pigs. Following live virus challenge of immune animals, an anamnestic response of nsp7-specific memory B cells and neutralizing antibodies was observed. This characterization of the functional humoral immune response to PRRSV answers key questions involved in regional specialization of the immune response following intramuscular inoculation of PRRSV MLV.

MinION sequencing of Streptococcus suis allows for functional characterization of bacteria by multilocus sequence typing and antimicrobial resistance profiling.

In recent years, high-throughput sequencing has revolutionized disease diagnosis by its powerful ability to provide high resolution genomic information. The Oxford Nanopore MinION sequencer has unparalleled potential as a rapid disease diagnostic tool due to its high mobility, accessibility, and short turnaround time. However, there is a lack of rigorous quality assessment and control processes standardizing the testing on the MinION, which is necessary for incorporation into a diagnostic workflow. Thus, our study examined the use of the MinION sequencer for bacterial whole genome generation and characterization. Using Streptococcus suis as a model, we optimized DNA isolation and treatments to be used for MinION sequencing and standardized de novo assembly to quickly generate a full-length consensus sequence achieving a 99.4% average accuracy. The consensus genomes from MinION sequencing were able to accurately predict the multilocus sequence type in 8 out of 10 samples and identified antimicrobial resistance profiles for 100% of the samples, despite the concern of a high error rate. The inability to unequivocally predict sequence types was due to difficulty in differentiating high identity alleles, which was overcome by applying additional error correction methods to increase consensus accuracy. This manuscript provides methods for the use of MinION sequencing for identification of S. suis genome sequence, sequence type, and antibiotic resistance profile that can be used as a framework for identification and classification of other pathogens.

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools.

Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 104 to 106 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.

Characterization of age-related susceptibility of macrophages to porcine reproductive and respiratory syndrome virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most economically important disease affecting swine production worldwide. The severity and susceptibility of PRRSV infection varies with age. Nursery pigs have been shown to be more susceptible to PRRSV infection and a more severe and prolonged infection is observed as compared to growing or adult pigs. However, antibody responses to PRRSV are observed independent of age. Swine are the only known hosts of PRRSV, infection is restricted to cells of monocytic lineage, and fully differentiated porcine alveolar macrophages are the primary target of natural infection. Pulmonary intravascular macrophages from young pigs have been shown to be more susceptible to infection than those from adult pigs. A better understanding of why young pigs and macrophages from young pigs are more susceptible to PRRSV infection is critical to identify mechanisms of infection that can be explored for enhanced treatment or prevention of disease. This study examined PRRSV susceptibility of porcine alveolar macrophages isolated from the lungs of pigs of different age groups, and the presence of cell surface receptors to determine if differences correlated with infection level. The younger the pigs were, the more susceptible the macrophage were to PRRSV infection, but no differences in cellular receptor expression were observed between pigs of different ages. Resistance to infection is likely related to intracellular innate immune mechanisms rather than receptor-mediated entry.

Porcine circovirus 2 infection induces IFNß expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways.

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNß, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.

Effect of Maternal Antibody Transfer on Antibody Dynamics and Control of Porcine Circovirus Type 2 Infection in Offspring.

Sow immunity plays an important role in preventing viral infection and disease in newborn piglets. Vertical transmission of porcine circovirus type 2 (PCV2) may perpetuate porcine circovirus associated disease (PCVAD) in newborn and growing pigs. Hence, the immunological effects of maternal immunoglobulin transfer of PCV2-specific antibodies on PCV2 viremia and immune response in piglets in commercial swine herds were evaluated. Sow vaccination has been shown to reduce viral shedding and viremia, and increases the neutralizing antibody (NA) titers. Since NAs are important for control of PCVAD and mammary secretions may contain high anti-PCV2 NA levels, we examined the PCV2 NA levels in colostrum, milk, sow serum, and piglet serum over time to investigate an association between NA levels and protection against infection. NA titers were remarkably high (up to 10-6 50% neutralizing titer) in sow serum and colostrum on all farms regardless of viremia levels. In piglets vaccinated at 3 weeks of age, NA titers peaked at 10 weeks of age and continued to maintain high viral neutralizing titers to slaughter. The impact of maternally derived neutralizing activity was most evident during the suckling period. Although PCV2 was transmitted from sows to piglets in colostrum, piglets were largely nonviremic at weaning. Thus, NAs appear to control or suppress initial infection even though they are unable to clear or prevent infection later in life.

An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine.

BACKGROUND: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases. METHODS: We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3. RESULTS: Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4-60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react. CONCLUSIONS: A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases.

Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions.

Porcine epidemic diarrhea virus (PEDV) causes a severe clinical enteric disease in suckling neonates with up to 100% mortality, resulting in devastating economic losses to the pork industry in recent years. Maternal immunity via colostrum and milk is a vital source to neonates of passive protection against diarrhea, dehydration and death caused by PEDV. Comprehensive information on neutralizing activity (NA) against PEDV in mammary secretions is critically important for assessing the protective capacity of sows. Therefore, the objectives of this study were to characterize anti-PEDV neutralizing activity in mammary secretions. Anti-PEDV NA was present in colostrum, milk and serum from PEDV-infected sows as determined both by immunofluorescence and ELISA-based neutralizing assays, with neutralization levels higher in colostrum and milk than in serum. The highest NA was observed in colostrum on day 1, and decreased rapidly in milk at day 3, then gradually declined from day 3 to day 19 post-farrowing. Notably, the NA in mammary secretions showed various patterns of decline over time of lactation that may contribute to variation in sow protective capacities. The kinetics of NA decline were associated with total IgA and IgG antibody levels. Neutralizing activity significantly correlated with specific IgA primarily to spike domain 1 (S1) and domain 2 (S2) proteins of PEDV rather than to specific IgG in colostrum. Subsequently, the NA in milk was mainly related to specific IgA to S1 and S2 during lactation.

National reduction in porcine circovirus type 2 prevalence following introduction of vaccination.

Porcine circovirus type 2 (PCV2), a small, single-stranded circular DNA virus and the causative agent of porcine circovirus associated disease (PCVAD), was first observed in the mid-1990s in pigs with a post-weaning wasting disease. In 2006 the number of PCVAD cases greatly increased, marking it as an important viral pathogen for the United States (US) swine industry. PCV2 vaccines were introduced to the US in 2006 in response to widespread outbreaks of PCVAD. These vaccines were effective in preventing disease, but did not eliminate virus from the animals. In 2006, prior to vaccine use, a study of PCV2 prevalence in pig herds across the US was performed in conjunction with the US National Animal Health Monitoring System. In 2012, 6 years after widespread PCV2 vaccination, this study was repeated. Since the introduction of PCV2 vaccines in 2006, viral presence and viral loads have greatly decreased, and a genotypic shift dominated by PCV2b has occurred. Antibody levels have decreased in the pig population, but approximately 95% of sites continue to be antibody-positive. Widespread vaccination has controlled PCVAD and decreased PCV2 prevalence to the point that viremia is not detected on many sites. Thus, continued vaccination may lead to PCV2 elimination in the national herd over time.

Diagnostic phylogenetics reveals a new Porcine circovirus 2 cluster.

Porcine circovirus 2 (PCV2) was prevalent in swine in the United States before PCV2-associated disease (PCVAD) appeared in 2006. Limited nucleotide sequencing of open reading frame 2 (ORF2) encoding capsid, the only structural protein, revealed the presence of two genotypes, PCV2a and PCV2b. Later, PCV2c and mutant PCV2b, or PCV2d, were also described. However, extensive PCV2 ORF2 sequence databases in veterinary diagnostic laboratories have not been analyzed systematically to determine the genetic diversity of field isolates. Here, we interrogated >1100 PCV2 ORF2 nucleotide sequences to assess population diversity and genetic variation. We detected a novel PCV2 genotype that is substantially different, primarily in ORF2, from all known PCV2. Notably, ORF2 contains a unique carboxyl terminal amino acid insertion resulting in a 238 amino acid ORF2. All other PCV2 ORF2 proteins are 233 or 234 aa in length. Phylogenetic analysis indicates that it is more ancient than other PCV2 genotypes. The findings demonstrate the value of analyzing routine diagnostic laboratory sequence databases in population genetic analyses of animal pathogens.

Multiple routes of porcine circovirus type 2 transmission to piglets in the presence of maternal immunity.

Porcine circovirus 2 (PCV2), the cause of porcine circovirus-associated disease (PCVAD), is widespread in swine farms throughout the United States with vaccine controlling disease, but not eliminating infection. We examined the PCV2 virological and immunological status of sows, pre-suckling piglets, and the farrowing environment of sow farms to determine PCV2 exposure risks, transmission dynamics, and immunological impacts at the time of farrowing. PCV2 was widely distributed in animals and the farrowing environment of 6 midwestern US sow farms irrespective of sow vaccination status. High levels of PCV2 capsid-specific antibodies were observed in sow serum and colostrum and had no apparent effect on PCV2 transmission to and infection in piglets. In 281 pre-suckling piglets from 59 sows, PCV2 DNA was detected in 63% of serum samples and on 93% of axillary skin swabs. PCV2 was present in one or more samples from 58 of 59 sows and in the farrowing environment. Isolated infectious virus samples from sows, presuckling piglets, and the environment were shown by sequencing to be genetically similar from all farms. In conclusion, piglets are readily infected with PCV2 in utero and are under constant challenge by PCV2 through contact with infected sows and a contaminated farrowing environment. However, maternal immunity did not affect PCV2 transmission to piglets or the viral load in sows. These findings illustrate the importance of maternal infection, despite robust anti-PCV2 immunity, in early infection of newborn piglets, and the need to develop appropriate infection models for elucidation of mechanisms of protective immunity.

Cellular pathogenesis of porcine circovirus type 2 infection.

Porcine circovirus associated disease (PCVAD) and the associated histological lesions are thought to appear due to an increase in the amount of porcine circovirus type 2 (PCV2) present in an infected animal. However, examination of the cellular and molecular pathogenesis of PCVAD is complicated by the lack of a consistent cell culture model that replicates the animal phenotypes of persistent, asymptomatic infection, and acute, pathological disease typified by lymphocyte depletion. The porcine fetal retina cell line, VR1BL, shows a high permissiveness to PCV2 infection, 40 times higher than the alternative PK15 culture model, allowing for high titer viral production, with PCV2b growth higher than PCV2a growth. Cytopathic effect due to apoptosis is observed after challenge with high amounts of PCV2, but at low levels, infection is maintained in passaged cells. Thus, VR1BL cells may be used as a model system to examine both acute viral pathogenesis and cellular innate defense, as well as persistent PCV2 infection.

Genomic analysis of mucosal immunobiology in the porcine small intestine.

The vast majority of infectious diseases of animals and humans occur at mucosal surfaces, especially in the intestinal tract. However, vaccines to stimulate durable immunity to intestinal pathogens often do not produce effective protection. A better understanding of the molecules and molecular mechanisms that mediate effective immune induction at mucosal surfaces may facilitate the development of more effective mucosal vaccines. Characterization of the mRNA expression profile of Peyer's patch, a key immunological tissue in immune surveillance and response, would help to identify important proteins and pathways required for the development of effective mucosal immunity in swine. We produced a cDNA library of about 2400 genes differentially expressed in jejunal Peyer's patch, of which approximately 900 had unknown functions or were novel to public databases. We hypothesize that knowledge of the roles and regulation of the encoded proteins under conditions of substantial immune stimulation will help to understand mucosal immunoregulatory mechanisms in the Peyer's patch. Cholera toxin is a potent mucosal adjuvant and antigen. The response to cholera toxin is amenable to dissection at a variety of levels from local to systemic and molecular to cellular in whole animals, tissue explants, and cultured cells. The powerful tools of genomics and genetics that are now available provide veterinary immunologists with new opportunities to elucidate and dissect the mysteries of host defense. By building on the findings from multiple systems and models we can develop a better understanding of complex interactions of immune and absorptive tissues in the regulation of immunity in the small intestine.

Presence of free haptoglobin alpha 1S-subunit in acute porcine reproductive and respiratory syndrome virus infection.

The biochemical events triggered by viral infection are critical to the outcome of a host immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) causes the most significant disease of swine worldwide. Onset of infection is insidious and subclinical. Clinical signs may not appear for days and antibodies cannot be detected for a week or more. To understand better the early pathophysiological response of swine to PRRSV infection and its inapparent onset, we examined serum samples in the first days of infection for evidence of early biochemical changes. Sera from pigs infected with various isolates of PRRSV were extracted to remove high molecular mass proteins, desalted and analysed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MS). Comparative analysis of low molecular mass serum protein profiles revealed that one protein, with an m/z value of 9244+/-2, appeared within 1 day of infection. The 9244+/-2 peak was identified as the alpha 1S (alpha1S)-subunit of porcine haptoglobin (Hp) by tandem MS sequencing and confirmed by immunoblotting with anti-porcine Hp antibody. Hp is an acute phase haem-binding protein consisting of alpha-beta heterodimers that is secreted from the liver in response to stresses, including infection. However, the presence of free alpha1S-subunit in response to infection is novel and may provide new insights into biochemical processing of Hp and its role in disease pathogenesis, including PRRS.

Genomic dissection of mucosal immunobiology in the porcine small intestine.

The enteric immune system of swine protects against infectious and noninfectious environmental insults and discriminates ingested nutrients, food, and commensal microflora from pathogenic agents. The molecular and cellular elements of the immune system have been selected over evolutionary time in response to the specific environment of pigs. Thus, models of immune function based on mouse and human need to be applied cautiously in the pig. To better understand how the mucosal immune system of the small intestine accomplishes the conflicting functions of food tolerance and immunity to enteric infection, we used a genomic approach to profile gene expression in the Peyer's patch. More than 40% of mRNA enriched by differential subtraction for Peyer's patch-specific expressed sequences represented genes of unknown function or had no match in GenBank. Microarray analysis and radiation hybrid mapping validated their porcine origin and provided additional insights into putative functions. The abundance of expressed genes of unknown function indicates that a substantial fraction of the immunological and physiological processes of the Peyer's patch remains to be discovered. It further suggests that swine have evolved specialized biochemical and immunological processes in the small intestine. Further elucidation of these processes are expected to provide novel insights into swine enteric mucosal immune function.