Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates.

Streptococcus suis is a significant economic and welfare concern in the swine industry. Pan-genome analysis provides an in-silico approach for the discovery of genes involved in pathogenesis in bacterial pathogens. In this study, we performed pan-genome analysis of 208 S. suis isolates classified into the pathogenic, possibly opportunistic, and commensal pathotypes to identify novel candidate virulence-associated genes (VAGs) of S. suis. Using chi-square tests and LASSO regression models, three accessory pan-genes corresponding to S. suis strain P1/7 markers SSU_RS09525, SSU_RS09155, and SSU_RS03100 (>95% identity) were identified as having a significant association with the pathogenic pathotype. The proposed novel SSU_RS09525 + /SSU_RS09155 + /SSU_RS03100 + genotype identified 96% of the pathogenic pathotype strains, suggesting a novel genotyping scheme for predicting the pathogenicity of S. suis isolates in North America. In addition, mobile genetic elements carrying antimicrobial resistance genes (ARGs) and VAGs were identified but did not appear to play a major role in the spread of ARGs and VAGs.

Pseudocowpox virus infection in an American bison (Bison bison).

BACKGROUND: The present report describes a case of pseudocowpox virus (PCPV) infection in a seven-year-old female bison euthanized due to a history of declining condition and sores on the vulva and udder. CASE PRESENTATION: External examination revealed multifocal, raised, keratinized plaques (0.5-2 cm) covering the skin of the ventral surface of the tail, perineum, caudoventral abdomen, udder, both inguinal recesses, and the medial aspects of both thighs. No significant gross lesions were present in the reminder of the tissues examined. Histopathological examination of the affected skin showed moderate epidermal hyperplasia with rete pegs, marked parakeratotic hyperkeratosis with crusts of degenerate neutrophils and cell debris, and few epithelial cells undergoing ballooning degeneration with occasional eosinophilic intracytoplasmic inclusion bodies (3-5 µm Bollinger body). Negative staining electron microscopy from skin revealed typical Parapoxvirus (PPV) particles, which were also confirmed by real-time PCR (Ct =18.6). Metagenomic analysis of the skin samples revealed only poxviruses. The bison parapox B2L envelope gene clustered with other parapox sequences identified from ruminants. CONCLUSIONS: This is the first report of PCPV virus infection in an American bison. Identification of novel susceptible hosts of parapox viruses sheds light on the viral evolution and highlights the importance of potential economic impact of this disease to the bison industry.

Serotype and Genotype (Multilocus Sequence Type) of Streptococcus suis Isolates from the United States Serve as Predictors of Pathotype.

Streptococcus suis is a significant cause of mortality in piglets and growing pigs worldwide. The species contains pathogenic and commensal strains, with pathogenic strains causing meningitis, arthritis, endocarditis, polyserositis, and septicemia. Serotyping and multilocus sequence typing (MLST) are primary methods to differentiate strains, but the information is limited for strains found in the United States. The objective of this study was to characterize the diversity of 208 S. suis isolates collected between 2014 and 2017 across North America (mainly the United States) by serotyping and MLST and to investigate associations between subtype and pathotype classifications (pathogenic, possibly opportunistic, and commensal), based on clinical information and site of isolation. Twenty serotypes were identified, and the predominant serotypes were 1/2 and 7. Fifty-eight sequence types (STs) were identified, and the predominant ST was ST28. Associations among serotypes, STs, and pathotypes were investigated using odds ratio and clustering analyses. Evaluation of serotype and ST with pathotype identified a majority of isolates of serotypes 1, 1/2, 2, 7, 14, and 23 and ST1, ST13, ST25, ST28, ST29, ST94, ST108, ST117, ST225, ST373, ST961, and ST977 as associated with the pathogenic pathotype. Serotypes 21 and 31, ST750, and ST821 were associated with the commensal pathotype, which is composed of isolates from farms with no known history of S. suis-associated disease. Our study demonstrates the use of serotyping and MLST to differentiate pathogenic from commensal isolates and establish links between pathotype and subtype, thus increasing the knowledge about S. suis strains circulating in the United States.

Complete genome characterization of a rotavirus B (RVB) strain identified in Alpine goat kids with enteritis reveals inter-species transmission with RVB bovine strains.

Rotavirus B (RVB) has been associated with enteric disease in many animal species. An RVB strain was identified in pooled intestinal samples from Alpine caprine kids (between 2 and 3 days of age) experiencing high (>90 %) morbidity, and the complete caprine RVB genome was characterized. Histology revealed villus atrophy, the samples tested positive for RVB by real-time RT-PCR and metagenomic next-generation sequencing identified only RVB and orf virus. In the VP4 gene segment, the caprine RVB strain had a higher percentage nucleotide identity to the Indian bovine RVB strains than to the Japanese bovine RVB strains, but the VP7, VP6, VP2, NSP1, NSP2 and NSP5 gene segments of the American caprine RVB strain were genetically related to the Japanese bovine RVB strains. The results indicate a lack of RVB sequences to understand reassortment or the evolutionary relationship of RVB strains from cattle and goats.

A porcine enterovirus G associated with enteric disease contains a novel papain-like cysteine protease.

Identification of unknown pathogens in pigs displaying enteric illness is difficult due to the large diversity of bacterial and viral species found within faecal samples. Current methods often require bacterial or viral isolation, or testing only a limited number of known species using quantitative PCR analysis. Herein, faeces from two 25-day-old piglets with diarrhoea from Texas, USA, were analysed by metagenomic next-generation sequencing to rapidly identify possible pathogens. Our analysis included a bioinformatics pipeline of rapid short-read classification and de novo genome assembly which resulted in the identification of a porcine enterovirus G (EV-G), a complete genome with substantial nucleotide differences (>30 %) among current sequences, and a novel non-structural protein similar in sequence to the Torovirus papain-like cysteine protease (PLpro). This discovery led to the identification and circulation of an EV-G with a novel PLpro in the USA that has not been previously reported.

Genome characterization of Turkey Rotavirus G strains from the United States identifies potential recombination events with human Rotavirus B strains.

Rotavirus G (RVG) strains have been detected in a variety of avian species, but RVG genomes have been published from only a single pigeon and two chicken strains. Two turkey RVG strains were identified and characterized, one in a hatchery with no reported health issues and the other in a hatchery with high embryo/poult mortality. The two turkey RVG strains shared only an 85.3 % nucleotide sequence identity in the VP7 gene while the other genes possessed high nucleotide identity among them (96.3-99.9 %). Low nucleotide percentage identities (31.6-87.3 %) occurred among the pigeon and chicken RVG strains. Interestingly, potential recombination events were detected between our RVG strains and a human RVB strain, in the VP6 and NSP3 segments. The epidemiology of RVG in avian flocks and the pathogenicity of the two different RVG strains should be further investigated to understand the ecology and impact of RVG in commercial poultry flocks.

Longitudinal study of Staphylococcus aureus colonization and infection in a cohort of swine veterinarians in the United States.

BACKGROUND: People working with pigs are at elevated risk of harboring methicillin resistant S. aureus (MRSA) in their nose, which is attributable to occupational exposure to animals harboring livestock adapted S. aureus. To obtain insight into the biological nature of occupationally related nasal culture positivity, we conducted a longitudinal study of 66 swine veterinarians in the USA. METHODS: The study cohort resided in 15 US states and worked predominantly with swine. Monthly for 18 months, participants self-collected nasal swabs and completed a survey to report recent exposure to pigs and other animals; the occurrence of work related injuries; and any relevant health events such as skin and soft tissue infections or confirmed staphylococcal infections. Nasal swabs were cultured using selective methods to determine the presence of MRSA and methicillin susceptible S. aureus (MSSA), and isolates were characterized by spa typing and MLST. RESULTS: Prevalences of S. aureus (64%, monthly range from 58 to 82%) and MRSA (9.5%; monthly range from 6 to15%) were higher than reported for the US population (30% and 1.5% respectively). Predominant spa types were t034 (ST398, 37%), t002 (ST5, 17%) and t337 (ST9/ST398 13%), a distribution similar to that found in a concurrent study in pigs in the USA. Veterinarians were classified into three groups: Persistent carriers (PC, 52%), Intermittent carriers (IC, 47%) and Non-carriers (NC, 1%). Persistent carriage of a single spa type was observed in 14 (21%) of participants, and paired (first and last) isolates from PC subjects had minor genetic differences. Swabs from PC veterinarians carried higher numbers of S. aureus. Among IC veterinarians, culture positivity was significantly associated with recent contact with pigs. CONCLUSIONS: Exposure to pigs did not lead to prolonged colonization in most subjects, and the higher numbers of S. aureus in PC subjects suggests that unknown host factors may determine the likelihood of prolonged colonization by S. aureus of livestock origin. Exposure to S. aureus and persistent colonization of swine veterinarians was common but rarely associated with S. aureus disease.

Longitudinal study of Senecavirus a shedding in sows and piglets on a single United States farm during an outbreak of vesicular disease.

BACKGROUND: The study highlights the shedding pattern of Senecavirus A (SVA) during an outbreak of vesicular disease in a sow farm from the South-central Minnesota, USA. In this study, 34 individual, mixed parity sows with clinical signs of vesicular lesions and 30 individual piglets from 15 individual litters from sows with vesicular lesions were conveniently selected for individual, longitudinal sampling. Serum, tonsil, rectal, and vesicular swabs were collected on day1 post outbreak, and then again at 1, 2, 3, 4, 6, and 9 weeks post outbreak. Samples were tested at the University of Minnesota Veterinary Diagnostic Laboratory for SVA via Real Time Polymerase Chain Reaction (RT-PCR) RESULTS: In sows, vesicular lesions had the highest concentration of SVA, but had the shortest duration of detection lasting only 2 weeks. Viremia was detected for 1 week post outbreak, and quickly declined thereafter. SVA was detected at approximately the same frequency for both tonsil and rectal swabs with the highest percentage of SVA positive samples detected in the first 6 weeks post outbreak. In suckling piglets, viremia quickly declined 1 week post outbreak and was prevalent in low levels during the first week after weaning (4 weeks post outbreak) and was also detected in piglets that were co-mingled from a SVA negative sow farm. Similar to sows, SVA detection on rectal and tonsil swabs in piglets lasted approximately 6 weeks post outbreak. CONCLUSION: The study illustrates the variation of SVA shedding patterns in different sample types over a 9 week period in sows and piglets, and suggests the potential for viral spread between piglets at weaning.

A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials.

BACKGROUND: The use of porcine islets to replace insulin-producing islet ß-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. METHODS: A four-checkpoint program systematically screens the donor herd (Large White - Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. RESULTS: Of 1207 monthly samples taken from random animals over a 2-year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2-year period, were negative for the presence of viruses when co-cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2-year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non-immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV-A, PERV-B, and PERV-C by PCR analysis in all six recipient animals. CONCLUSION: The four-checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.

Evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral RNA at long distances from infected herds.

Porcine epidemic diarrhea virus (PEDV) spread rapidly after being diagnosed in the USA in April 2013. In this study we assessed whether PEDV could become airborne and if so, whether the virus was infectious. Air samples were collected both from a room containing experimentally infected pigs and at various distances from the outside of swine farms experiencing acute PEDV outbreaks. Results indicated presence of infectious PEDV in the air from experimentally infected pigs and genetic material of PEDV was detected up to 10 miles downwind from naturally infected farms. Airborne transmission should be considered as a potential route for PEDV dissemination.

Role of Pre-Farrow Natural Planned Exposure of Gilts in Shaping the Passive Antibody Response to Rotavirus A in Piglets.

Natural planned exposure (NPE) remains one of the most common methods in swine herds to boost lactogenic immunity against rotaviruses. However, the efficacy of NPE protocols in generating lactogenic immunity has not been investigated before. A longitudinal study was conducted to investigate the dynamics of genotype-specific antibody responses to different doses (3, 2 and 1) of Rotavirus A (RVA) NPE (genotypes G4, G5, P[7] and P[23]) in gilts and the transfer of lactogenic immunity to their piglets. Group 1 gilts received three doses of NPE at 5, 4 and 3 weeks pre-farrow (WPF), group 2 received two doses at 5 and 3 WPF, group 3 received one dose at 5 WPF, and group 4 received no NPE (control group). VP7 (G4 and G5) and truncated VP4* (P[7] and P[23]) antigens of RVA were expressed in mammalian and bacterial expression systems, respectively, and used to optimize indirect ELISAs to determine antibody levels against RVA in gilts and piglets. In day-0 colostrum samples, group 1 had significantly higher IgG titers compared to the control group for all four antigens, and either significantly or numerically higher IgG titers than groups 2 and 3. Group 1 also had significantly higher colostrum IgA levels than the control group for all antigens (except G4), and either significantly or numerically higher IgA levels compared to groups 2 and 3. In piglet serum, group 1 piglets had higher IgG titers for all four antigens at day 0 than the other groups. Importantly, RVA NPE stimulated antibodies in all groups regardless of the treatment doses and prevented G4, G5, P[7] and P[23] RVA fecal shedding prior to weaning in piglets in the absence of viral challenge. The G11 and P[34] RVA genotypes detected from pre-weaning piglets differed at multiple amino acid positions with parent NPE strains. In conclusion, the results of this study suggest that the group 1 NPE regimen (three doses of NPE) resulted in the highest anti-RVA antibody (IgG and IgA) levels in the colostrum/milk, and the highest IgG levels in piglet serum.

Frequency of porcine circovirus 3 detection and histologic lesions in clinical samples from swine in the United States.

Porcine circovirus 3 (PCV3) is widespread in pigs worldwide. Diverse clinical signs and lesions have been associated with PCV3, but the role of PCV3 as a cause of disease in swine remains unclear. We investigated the association of PCV3 with clinical signs and histologic lesions in 730 diagnostic swine cases between February 2016 and January 2018. The cases contained 2,177 samples submitted from 474 sites located in 21 states in the United States. PCR assay results were positive for PCV3 for 577 of 2,177 (27%) samples, 255 of 730 (35%) cases, 181 of 474 (38%) sites, and 17 of 21 (81%) states. We detected PCV3 in 19 of 28 specimen types and in pigs of all ages and clinical presentations, including healthy pigs, with the highest detection rate in adult pigs. PCV3 detection was not associated with respiratory, gastrointestinal, or CNS signs, weight loss, or sudden death. Of 58 types of histologic lesions evaluated, PCV3 detection was associated with myocarditis, cardiac vasculitis, and interstitial pneumonia in growing pigs. A high PCV3 detection rate was observed in aborted fetuses.

Antibody Response to Rotavirus C Pre-Farrow Natural Planned Exposure to Gilts and Their Piglets.

A longitudinal study was conducted to investigate the dynamics of genotype-specific (G6 and P[5]) antibody response to different doses (3, 2 and 1) of rotavirus C (RVC) natural planned exposure (NPE) in gilt serum, colostrum/milk and piglet serum, and compare with antibody response to rotavirus A NPE (RVA genotypes G4, G5, P[7] and P[23]). G6 and P[5] antigens of RVC were expressed in mammalian and bacterial cells, and used to develop individual indirect ELISAs. For both antigens, group 1 with 3 doses of NPE resulted in significantly higher IgG and IgA levels in colostrum compared to other groups. In piglet serum, group 1 P[5] IgG levels were significantly higher than other study groups at day 0 and 7. Piglet serum had higher IgA levels for group 1 piglets compared to other groups for both antigens. A comparison of colostrum antibody levels to rotavirus A (RVA) and RVC revealed that colostrum RVC IgG and IgA titers were lower than RVA titers irrespective of the G and P-type. Next generation sequencing (NGS) detected same RVC genotypes (G6 and P[5]) circulating in the piglet population under the window of lactogenic immunity. We conclude that the low RVC load in NPE material (real-time PCR Ct-values 32.55, 29.32 and 30.30) failed to induce sufficient maternal immunity in gilts (low colostrum RVC antibody levels) and passively prevent piglets from natural RVC infection in the farrowing room. To the best of our knowledge, this is the first study comparing differences in antibody response to porcine RVA and RVC in a commercial setting.

Rotavirus Infection in Swine: Genotypic Diversity, Immune Responses, and Role of Gut Microbiome in Rotavirus Immunity.

Rotaviruses (RVs) are endemic in swine populations, and all swine herds certainly have a history of RV infection and circulation. Rotavirus A (RVA) and C (RVC) are the most common among all RV species reported in swine. RVA was considered most prevalent and pathogenic in swine; however, RVC has been emerging as a significant cause of enteritis in newborn piglets. RV eradication from swine herds is not practically achievable, hence producers' mainly focus on minimizing the production impact of RV infections by reducing mortality and diarrhea. Since no intra-uterine passage of immunoglobulins occur in swine during gestation, newborn piglets are highly susceptible to RV infection at birth. Boosting lactogenic immunity in gilts by using vaccines and natural planned exposure (NPE) is currently the only way to prevent RV infections in piglets. RVs are highly diverse and multiple RV species have been reported from swine, which also contributes to the difficulties in preventing RV diarrhea in swine herds. Human RV-gut microbiome studies support a link between microbiome composition and oral RV immunogenicity. Such information is completely lacking for RVs in swine. It is not known how RV infection affects the functionality or structure of gut microbiome in swine. In this review, we provide a detailed overview of genotypic diversity of swine RVs, host-ranges, innate and adaptive immune responses to RVs, homotypic and heterotypic immunity to RVs, current methods used for RV management in swine herds, role of maternal immunity in piglet protection, and prospects of investigating swine gut microbiota in providing immunity against rotaviruses.

Effect of hay type on cecal and fecal microbiome and fermentation parameters in horses.

The effect of hay type on the microbiome of the equine gastrointestinal tract is relatively unexplored. Our objective was to characterize the cecal and fecal microbiome of mature horses consuming alfalfa or Smooth Bromegrass (brome) hay. Six cecally cannulated horses were used in a split-plot design run as a crossover in two periods. The whole plot treatment was ad libitum access to brome or alfalfa hay fed over two 21-d acclimation periods with subplots of sampling location (cecum and rectum) and sampling hour. Each acclimation period was followed by a 24-h collection period where cecal and fecal samples were collected every 3 h for analysis of pH and volatile fatty acids (VFA). Fecal and cecal samples were pooled and sent to a commercial lab (MR DNA, Shallowater, TX) for the amplification of the V4 region of the 16S rRNA gene and sequenced using Illumina HiSeq. The main effects of hay on VFA, pH, and taxonomic abundances were analyzed using the MIXED procedure of SAS 9.4 with fixed effects of hay, hour, location, period, and all possible interactions and random effect of horse. Alpha and beta diversities were analyzed using the R Dame package. Horses fed alfalfa had greater fecal than cecal pH (P ≤ 0.05), whereas horses fed brome had greater cecal than fecal pH (P ≤ 0.05). Regardless of hay type, total VFA concentrations were greater (P ≤ 0.05) in the cecum than in feces, and alfalfa resulted in greater (P ≤ 0.05) VFA concentrations than brome in both sampling locations. Alpha diversity was greater (P ≤ 0.05) in fecal compared with cecal samples. Microbial community structure within each sampling location and hay type differed from one another (P ≤ 0.05). Bacteroidetes were greater (P ≤ 0.05) in the cecum compared with the rectum, regardless of hay type. Firmicutes and Firmicutes:Bacteroidetes were greater (P ≤ 0.05) in the feces compared with cecal samples of alfalfa-fed horses. In all, fermentation parameters and bacterial abundances were impacted by hay type and sampling location in the hindgut.

Proposed virulence-associated genes of Streptococcus suis isolates from the United States serve as predictors of pathogenicity.

BACKGROUND: There is limited information on the distribution of virulence-associated genes (VAGs) in U.S. Streptococcus suis isolates, resulting in little understanding of the pathogenic potential of these isolates. This lack also reduces our understanding of the epidemiology associated with S. suis in the United States and thus affects the efficiency of control and prevention strategies. In this study we applied whole genome sequencing (WGS)-based approaches for the characterization of S. suis and identification of VAGs. RESULTS: Of 208 S. suis isolates classified as pathogenic, possibly opportunistic, and commensal pathotypes, the genotype based on the classical VAGs (epf, mrp, and sly encoding the extracellular protein factor, muramidase-release protein, and suilysin, respectively) was identified in 9% (epf+/mrp+/sly+) of the pathogenic pathotype. Using the chi-square test and LASSO regression model, the VAGs ofs (encoding the serum opacity factor) and srtF (encoding sortase F) were selected out of 71 published VAGs as having a significant association with pathotype, and both genes were found in 95% of the pathogenic pathotype. The ofs+/srtF+ genotype was also present in 74% of 'pathogenic' isolates from a separate validation set of isolates. Pan-genome clustering resulted in the differentiation of a group of isolates from five swine production companies into clusters corresponding to clonal complex (CC) and virulence-associated (VA) genotypes. The same CC-VA genotype patterns were identified in multiple production companies, suggesting a lack of association between production company, CC, or VA genotype. CONCLUSIONS: The proposed ofs and srtF genes were stronger predictors for differentiating pathogenic and commensal S. suis isolates compared to the classical VAGs in two sets of U.S. isolates. Pan-genome analysis in combination with metadata (serotype, ST/CC, VA genotype) was illustrated to be a valuable subtyping tool to describe the genetic diversity of S. suis.

Several lineages of porcine epidemic diarrhea virus in Colombia during the 2014 and 2016 epidemic.

Porcine epidemic diarrhea virus (PEDV) is a significant global, enteric coronavirus in pigs and was first reported in Colombia in 2014. However, the epidemiology, genetic and antigenic characteristics of the virus have yet to be investigated. In this study, we investigated the dissemination of PEDV by testing 536 samples by RT-PCR over a 33-month period. The 35.8% of positive samples (n = 192) was significantly different (p < .01) between months over time, with a higher number of positives samples occurring at the beginning of the epidemic and during the second epidemic wave within the main pork producing region. The complete PEDV genomes were generated for 21 strains, which shared a high nucleotide and amino acid sequence identity, except for the spike (S) gene. Recombinant regions were identified within the Colombian strains and between Colombian and Asian PEDV strains. Phylogenetic analysis of the 21 Colombian strains demonstrated the presence of 7 lineages that shared common ancestors with PEDV strains from the United States. Moreover, the antigenic analysis demonstrated residue differences in the neutralizing epitopes in the spike and nucleocapsid proteins. Our results illustrated the simultaneous introduction of the two PEDV genotypes (GIIa American pandemic and S-INDEL) into the Colombian swine industry during the 2014 PEDV epidemic and enhanced our understanding of the epidemiology and molecular diversity of PEDV in Colombia.

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.

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.

Decline of transmissible gastroenteritis virus and its complex evolutionary relationship with porcine respiratory coronavirus in the United States.

The epidemiology and genetic diversity of transmissible gastroenteritis virus (TGEV) in the United States (US) was investigated by testing clinical cases for TGEV by real time RT-PCR between January 2008 and November 2016. Prevalence of TGEV ranged between 3.8-6.8% and peaked during cold months until March 2013, in which prevalence decreased to < 0.1%. Nineteen complete TGEV genomes and a single strain of porcine respiratory coronavirus (PRCV) from the US were generated and compared to historical strains to investigate the evolution of these endemic coronaviruses. Sixteen of our TGEV strains share 8 unique deletions and 119 distinct amino acid changes, which might greatly affect the biological characteristics of the variant TGEV, and resulted in a "variant" genotype of TGEV. The "variant" genotype shared similar unique deletions and amino acid changes with the recent PRCV strain identified in this study, suggesting a recombination event occurred between the ''variant'' TGEV and PRCV. Moreover, the results indicate the "variant" genotype is the dominant genotype circulating in the US. Therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of TGEV and PRCV circulating in the US.