The objective of this study was to compare rotavirus shedding and performance of piglets from gilts immunized using natural planned exposure (NPE) or an RNA particle vaccine (RPV) prior to farrowing following initial NPE pre-breeding. A final total of 117 farrowed gilts and their piglets were enrolled into 4 groups. All gilts received two administrations of NPE prior to breeding. Gilts in group 1 were later given three NPE administrations at 5, 4, and 3 weeks prior to farrowing (WPF). Group 2 was dosed with an RNA particle vaccine (RPV) at 5 and 3 WPF and group 3 at 1 WPF only. Group 4 (control group) did not receive any NPE or RPV. Fecal samples from gilts and fecal swabs from their piglets were tested for rotavirus A (RVA), rotavirus B (RVB), and rotavirus C (RVC) by qRT-PCR. The 117 gilt samples were tested individually at 5 sampling points from pre-breeding to entry into the farrowing rooms. Piglet samples were pooled by litter (3 piglets sampled per litter) and tested at 3, 7, 14, and 21 days of age. The clinical and production impact of the treatments were assessed by comparing average adjusted weaning weights, the percentage of piglets weighing in the bottom 10% of study piglets at weaning, the percentage of piglets placed in the nursery, and the percentage of litters with the presence of diarrhea. No statistically significant differences were identified but there appeared to be several interesting numerical trends that are both biologically plausible and consistent among treatment groups. The control group, which received no pre-farrow immunization, had the highest percentage of litters with diarrhea at all time points and overall in the farrowing house. A numerical trend was also observed on average cycle threshold (Ct) values for RVC in piglets. Interestingly, the control group had the lowest numerical average Ct value at 7, 14, and 21 days of age. Ct values for RVA were consistently lower than for RVC in the stock solution and natural planned exposure gruel. This likely correlated to a lower level of RVC exposure in all gilts pre-breeding and group 1 gilts pre-farrow. This may have an inverse correlation to the higher levels of shedding of RVC in piglets. The lack of statistical significance between treatment groups with regard to diarrhea and weights in the piglets may be attributed to sample size, lack of sufficient natural rotavirus challenge, pre-breeding NPE in all gilts, or a variety of other reasons. Replication of this study with a larger sample size, using a challenge model, or relocated to a farm with increased natural rotavirus clinical challenges may yield different results. If beneficial under different conditions, the RPV would eliminate many of the risks associated with NPE administration, including continuous introduction of live virus on-farm, potential spread of other pathogens, difficulty of isolating on-farm strains, and labor and cost of producing NPE material. Limitations of the original research include the use of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to measure the levels of RVA and RVC in the stock solution and gruel and to monitor RV shedding, rather than techniques with the ability to differentiate live and non-viable virus. Additionally, these studies were performed on an all-gilt breeding farm. It may be more challenging to appreciate differences in treatment groups on a multiparous sow farm due to the higher level of immunity correlated to increasing parity. These immunization protocols were designed to be applicable in a field setting for routine rotavirus immunization, though their feasibility must be carefully considered before implementation on each individual farm.

Rotaviruses (RVs) are significant enteric pathogens of humans and animals. In March 2021, the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) received a fecal sample from a 1-week-old goat kid with diarrhea from a farm with 5 additional diarrheic kids. The fecal sample was tested negative for Bovine coronavirus, Bovine rotavirus Group A, E. coli K99+, Cryptosporidium parvum and Salmonella spp by multiplex real-time PCR assays. Interestingly, a novel Equine Rotavirus B (ERVB) in Kentucky was identified from neonatal foals also with watery diarrhea in the Spring of 2021. Once the ERVB-specific real-time PCR assay became available, the fecal sample from the goat kid was tested and found positive for RVB. Genome sequence of the caprine RVB from fecal sample was obtained using shotgun metagenomic sequencing by Illumina MiSeq. All of the eleven viral segments of caprine RVB were sequenced either completely or partially. Genetic and phylogenetic analysis of VP7, VP4, VP6, VP1–VP3, and NSP1- NSP5 genes indicated the caprine RVB strain had the genotype constellation of G3-P[3]-I3- R3-C3-M3-A4-N3-T3-E3-H3. In addition, the caprine RVB sequence showed the highest nucleotide identity and evolutionary relationship to the ERVB and previously sequenced caprine RVB strains. Given the similar geographical location of the equine and caprine strains from our study, these findings suggest a possible common source of infection.

Objective: The objectives of this study were to determine the pattern of rotavirus A (RVA), rotavirus B (RVB), and rotavirus C (RVC) shedding in gilts after natural planned exposure (NPE) administration and assess the effects on piglet weaning weight, preweaning mortality, and RV shedding. Materials and methods: A total of 70 pregnant gilts were enrolled and allocated into 4 groups. Group 1 was given NPE at 5, 4, and 3 weeks prefarrowing (WPF); Group 2 at 5 and 3 WPF; and Group 3 at 5 WPF only. Group 4 (control group) did not receive any NPE. Samples from 46 gilts and litters (5 piglets/litter) were tested at 12 sample times. Piglets were sampled weekly from 24 hours of age until 6 weeks of age and tested by quantitative reverse transcriptase-polymerase chain reaction for RVA, RVB, and RVC. Results: There was a significant improvement in weaning weight of piglets born to gilts that received 3 NPE administrations compared to fewer or no NPE administrations. Shedding of RVA and RVB from piglets were well controlled in the farrowing room regardless of treatment group, but RVC was observed as early as 1 week old. This study was conducted on a single farm, and the results should be carefully interpreted with knowledge of variations in farms and systems. Implications: Three administrations of NPE to gilts prefarrowing had valuable production and economic benefits for the producer. Circulation patterns of RVA, RVB, and RVC appear to correlate; interventions for one have value against the others.

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.

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.

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.

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.

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.

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.

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.

An emerging porcine sapelovirus was isolated in a diagnostic specimen from a US swine farm, designated as PSV KS18-01. Full-length genome sequence was obtained through next-generation sequencing. Phylogenetic analysis showed that the virus is more closely related to two Japanese strains but is distantly related to two known US strains. PSV specific diagnostic tools were developed, including the monoclonal antibodies again VP1 and VP2, and a VP1-VP2 antigen-based indirect ELISA. Using this assay, the dynamic response of PSV antibody was investigated in a group of post-weaned pigs that naturally exposed with PSV. The availability of the PSV isolate (KS18-01) and the specific diagnostic reagents and assays provide important tools for PSV control and prevention. Porcine Sapelovirus (PSV), previously named as porcine enterovirus 8, belongs to the genus Sapelovirus in the family Picornaviridae (1). PSV is a non-enveloped, positive-sense singlestranded RNA virus. It has a genome of approximately 7.5 kb, with a typical genomic organization of 5’ untranslated region -Leader-polyprotein (P1-P2-P3)-3’UTR. The polyprotein is synthesized and proteolytically cleaved into seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D) and four structural proteins (VP4, VP2, VP3, VP1) (2). The PSV capsid is icosahedral arrangement with VP1, VP2, and VP3 on the surface, while VP4 embedded in the internal side of the virion. Host antibodies are mainly directed toward the viral capsid proteins, in which VP1 was reported having the ability to stimulate strong antibody response during the infection (2). PSV infection is commonly asymptomatic, but clinical disease of respiratory failure, diarrhea, reproductive disorder, and polioencephlamyelitis have been reported in swine farms from many countries (3-5). Neurotropic strains have been reported from China, Indian, the United Kingdom, and the United States (US). However, little is known about the disease prevalence, morbidity and mortality caused by PSV infection. PSV isolates and diagnostic tools are need for the studies of PSV pathogenesis and epidemiology. In this study, we isolated an emerging PSV in a fecal sample from the US swine farm. The virus isolation was performed on ST cells using the method as described previously (6). The new isolate was designated as PSV KS18-01. The full-length genome sequence was obtained through metagenomic sequencing (7). The genome size of KS18-01 was 7,453 bp, encoding a polyprotein of 2,316 amino acids (aa). Compared to other PSV strains, KS18-01 genome has 79.88-87.91% nucleotide (nt) identity. The 5’-untranslated region (5’-UTR) is the most

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.

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.

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 escapein 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.

We developed a model to predict the cyclic pattern of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by reverse-transcription real-time PCR (RT-rtPCR) from 4 major swine-centric veterinary diagnostic laboratories (VDLs) in the United States and to use historical data to forecast the upcoming year’s weekly percentage of positive submissions and issue outbreak signals when the pattern of detection was not as expected. Standardized submission data and test results were used. Historical data (2015–2017) composed of the weekly percentage of PCR-positive submissions were used to fit a cyclic robust regression model. The findings were used to forecast the expected weekly percentage of PCR-positive submissions, with a 95% confidence interval (CI), for 2018. During 2018, the proportion of PRRSV-positive submissions crossed 95% CI boundaries at week 2, 14–25, and 48. The relatively higher detection on week 2 and 48 were mostly from submissions containing samples from wean-to-market pigs, and for week 14–25 originated mostly from samples from adult/sow farms. There was a recurring yearly pattern of detection, wherein an increased proportion of PRRSV RNA detection in submissions originating from wean-to-finish farms was followed by increased detection in samples from adult/sow farms. Results from the model described herein confirm the seasonal cyclic pattern of PRRSV detection using test results consolidated from 4 VDLs. Wave crests occurred consistently during winter, and wave troughs occurred consistently during the summer months. Our model was able to correctly identify statistically significant outbreak signals in PRRSV RNA detection at 3 instances during 2018.

Rotaviruses (RVs) are the leading cause of the acute viral gastroenteritis in young children and livestock animals worldwide. Although live attenuated vaccines have been applied to control RV infection for many years, the underlying mechanisms of RV attenuation following cell culture adaption are unknown. To study these mechanisms at the genomic level, we have sequenced and conducted a comparative analysis of two virulent human (Wa, G1P[8] and M, G3P[8]) and two virulent porcine (Gottfried, G4P[6] and OSU, G5P[7]) RV strains maintained in gnotobiotic piglets for 22, 11, 12 and 9 serial passages, respectively, with their attenuated counterparts serially passaged in MA-104 cell cultures for 25, 43, 54 and 43 passages, respectively. We showed that most of the mutations were clustered in the VP4 gene, with a relatively high nonsynonymous substitution rate (81.2%). Moreover, two amino acid substitutions observed in the VP4 gene were conserved between two or more strain pairs. D385N substitution was found in M, Wa and Gottfried strains, and another one, S471H/L was present in Wa and Gottfried strains. Importantly, D385 was reported previously in another study and may be involved in regulation of virus entry. Of interest, although no 385 substitution was found in OSU strains, the attenuated OSU strain contained a unique D393H substitution within the same VP4 hydrophobic domain. Collectively, our data suggest that the VP4 hydrophobic region may play an important role in RV attenuation and aa385 and aa393 may represent potential targets for RV vaccine development using reverse genetics and site-specific mutagenesis.

The increased prevalence of porcine group C rotavirus (PRVC) in suckling piglets and the emergence of new genetically distinct PRVC strains are concerning due to the associated significant economic losses they cause to the swine industry. We sequenced and analyzed two new PRVC strains, RV0104 (G3), and RV0143 (G6) and compared their pathogenesis with that of the historic strain Cowden (G1) in gnotobiotic (Gn) pigs. Near complete genome sequence analysis confirmed that these two strains were distinct from one another and the Cowden strain. VP1, VP2, VP6, NSP1-NSP3, and NSP5 genes were more similar between Cowden and RV0143, whereas VP3, VP7, and NSP4 shared higher nucleotide identity between Cowden and RV0104. Three-day-old and 3-week-old Gn piglets were inoculated with 105 FFU/piglet of Cowden, RV0104 or RV0143, or mock. All 3-day-old piglets developed severe diarrhea, anorexia, and lethargy, with mean PRVC fecal shedding titers peaking and numerically higher in RV0104 and RV0143 piglets on post infection day (PID) 2. Histopathological examination of the small intestine revealed that the 3-day-old Cowden and RV0104 inoculated piglets were mildly affected, while significant destruction of small intestinal villi was observed in the RV0143 inoculated piglets. Consistent with the highest degree of pathological changes in the small intestines, the RV0143 inoculated piglets had numerically higher levels of serum IL-17 and IFN-α cytokines and numerically lower PRVC IgA geometric mean antibody titers. Milder pathological changes and overall higher titers of PRVC IgA antibodies were observed in 3-week-old vs. 3-day-old piglets. Additionally, diarrhea was only observed in RV0104 and RV0143 (but not Cowden) inoculated 3-week-old piglets, while levels of serum IL-10 and PRVC IgA antibodies were higher in Cowden inoculated pigs, consistent with the lack of diarrhea. Thus, we confirmed that these current, genetically heterogeneous PRVC strains possess distinct pathobiological characteristics that may contribute to the increased prevalence of PRVC diarrhea in neonatal suckling piglets.

The genus Rotavirus comprises eight species, designated A to H, and two recently identified tentative species I in dogs and J in bats. Species Rotavirus A, B, C and H (RVA, RVB, RVC and RVH) have been detected in humans and animals. While human and animal RVA are well characterized and defined, complete porcine genome sequences in the GenBank are limited compared to human strains. Here, we used a metagenomic approach to sequence the 11 segments of RVA, RVC and RVH strains from piglets in the United States (US) and explore the evolutionary relations of these RV species. Metagenomics identified Astroviridae, Picornaviridae, Caliciviridae, Coronoviridae in samples MN9.65 and OK5.68 while Picobirnaviridae and Arteriviridae were only identified in sample OK5.68. Whole genome sequencing and phylogenetic analyses identified multiple genotypes with the RVA of strain MN9.65 and OK5.68, with the genome constellation of G5/G9-P[7]/P[13]-I5/I5- R1/R1-C1-M1-A8-N1-T7-E1/E1-H1 and G5/G9-P[6]/P[7]-I5-R1/R1-C1-M1-A8-N1-T1/T7-E1/E1-H1, respectively. The RVA strains had a complex evolutionary relationship with other mammalian strains. The RVC strain OK5.68 had a genome constellation of G9-P[6]-I1-R1-C5-M6-A5-N1-T1-E1-H1, and shared an evolutionary relationship with porcine strains from the US. The RVH strains MN9.65 and OK5.68 had the genome constellation of G5-P1-I1-R1-C1-M1-A5-N1-T1-E4-H1 and G5-P1-I1-R1-C1-M1-A5-N1-T1-E1-H1, indicating multiple RVH genome constellations are circulating in the US. These findings allow us to understand the complexity of the enteric virome, develop improved screening methods for RVC and RVH strains, facilitate expanded rotavirus surveillance in pigs, and increase our understanding of the origin and evolution of rotavirus species.

An experiment was conducted with 6 cecally cannulated horses (524 ± 65.5 kg BW) to evaluate effects of increasing dietary starch on equine cecal microbiota. Starch was supplied via corn pellets and was increased by 0.5 g starch/kg BW/meal every 7 d until horses received 3.5 g starch/kg BW/meal. Throughout the experiment, Smooth Bromegrass hay and water were offered ad libitum. Meals were fed every 6 h, starting at 0600 h. On d 7 of each period, cecal digesta was collected every 2 h for 12 h. Within period, cecal samples for each horse were pooled and DNA was extracted for PCR amplification of the 16 S rRNA gene (V3 and V4 regions) and sequencing via Illumina MiSeq. Alpha and beta diversity were measured. Data were analyzed as a randomized complete block with fixed effect of starch level and random effect of horse (SAS version 9.4). If a horse presented with colic, it was removed from the experiment. Parameters when feeding 1.5 g starch/kg BW/meal were compared between horses which completed the trial and those removed using covariate of 0 g starch/kg BW/meal. Feeding 1.5 g starch/kg BW/meal elicited the greatest changes in microbiota, indicated by reduced (P ≤ 0.02) operational taxonomical units and Faith phylogenetic diversity and different (P ≤ 0.017) beta diversity compared to all other treatments. Across treatments, Firmicutes was the most abundant phyla, followed by Bacteroidetes. Feeding 1.5 g starch/kg BW/meal led to lowest (P ≤ 0.0342) relative abundance (RA) of Prevotella, Treponema, Phascolarctobacterium, and [Prevotella]. Horses that persisted throughout the experiment had reduced (P = 0.0163) RA of Ruminococcus and greater RA of Phascolarctobacterium (P = 0.0057) when consuming 1.5 g starch/kg BW/meal compared to those removed. This is the first report describing effects of gradually increasing dietary starch on equine cecal microbiota in vivo.