Experimental infection of pregnant goats with bovine viral diarrhea virus (BVDV) 1 or 2.

Infections with bovine viral diarrhea virus (BVDV) of the genus pestivirus, family Flaviviridae, are not limited to cattle but occur in various artiodactyls. Persistently infected (PI) cattle are the main source of BVDV. Persistent infections also occur in heterologous hosts such as sheep and deer. BVDV infections of goats commonly result in reproductive disease, but viable PI goats are rare. Using 2 BVDV isolates, previously demonstrated to cause PI cattle and white-tailed deer, this study evaluated the outcome of experimental infection of pregnant goats. Pregnant goats (5 goats/group) were intranasally inoculated with BVDV 1b AU526 (group 1) or BVDV 2 PA131 (group 2) at approximately 25-35 days of gestation. The outcome of infection varied considerably between groups. In group 1, only 3 does became viremic, and 1 doe gave birth to a stillborn fetus and a viable PI kid, which appeared healthy and shed BVDV continuously. In group 2, all does became viremic, 4/5 does aborted, and 1 doe gave birth to a non-viable PI kid. Immunohistochemistry demonstrated BVDV antigen in tissues of evaluated fetuses, with similar distribution but reduced intensity as compared to cattle. The genetic sequence of inoculated viruses was compared to those from PI kids and their dam. Most nucleotide changes in group 1 were present during the dam's acute infection. In group 2, a similar number of mutations resulted from fetal infection as from maternal acute infection. Results demonstrated that BVDV may cause reproductive disease but may also be maintained in goats.

Rapid detection of high consequence and emerging viral pathogens in pigs.

Introduction: An increasing emergence of novel animal pathogens has been observed over the last decade. Viruses are a major contributor to the increased emergence and therefore, veterinary surveillance and testing procedures are greatly needed to rapidly and accurately detect high-consequence animal diseases such as Foot and Mouth Disease, Highly Pathogenic Avian Influenza, Classical Swine Fever, and African Swine Fever. The major detection methods for such diseases include real-time PCR assays and pathogen-specific antibodies among others. However, due to genetic drift or -shift in virus genomes, failure to detect such pathogens is a risk with devastating consequences. Additionally, the emergence of novel pathogens with no prior knowledge requires non-biased detection methods for discovery. Methods: Utilizing enrichment techniques coupled with Oxford Nanopore Technologies MinION™ sequencing platform, we developed a sample processing and analysis pipeline to identify DNA and RNA viruses and bacterial pathogens from clinical samples. Results and discussion: The sample processing and analysis pipeline developed allows the identification of both DNA and RNA viruses and bacterial pathogens simultaneously from a single tissue sample and provides results in less than 12 h. Preliminary evaluation of this method using surrogate viruses in different matrices and using clinical samples from animals with unknown disease causality, we demonstrate that this method can be used to simultaneously detect pathogens from multiple domains of life simultaneously with high confidence.

The Role of Histopathology in Ruminant Diagnostics.

Histopathology remains an important tool for ruminant disease diagnostic investigations. Some ruminant diseases require histopathology to make a definitive diagnosis. Clinical history, proper tissue sampling and handling, and proper fixation all increase the efficiency of a histopathologic examination and the likelihood of an accurate diagnosis. This article discusses some of the main organ systems of ruminants and highlights common ruminant diseases encountered by diagnosticians where histopathology is particularly important. Where applicable, correlative gross lesions, special considerations regarding tissue sampling, and histologic report interpretation are discussed.

Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis.

Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real-time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4°C or 25°C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4°C for 5 days with a mean Cq 26.34 (95% CI: 23.11-29.58) and detected at -20°C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73-31.37). A significant decrease in detectable RNA was observed in samples containing <10 parasites/extraction at -20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs.

DETECTION OF SARS-COV-2 NEUTRALIZING ANTIBODIES IN RETROPHARYNGEAL LYMPH NODE EXUDATES OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) FROM NEBRASKA, USA.

Disease surveillance testing for emerging zoonotic pathogens in wildlife is a key component in understanding the epidemiology of these agents and potential risk to human populations. Recent emergence of SARS-CoV-2 in humans, and subsequent detection of this virus in wildlife, highlights the need for developing new One Health surveillance strategies. We used lymph node exudate, a sample type that is routinely collected in hunter-harvested white-tailed deer (WTD, Odocoileus virginianus) for surveillance of chronic wasting disease, to assess anti-SARS-CoV-2 neutralizing antibodies. A total of 132 pairs of retropharyngeal lymph nodes collected from Nebraska WTD harvested in Nebraska, US, in 2019 (pre-SARS-CoV-2 pandemic) and 2021 (post-SARS-CoV-2 pandemic) were tested for SARS-CoV-2 with reverse transcription PCR. Thereafter, exudates obtained from these same lymph nodes were tested for SARS-CoV-2 neutralizing antibodies using a surrogate virus neutralization test. Neutralizing antibodies were detected in the exudates with high diagnostic specificity (100% at proposed cutoff of 40% inhibition). Application of this testing approach to samples collected for use in other disease surveillance activities may provide additional epidemiological data on SARS-CoV-2 exposure, and there is further potential to apply this sample type to detection of other pathogens of interest.

Detection of Mannheimia haemolytica-Specific IgG, IgM and IgA in Sera and Their Relationship to Respiratory Disease in Cattle.

Mannheimia haemolytica is one of the major causes of bovine respiratory disease in cattle. The organism is the primary bacterium isolated from calves and young cattle affected with enzootic pneumonia. Novel indirect ELISAs were developed and evaluated to enable quantification of antibody responses to whole cell antigens using M. haemolytica A1 strain P1148. In this study, the ELISAs were initially developed using sera from both M. haemolytica-culture-free and clinically infected cattle, then the final prototypes were tested in the validation phase using a larger set of known-status M. haemolytica sera (n = 145) collected from feedlot cattle. The test showed good inter-assay and intra-assay repeatability. Diagnostic sensitivity and specificity were estimated at 91% and 87% for IgG at a cutoff of S/P ≥ 0.8. IgM diagnostic sensitivity and specificity were 91% and 81% at a cutoff of sample to positive (S/P) ratio ≥ 0.8. IgA diagnostic sensitivity was 89% whereas specificity was 78% at a cutoff of S/P ≥ 0.2. ELISA results of all isotypes were related to the diagnosis of respiratory disease and isolation of M. haemolytica (p-value < 0.05). These data suggest that M. haemolytica ELISAs can be adapted to the detection and quantification of antibody in serum specimens and support the use of these tests for the disease surveillance and disease prevention research in feedlot cattle.

The pseudorabies virus R2 non-neuroinvasive vaccine: A proof-of-concept study in pigs.

Neurotropic alpha-herpesviruses that infect mammals establish life-long latent infections in the peripheral nervous system after initial infection of exposed mucosal tissues. The neuroinvasive properties can lead to severe complications both with clinical and veterinary alpha-herpesviruses, and vaccines are often unavailable or provide limited protection. Here we assess the properties and efficacy of an R2 vaccine derived from the alpha-herpesvirus, pseudorabies virus (PRV), in pigs. We demonstrate that the PRV R2 vaccine does not invade the porcine peripheral nervous system within the limits of detection. Furthermore, after a single intranasal vaccination, R2 conferred protection to pigs subsequently challenged with a virulent PRV field strain (NIA-3). These findings support that the R2 vaccine design is non-neuroinvasive and is an effective vaccine in the context of a natural host.

Geographic variation in the PRNP gene and its promoter, and their relationship to chronic wasting disease in North American deer.

PRNP genotypes, number of octarepeats (PHGGGWGQ) and indels in the PRNP promoter can influence the progression of prion disease in mammals. We found no relationship between presence of promoter indels in white-tailed deer and mule deer from Nebraska and CWD presence. White-tailed deer with the 95 H allele and G20D mule deer were more likely to be CWD-free, but unlike other studies white-tailed deer with the 96S allele(s) were equally likely to be CWD-free. We provide the first information on PRNP genotypes and indels in the promoter for Key deer (all homozygous 96SS) and Coues deer (lacked 95 H and 96S alleles, but possessed a uniquely high frequency of 103 T). All deer surveyed were homozygous for three tandem octarepeats.

Upregulation of the type I interferon pathway in feedlot cattle persistently infected with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) has a profound economic impact on the cattle industry. Calves infected in utero and born persistently infected (PI) with BVDV have increased morbidity, mortality, and reduced productivity. Further, they serve as a continual source of viral exposure to herd mates and thereby pose a significant risk to animal wellbeing and production efficiency. Understanding the mechanisms through which PI is established and maintained is therefore important in working toward finding means to prevent or mitigate losses due to infection. Early studies of acute infection suggested BVDV infection alters the host's ability to mount a type I interferon (IFN) response, thereby allowing for the establishment of PI. More recently, however, animals experimentally challenged with the virus demonstrated a chronic yet modest upregulation of the IFN pathway. To identify if the IFN or other pathways are altered due to PI by BVDV in a natural infection, the circulating blood transcriptome was analyzed from PI feedlot cattle (N = 10 BVDV1a, 8 BVDV1b, 8 BVDV2), cattle co-mingling with PI cattle but not themselves infected (N = 9), and a group of unrelated, unexposed controls (N=10). Differential expression analyses included contrasts among BVDV subtypes, and all pair-wise comparisons of PI, co-mingled non-PI, and unexposed animals. Analyses in limma-voom revealed no difference in the transcriptome based upon the BVDV genotype with which the animal was infected. However, gene expression did differ (adj P < 0.05 and |logFC|> 1) at 175 loci between the PI and co-housed, non-PI contemporaries and when compared to the unexposed controls, 489 loci were differentially expressed. Pathway analyses predict that alterations in the transcriptome of the PI cattle indicate significant upregulation of innate immune function including IFN signaling. These data support prior work suggesting IFN signaling is not completely suppressed in cattle naturally PI with BVDV.

Prevalence of bovine viral diarrhea virus infections in alpacas in the United States.

OBJECTIVE: To determine the prevalence of bovine viral diarrhea virus (BVDV)-infected alpaca herds in the United States and investigate factors associated with seropositive herd status and, subsequently, determine the proportion of animals within seropositive alpaca herds that are persistently infected (PI) carriers for BVDV, obtain information regarding previous herd exposure to BVDV, determine titers of anti-BVDV antibodies of dams, and ascertain whether individual seropositive crias had received supplemental colostrum at birth. DESIGN: Prevalence study. ANIMALS: 63 alpaca herds with >or= 12 registered female alpacas. PROCEDURES: 250 alpaca breeders were randomly selected from 562 eligible herds listed in the Alpaca Owner and Breeders Association membership directory and mailed a voluntary participation request. Sixty-three alpaca breeders participated in the study. From each herd, blood samples from >or= 4 crias were tested for BVDV, BVDV RNA, and serum neutralizing antibodies against BVDV. A region of the genome of BVDV recovered from PI crias was sequenced to determine genetic homology. RESULTS: Among the 63 herds, 16 (25.4%) had seropositive crias and 4 (6.3%) had PI crias. Infections in 3 of the 4 herds with PI crias were linked as evidence by the genetic homologies of viruses. In addition to PI crias, feeding supplemental colostrum was associated with herd seropositivity. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the importance of BVDV infections in alpacas in the United States and highlighted the importance of determining the BVDV infection status of animals before they are commingled to limit exposure of herds to BVDV infection.

Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories.

This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Reproductive tract disease associated with inoculation of pregnant white-tailed deer with bovine viral diarrhea virus.

OBJECTIVE: To inoculate white-tailed deer (Odocoileus virginianus) during the sixth or seventh week of gestation with bovine viral diarrhea virus (BVDV) and observe for signs of reproductive tract disease during a 182-day period. ANIMALS: 10 pregnant white-tailed deer (8 seronegative and 2 seropositive [control deer] for BVDV). PROCEDURES: Deer were inoculated with 1 of 2 deer-derived BVDV strains (RO3-20663 or RO3-24272). Serum anti-BVDV antibody titers were determined prior to and 21 or 35 days after inoculation. Virus isolation (VI) procedures were performed on tissues from fetuses and does that died and on blood samples collected from live fawns. Ear notch specimens obtained from live fawns were assessed by use of BVDV antigen-capture ELISA (ACE). RESULTS: Both RO3-20663-inoculated seropositive deer gave birth to apparently normal fawns. Among the RO3-24272-inoculated seronegative deer, 1 died, and 1 aborted and 1 resorbed their fetuses; among the RO3-20663-inoculated seronegative deer, 3 died, 1 aborted its fetus, and 1 gave birth to 2 fawns that were likely persistently infected. On the basis of VI and ACE results, those 2 fawns were positive for BVDV; both had no detectable neutralizing anti-BVDV antibodies in serum. CONCLUSIONS AND CLINICAL RELEVANCE: Reproductive tract disease that developed in pregnant white-tailed deer following BVDV inoculation was similar to that which develops in BVDV-exposed cattle. Methods developed for BVDV detection in cattle (VI, immunohistochemical evaluations, and ACE) can be applied in assessments of white-tailed deer. Fawns from does that had serum anti-BVDV antibodies prior to inoculation were protected against BVDV infection in utero.

Efficacy of tulathromycin or enrofloxacin for initial treatment of naturally occurring bovine respiratory disease in feeder calves.

After undergoing arrival processing at one of two commercial feedlots, feeder calves with clinical signs of bovine respiratory disease (BRD) were randomly assigned to receive either tulathromycin (2.4 mg/kg SC) or enrofloxacin (12.5 mg/kg SC). Additional therapy for calves that did not respond to initial treatment followed a prescribed course. Initial treatment with tulathromycin resulted in significantly higher (P = .009 and P = .031 at sites 1 and 2, respectively) therapeutic success (87.9% and 80%, respectively) than did initial treatment with enrofloxacin (70.2% and 62.5%, respectively). Animals treated with tulathromycin also had fewer subsequent treatments and higher weight gains compared with those treated with enrofloxacin.

Factors associated with practice decisions of Nebraska veterinarians regarding type of practice and community size.

Nebraska veterinary practitioners were surveyed to collect data about background characteristics and other factors related to veterinarians' decision to include or not include food animals in their practices and to practice in rural versus urban communities. Background characteristics that were significantly (p < or = 0.05) associated with choosing food-animal practice included growing up on a working farm or ranch; having parents who owned livestock; growing up in a town with a population of less than 10,000; majoring in animal science at university; being male; and having a primary interest, at the time of entering veterinary college, in food animal-exclusive or mixed-animal veterinary practice. The primary factor for choosing the community in which to practice was rural/urban lifestyle for rural veterinarians, while this factor was second for urban veterinarians. For all groups of veterinarians, the primary consideration in selecting their current practice was the species orientation of the practice. The primary reason for not choosing food-animal practice was better working conditions and lifestyle in companion-animal practice, followed by greater interest elsewhere.

Effectiveness of composting as a biosecure disposal method for porcine epidemic diarrhea virus (PEDV)-infected pig carcasses.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is an enteric disease of swine that has emerged as a worldwide threat to swine herd health and production. Substantial research has been conducted to assess viability of the virus on surfaces of vehicles and equipment, in feed and water, and on production building surfaces, but little is known about the persistence in PEDV-infected carcasses and effective disposal methods thereof. This study was conducted to quantify the persistence of PEDV RNA via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) at various time-temperature combinations and in infected piglet carcasses subjected to composting. Although this method does not distinguish between infectious and noninfectious virus, it is a rapid and sensitive test to evaluate materials for evidence of virus genome. RESULTS: In the first study, PEDV was suspended in cell culture media at 1 × 105 TCID50 per sample (1 mL sample size) and subjected to various time and temperature combinations in triplicate including temperatures of 37, 45, 50, 55, 60, 65, 70 °C and exposure times of 0, 1, 2, 3, 4, 5, 7, and 14 days. At all temperatures, viral RNA copies declined over time, with the decline most marked and rapid at 65 and 70 °C. Detectable RNA did persist throughout the trial in all but the most extreme condition, where two of three samples incubated at 70 °C yielded undetectable viral RNA after 14 days. In the second study, PEDV-infected piglet carcasses were subjected to two cycles of composting lasting 36 and 37 days, respectively, for a total compost time of 73 days. Composting was performed in triplicate windrow sections housed inside biosecure, climate-controlled rooms using insulated bins designed to represent a continuous windrow compost pile. Temperatures reached 35-57 °C for 26 days of cycle 1 and 35-45 °C for 3 days of cycle 2. Samples consisting of carbon material with or without decomposed tissue as available per sample site collected at ten locations throughout the cross-section of each windrow section following the primary and secondary compost cycles yielded no detectable viral RNA. CONCLUSIONS: Composting appears to be an effective disposal method for PEDV-infected piglet carcasses under the conditions examined. The combination of time and high temperature of the compost cycle effectively degraded viral RNA in cell culture media that should provide optimum stability. Complex compost material matrices collected from windrow sections yielded undetectable PEDV RNA by qRT-PCR after one 36-day compost cycle despite incomplete decomposition of soft tissue.

Evaluation of reproductive protection against bovine viral diarrhea virus and bovine herpesvirus-1 afforded by annual revaccination with modified-live viral or combination modified-live/killed viral vaccines after primary vaccination with modified-live viral vaccine.

The objective of this study was to compare reproductive protection in cattle against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) provided by annual revaccination with multivalent modified-live viral (MLV) vaccine or multivalent combination viral (CV) vaccine containing temperature-sensitive modified-live BoHV-1 and killed BVDV when MLV vaccines were given pre-breeding to nulliparous heifers. Seventy-five beef heifers were allocated into treatment groups A (n=30; two MLV doses pre-breeding, annual revaccination with MLV vaccine), B (n=30; two MLV doses pre-breeding, annual revaccination with CV vaccine) and C (n=15; saline in lieu of vaccine). Heifers were administered treatments on days 0 (weaning), 183 (pre-breeding), 366 (first gestation), and 738 (second gestation). After first calving, primiparous cows were bred, with pregnancy assessment on day 715. At that time, 24 group A heifers (23 pregnancies), 23 group B heifers (22 pregnancies), and 15 group C heifers (15 pregnancies) were commingled with six persistently infected (PI) cattle for 16days. Ninety-nine days after PI removal, cows were intravenously inoculated with BoHV-1. All fetuses and live offspring were assessed for BVDV and BoHV-1. Abortions occurred in 3/23 group A cows, 1/22 group B cows, and 11/15 group C cows. Fetal infection with BVDV or BoHV-1 occurred in 4/23 group A offspring, 0/22 group B offspring, and 15/15 group C offspring. This research demonstrates efficacy of administering two pre-breeding doses of MLV vaccine with annual revaccination using CV vaccine to prevent fetal loss due to exposure to BVDV and BoHV-1.

Use of a modified-live vaccine to prevent persistent testicular infection with bovine viral diarrhea virus.

A commercial vaccine containing modified-live bovine viral diarrhea virus (BVDV; types 1 and 2) was administered to one group of 22 peripubertal bulls 28 days before intranasal inoculation with a type 1 strain of BVDV. A second group of 23 peripubertal bulls did not receive the modified-live BVDV vaccine before intranasal inoculation. Ten of 23 unvaccinated bulls--but none of the vaccinated bulls--developed a persistent testicular infection as determined by immunohistochemistry and polymerase chain reaction. Results of this study indicate that administration of a modified-live vaccine containing BVDV can prevent persistent testicular infection if peripubertal bulls are vaccinated before viral exposure.

Resolving Bovine viral diarrhea virus subtypes from persistently infected U.S. beef calves with complete genome sequence.

Bovine viral diarrhea virus (BVDV) is classified into 2 genotypes, BVDV-1 and BVDV-2, each of which contains distinct subtypes with genetic and antigenic variation. To effectively control BVDV by vaccination, it is important to know which subtypes of the virus are circulating and how their prevalence is changing over time. Accordingly, the purpose of our study was to estimate the current prevalence and diversity of BVDV subtypes from persistently infected (PI) beef calves in the central United States. Phylogenetic analysis of the 5'-UTR (5' untranslated region) for 119 virus strains revealed that a majority (82%) belonged to genotype 1b, and the remaining strains were distributed between genotypes 1a (9%) and 2 (8%); however, BVDV-2 subtypes could not be confidently resolved. Therefore, to better define the variability of U.S. BVDV isolates and further investigate the division of BVDV-2 isolates into subtypes, complete genome sequences were obtained for these isolates as well as representatives of BVDV-1a and -1b. Phylogenetic analyses of the complete coding sequence provided more conclusive genetic classification and revealed that U.S. BVDV-2 isolates belong to at least 3 distinct genetic groups that are statistically supported by both complete and individual coding gene analyses. These results show that a more complex set of BVDV-2 subtypes has been circulating in this region than was previously thought.

Experimental infection of conventional nursing pigs and their dams with Porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2-4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1-8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3-4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14-35 dpi.