Darin M. Madson

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90–95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6–100% identity among the PCR amplicons from the 4 farms and 97–99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6–99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011–2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6–100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States

ABSTRACT Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 102 to 2 × 105 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

Differences in virulence among porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and prior infection provides heterologous protection

Porcine circovirus type 2 (PCV2) is divided into two genetic clusters designated PCV2a and PCV2b. The objectives of this study were to determine whether isolates from different clusters vary in virulence and to determine whether infection with PCV2a isolates induces protective immunity against subsequent infection with a recent PCV2b isolate. One-hundred and thirteen conventional specific-pathogen-free (SPF) pigs were assigned randomly to treatment groups and rooms: pigs inoculated with PCV2a cluster isolates (ISU-40895 or ISU-4838), pigs inoculated with PCV2b cluster isolates (NC-16845 or Can-17639) and uninoculated pigs. Necropsies were performed at 16 or 51 days post-inoculation (p.i.). There were no significant differences in PCV2-associated lymphoid lesions between PCV2a and PCV2b clusters; however, within the same cluster, significant differences were found between isolates: ISU-4838- and Can-17639-inoculated pigs had significantly (P<0.05) less severe lesions compared with ISU-40895- and NC-16845-inoculated pigs. To evaluate cross-protection, six pigs within each group were challenged at 35 days p.i. with an isolate from the heterologous cluster and were necropsied 51 days p.i. The severity of PCV2-associated lesions was reduced in pigs with prior exposure to an isolate from the heterologous cluster in comparison with singly inoculated pigs. Results indicate that the virulence of PCV2a and PCV2b isolates is not different in the conventional SPF pig model; however, the virulence of isolates within the same cluster differs. Increased virulence as reported to be associated with PCV2b isolates in the field was not observed under the conditions of this study. Moreover, cross-protection between PCV2a and PCV2b exists.

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1 mL of 1 × 10(3) PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.

Comparison of efficacy of commercial one dose and two dose PCV2 vaccines using a mixed PRRSV–PCV2–SIV clinical infection model 2–3-months post vaccination

The study objectives were to compare the duration of immunity of commercially available, one and two dose, killed porcine circovirus type 2 (PCV2) vaccines. Sixty, 3.5-week-old pigs were randomly divided into six treatment groups: one dose vaccines (FDAH-1, BIVI-1), two dose vaccines (Intervet-2, FDAH-2), and non-vaccinated negative and positive controls. Tissue homogenate challenge was conducted 63 (two doses) or 84 (one dose) days post vaccination. Viremia was reduced by 78.5% in pigs vaccinated with one dose and by 97.1% in pigs vaccinated with two dose products and overall microscopic lymphoid lesions were reduced by 78.7% and 81.8%, respectively.

Reproductive Failure Experimentally Induced in Sows via Artificial Insemination with Semen Spiked with Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) is associated with reproductive failure in female pigs. However, the association of PCV2-positive semen in the pathogenesis has not been elucidated. The objectives of this study were to determine whether semen spiked with PCV2 causes infection in PCV2-naïve, mature female pigs and whether delivery of PCV2 via artificial insemination causes reproductive failure or fetal infection. Nine sows were randomly allocated into 3 groups of 3 sows each and artificially inseminated with PCV2 DNA-negative semen (group 1), PCV2 DNA-negative semen spiked with PCV2a (group 2), or PCV2b (group 3). All sows in groups 2 and 3 developed PCV2 viremia 7 to 14 days after insemination. None of the group 2 sows became pregnant, whereas all group 3 sows (3/3) farrowed at the expected date. At parturition, presuckle serum samples were collected, and live-born piglets, stillborn fetuses, and mummified fetuses were necropsied. All live-born piglets (n = 8) in group 3 were PCV2 viremic at birth. Stillborn fetuses (n = 2) had gross lesions of congestive heart failure. Mummified fetuses (n = 25) varied in crown-rump length from 7 to 27 cm, indicating fetal death between 42 and 105 days of gestation. PCV2 antigen was detected in the myocardium by immunohistochemistry of 7/8 (88%) live-born piglets, 2/2 (100%) of the stillborn fetuses, and 25/25 (100%) of the mummified fetuses. In addition, 4/25 mummified fetuses had PCV2 antigen associated with smooth muscle cells and fibroblasts. The results of this study indicate that intrauterine administration of PCV2 causes reproductive failure in naïve sows.

Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection

The objectives were to determine if PCV2 vaccination is effective in reducing disease and lesions associated with PRRSV and PCV2 coinfection and if there is a difference between intradermal (ID) and intramuscular (IM) route of PCV2 vaccination. Seventy-four, 21-day-old pigs were randomly allocated into one of six groups. On day 0, pigs were vaccinated with 2ml Suvaxyn PCV2 One Dose (Fort Dodge Animal Health, Inc.) by intramuscular (VAC-M-COINF) or intradermal (VAC-D-COINF) routes. On day 28, pigs were either singularly (PRRSV-only, PCV2-only) or coinfected (COINF) with PRRSV and PCV2. All pigs in all groups were necropsied on day 42. All vaccinated pigs seroconverted (IgM, IgG, and neutralizing antibodies) to PCV2 between 14 and 28 days post-vaccination. After challenge, all groups inoculated with PRRSV had reduced average daily gain compared to CONTROLS and PCV2-only (P<0.001). COINF pigs had significantly (P<0.05) reduced anti-PCV2-IgG antibody levels and neutralizing antibody levels compared to both vaccinated groups. COINF pigs had more severe lung lesions compared to VAC-M-COINF (P<0.05). COINF pigs had higher amounts of PCV2 DNA in serum samples and feces (P<0.05) and increased amounts of PCV2 in lymphoid tissues (P<0.05) compared to both vaccinated groups. In summary, PCV2 vaccination was effective at inducing a neutralizing antibody response and significantly reducing PCV2-associated lesions and PCV2 viremia in pigs coinfected with PCV2 and PRRSV. Differences between intradermal and intramuscular routes of vaccine administration were not observed.

Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets

Abstract Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2009–2010 and reported in United States swine for the first time in February 2014. However, diagnostic tools other than polymerase chain reaction for PDCoV detection were lacking and Koch׳s postulates had not been fulfilled to confirm the pathogenic potential of PDCoV. In the present study, PDCoV peptide-specific rabbit antisera were developed and used in immunofluorescence and immunohistochemistry assays to assist PDCoV diagnostics. The pathogenicity and pathogenesis of PDCoV was investigated following orogastric inoculation of 5-day-old piglets with a plaque-purified PDCoV cell culture isolate (3×104 TCID50 per pig). The PDCoV-inoculated piglets developed mild to moderate diarrhea, shed increasing amount of virus in rectal swabs from 2 to 7 days post inoculation, and developed macroscopic and microscopic lesions in small intestines with viral antigen confirmed by immunohistochemistry staining. This study experimentally confirmed PDCoV pathogenicity and characterized PDCoV pathogenesis in neonatal piglets.

Comparison of the effectiveness of passive (dam) versus active (piglet) immunization against porcine circovirus type 2 (PCV2) and impact of passively derived PCV2 vaccine-induced immunity on vaccination.

The objectives of this study were (1) to compare the efficacy of two different PCV2 vaccination protocols (colostrum-derived immunity versus piglet vaccination) in a conventional PCV2 growing pig challenge model and (2) to evaluate the efficacy of vaccinating piglets with the same vaccine used in the dams. Two different commercially available vaccines (VAC1; VAC2) were used in the same experiment. Seventy-eight piglets born to vaccinated or non-vaccinated sows were divided into 8 groups. A proportion of the pigs with and a proportion of the pigs without passively acquired immunity were vaccinated at 21 days of age. All pigs except negative controls were challenged with PCV2b at 35 days post-vaccination and necropsied at 21 days post-challenge (dpc). The data indicates that both dam vaccination and piglet vaccination had similar efficacies in reducing PCV2 viral loads and antigen levels in the growing pigs. Interestingly, dam vaccination alone did result in significantly (P<0.05) lower anti-PCV2-antibodies levels at challenge in piglets from dams immunized with VAC2 compared to piglets from VAC1 immunized dams. When data obtained from the growing piglets that were vaccinated with VAC1 or VAC2 were compared, antibody levels and reduction of incidence of PCV2-antigen were not different; however, piglets vaccinated with VAC2 had reduced PCV2-DNA genomic copies in serum by 21 dpc. Vaccination of piglets with the same vaccine as was used on their dams did not appear to affect vaccine efficacy as piglets in these groups had anti-PCV2-antibody levels and PCV2 genomic copies similar to the groups where vaccine was administered to the piglets only.

Effect of porcine circovirus type 2 (PCV2) infection on reproduction: disease, vertical transmission, diagnostics and vaccination

Abstract Porcine circovirus type 2 (PCV2) causes great economic losses in growing pigs and there are several reviews on disease manifestations and lesions associated with PCV2 in growing pigs. Reproductive failure in breeding herds, predominately associated with increased numbers of mummies and non-viable piglets at parturition, is one of the disease manifestations of PCV2 infection. Boars shed low amounts of infectious PCV2 in semen for extended time periods, and vertical transmission of PCV2 to fetuses during PCV2 viremia of the dam has been experimentally confirmed. However, intrauterine-infected piglets often are clinically normal. Nevertheless, pigs infected with PCV2 by the intrauterine route can be born viremic, possibly contributing to horizontal spread of PCV2 within the breeding herd and into the nursery. Shedding of PCV2 in semen and prevalence of intrauterine-infected piglets can both be greatly reduced by PCV2 vaccination well ahead of expected PCV2 exposure. This review is a discussion on current knowledge on the effects of PCV2 infection in the dam and in in utero fetuses, including clinical signs, lesions, diagnosis and prevention through vaccination. Infection of boars with PCV2, the potential for PCV2 transmission via semen and prevention of PCV2 shedding are also discussed.