Abby R. Patterson

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.

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.

Influence of Maternal Antibodies on Efficacy of Porcine Circovirus Type 2 (PCV2) Vaccination To Protect Pigs from Experimental Infection with PCV2

ABSTRACT Due to the ubiquitous nature of porcine circovirus type 2 (PCV2) in the pig population and the increasing use of PCV2 vaccines in breeding herds, the majority of dams have been exposed to field PCV2 or PCV2 vaccines, resulting in piglets with varied levels of passively acquired PCV2 maternal antibodies. The objective of the current research was to investigate the influence of passively acquired anti-PCV2 antibodies on PCV2 vaccine efficacy. Sixty 26-day-old pigs were divided into four groups: vaccinated pigs with no maternal PCV2 antibodies at the time of vaccination (VAC-NEG; n = 9), vaccinated pigs with maternal PCV2 antibodies at the time of vaccination (VAC-POS; n = 21), nonvaccinated pigs with no maternal antibodies at the time of challenge (NVAC-CNEG; n = 15), and nonvaccinated pigs with maternal antibodies at the time of challenge (NVAC-CPOS; n = 15). Vaccinations and challenges were performed on trial days 0 and 28, respectively, according to group designation. The pigs were monitored for clinical signs of disease daily and weighed weekly, and blood was collected weekly. All pigs were necropsied on trial day 49, and tissues were evaluated for macroscopic and microscopic lesions. Serum was evaluated using PCV2 immunoglobulin G (IgG) and PCV2 IgM enzyme-linked immunosorbent assays, quantitative PCV2 PCR, and a serum PCV2 neutralizing antibody test. In comparison to NVAC-CPOS pigs, VAC-POS animals had significantly (P < 0.01) less severe microscopic PCV2-associated lymphoid lesions and significantly (P < 0.04) reduced PCV2 genomic copies in serum following PCV2 challenge. These results indicate that vaccination with Suvaxyn PCV2 One Dose reduces viremia and prevents microscopic lesions associated with PCV2 in the presence of maternal antibodies.

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.

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.

Epidemiology and horizontal transmission of porcine circovirus type 2 (PCV2).

Abstract Porcine circovirus type 2 (PCV2) is a small, non-enveloped, circular, single-stranded DNA virus of economic importance in the swine industry worldwide. Based on the sequence analyses of PCV2 strains, isolates can be divided into five subtypes (PCV2a–e). PCV2 is an ubiquitous virus based on serological and viremia data from countries worldwide. In addition, PCV2 DNA was discovered in archived samples prior to the first recognition of clinical disease. Recently, a worldwide shift in PCV2 subtype from PCV2a to PCV2b occurred. PCV2 DNA can be detected in fecal, nasal, oral and tonsillar swabs as well as in urine and feces from both naturally and experimentally infected pigs. PCV2 DNA can be detected early in the infectious process and persists for extended periods of time. The effectiveness of disinfectants for reducing PCV2 in vitro is variable and PCV2 is very stable in the pig environment. Limited data exist on the horizontal transmission of PCV2. Direct transmission of PCV2 between experimentally or naturally infected animals and naïve animals has been documented and the incorporation of clinical or subclinically infected animals into a population represents a risk to the herd. Indirect transmission through the oral, aerosol or vaccine routes is likely a lesser risk for the transmission of PCV2 in most swine populations but may be worth evaluating in high heath herds. The objective of this review was to discuss data on the epidemiology and horizontal transmission of PCV2.

Effect of Porcine Circovirus Type 2 (PCV2) Vaccination of the Dam on PCV2 Replication In Utero

ABSTRACT The aims of this study were to determine if porcine circovirus type 2 (PCV2) vaccination of the dam is effective in preventing fetal PCV2 infection and reproductive failure. Twelve pregnant, PCV2-naïve sows were randomly divided into four groups, with three sows in each group. Group 1 sows served as noninoculated, nonvaccinated negative controls, group 2 sows were vaccinated with a commercially available PCV2 vaccine at 28 days of gestation and were not inoculated, group 3 sows were vaccinated at 28 days of gestation and inoculated with PCV2b at 56 days of gestation, and group 4 sows were inoculated with PCV2b but were not vaccinated. Serum samples from all sows were collected weekly throughout the gestation period, and sows were allowed to farrow naturally. At parturition, sow colostrum samples, presuckle serum samples, and tissues from the piglets were collected. Reproductive failure was not observed under the study conditions. PCV2 vaccination induced PCV2-specific immunoglobulin G and serum neutralizing antibodies in sows from groups 2 and 3 and prevented detectable PCV2 viremia in the dams after challenge. In group 3, PCV2 DNA was detected in colostrum samples, fetuses, and live-born pigs; however, microscopic lesions and PCV2-specific antigen were not present in any of the fetuses in this group. The results from this study indicate that vertical transmission of PCV2 can occur in PCV2-vaccinated dams.

Porcine circovirus type 2 in muscle and bone marrow is infectious and transmissible to naïve pigs by oral consumption.

Pork products are a possible source of introduction of PCV2 isolates into a pig population. However, limited work has been done on the transmission through meat of porcine circovirus type 2 (PCV2), a virus associated with several disease syndromes in pigs. The objectives of this study were to determine if pork products from PCV2-infected pigs contain PCV2 DNA/antigen and to determine if the PCV2 present in the tissues is infectious by performing in vitro and in vivo studies. Skeletal muscle, bone marrow, and lymphoid tissues from pigs experimentally inoculated with PCV2 were collected 14 days post-inoculation (DPI). The tissues were tested for presence of PCV2 DNA by quantitative real-time PCR, for PCV2 antigen by immunohistochemistry (IHC), and for presence of infectious PCV2 by virus isolation and inoculation of PCV2 naïve pigs. Lymphoid tissues contained the highest amount of PCV2 (positive by PCR, IHC, and virus isolation), bone marrow contained a lower amount of PCV2 (positive by PCR and IHC but negative by virus isolation), and skeletal muscle contained the lowest amount of PCV2 (positive by PCR but negative by IHC and virus isolation). Naïve pigs fed for three consecutive days with either skeletal muscle, bone marrow, or lymphoid tissues all became PCV2 viremic as determined by quantitative real-time PCR on serum starting at 7 DPI. The pigs also seroconverted to PCV2 as determined by PCV2 IgM and IgG ELISA. In addition, PCV2 antigen was detected by IHC stains in lymphoid tissues and intestines collected from the majority of these pigs. Results from this study indicate that uncooked PCV2 DNA positive lymphoid tissues, bone marrow, and skeletal muscle from PCV2 viremic pigs contain sufficient amount of infectious PCV2 to infect naïve pigs by the oral route.

Difference in severity of porcine circovirus type two-induced pathological lesions between Landrace and Pietrain pigs

Anecdotal information from the field suggests that there are host genetic differences in susceptibility to porcine circovirus type 2 (PCV2) associated disease among Landrace and Pietrain breeds. The objective of this study was to determine if a difference exists in PCV2 susceptibility between Landrace and Pietrain pigs under experimental conditions. Thirty-nine Landrace pigs and 39 Pietrain pigs were blocked by breed, sire, dam, and litter and randomly divided into the following 4 groups: Landrace noninoculated negative control (Landrace-NEG; n = 13), Pietrain noninoculated negative control (Pietrain-NEG; n = 13), Landrace-PCV2 (n = 26; Landrace), and Pietrain-PCV2 (n = 26; Pietrain). After waning of passively acquired anti-PCV2 antibodies, Landrace-PCV2 and Pietrain-PCV2 groups were inoculated with PCV2 isolate ISU-40895. The Landrace-NEG and Pietrain-NEG groups were housed in a separate room, remained noninoculated, and served as negative controls. All pigs in all groups were necropsied at 21 d post PCV2-inoculation. Onset of seroconversion and concentrations of anti-PCV2-IgM, anti-PCV2-IgG, and anti-PCV2 neutralizing antibodies were similar in Landrace-PCV2 and Pietrain-PCV2 groups. Furthermore, the amount of PCV2 DNA and cytokine concentrations in serum and plasma samples were not different between the 2 PCV2-inoculated groups. The severity of PCV2-associated microscopic lesions was different between Landrace and Pietrain pigs; Landrace-PCV2 pigs had significantly (P < 0.05) more severe lymphoid lesions than the Pietrain-PCV2 pigs. Although the pigs originated from the same farm where their dams were commingled, passively acquired anti-PCV2-antibodies waned in Pietrain pigs by approximately 12 wk of age, whereas the majority of the Landrace pigs remained PCV2 seropositive until 18 wk of age and beyond. The results from this study indicate that a genetic difference exists between these 2 breeds of pigs in susceptibility to PCV2-associated lesions.

Rescue of a porcine anellovirus (torque teno sus virus 2) from cloned genomic DNA in pigs.

ABSTRACT Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.