Priscilla Freitas Gerber

Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the U.S. concurrently infected with PPV2

Porcine circovirus (PCV) type 2b (PCV2b) emerged in North America in 2005-2006. During May of 2012, PCVAD occurred in 10-18-week-old pigs in two farms within a production system that routinely vaccinated against PCV2. Both farms received replacement gilts from the same multiplier. A mutant PCV2b strain not previously present in North America was identified. The strain was found to be 99.9% identical to a recently described mutant PCV2 isolate reported in China in 2010 and thought to be more virulent than classical PCV2a or PCV2b strains. It is possible that the current PCV2a-based commercial vaccines are not fully protective against this new strain. In addition, emerging porcine parvovirus type 2 (PPV2) was detected in 55% of the serum samples (73/132), perhaps implying that PPV2 could be a cofactor in cases of PCVAD.

A PCV2 vaccine based on genotype 2b is more effective than a 2a-based vaccine to protect against PCV2b or combined PCV2a/2b viremia in pigs with concurrent PCV2, PRRSV and PPV infection.

The predominant genotype of porcine circovirus (PCV) in the pig population today is PCV2b yet PCV2a-based commercial vaccines are considered effective in protecting against porcine circovirus associated disease. The objective of this study was to compare the ability of PCV2a- and PCV2b-based vaccines to control PCV2b viremia in a challenge model that mimics the U.S. field situation. Sixty-three pigs were randomly assigned to one of eight groups. Sixteen pigs were vaccinated with an experimental live-attenuated chimeric PCV1-2a vaccine based on genotype 2a and another 16 pigs with a chimeric PCV1-2b vaccine based on genotype 2b. Challenge was done 28 days post vaccination (dpv) using PCV2b (or a combination of PCV2a and PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) to mimic what commonly occurs in the field. The experiment was terminated 21 days post challenge (dpc) or 49dpv. Pigs vaccinated with the chimeric PCV1-2b vaccine had significantly higher levels of PCV1-2b viremia and shedding of the PCV1-2b vaccine virus in feces and nasal secretions but also a more robust humoral immune response as evidenced by significantly higher ELISA S/P ratios compared to the PCV1-2a vaccination. Regardless of challenge, the PCV1-2b vaccination significantly reduced the prevalence and amount of PCV2 viremia compared to the PCV1-2a vaccination. Interestingly, in the non-vaccinated pigs concurrent PCV2a infection resulted in clinical disease and increased macroscopic lung lesions compared to pigs challenged with PCV2b alone, further supporting the idea that concurrent PCV2a/PCV2b infection is necessary for optimal PCV2 replication.

Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA

Abstract An indirect porcine epidemic diarrhea virus (PEDV) anti-immunoglobulin (Ig) G ELISA based on the S1 portion of the spike protein was validated and compared with an indirect immunofluorescence assay. In serum samples from experimentally infected pigs (n = 35), anti-IgG PEDV antibodies were detected as early as 7 days post-infection. In field serum samples (n = 239), the diagnostic sensitivity of the S1 ELISA was 100% and the diagnostic specificity was 94%. The S1 ELISA showed no cross-reactivity with antibodies against other porcine coronaviruses. Colostrum samples (n = 133) were also tested for anti-PEDV IgG and IgA. The diagnostic sensitivity was 92% for IgG and 100% for IgA, and the diagnostic specificity was 90% for IgG and 99.4% for IgA. These data suggest that the S1 ELISA is a sensitive and specific test that could also be used to evaluate PEDV colostral immunity.

Porcine Epidemic Diarrhea Virus RNA Present in Commercial Spray-Dried Porcine Plasma Is Not Infectious to Naïve Pigs

Porcine epidemic diarrhea virus emerged in North America in April 2013 and has since been identified in 30 U.S. States, Canada and Mexico. The rapid spread of PEDV has raised concerns about the role of feed and particularly pork-by-product components such as spray-dried porcine plasma (SDPP) in PEDV transmission. The aim of this study was to determine the infectivity of PEDV RNA present in commercial SDPP. Specifically, 40 3-week-old PEDV naïve pigs were randomly assigned to one of five treatment groups. At day post inoculation (dpi) 0, NEG-CONTROL pigs were sham-inoculated, PEDV-CONTROL pigs received cell culture propagated PEDV, and SDPP-CONTROL pigs were switched to a diet with 5% SDPP containing 5.1±0.1 log10 PEDV RNA copies/g. To evaluate a potential positive effect of anti-PEDV antibodies in SDPP on PEDV challenge, four days prior to PEDV challenge the pigs in the SDPP-PEDV group were switched to and remained on a 5% SDPP diet through dpi 28. Another group, EGG-PEDV, was orally administered a commercial egg-derived liquid PEDV globulin product from dpi -4 through 6. All PEDV-CONTROL pigs began shedding PEDV in feces by dpi 3 and seroconverted between dpi 7 and 14, whereas pigs in NEG-CONTROL and SDPP-CONTROL groups remained PEDV RNA negative and did not seroconvert to PEDV for the study duration. This indicates no evidence of infectivity of the PEDV RNA in the SDPP lot utilized. Furthermore, under the study conditions SDPP or egg-derived liquid PEDV globulin addition did not significantly alter PEDV-shedding or overall disease course after experimental challenge.

Identification and characterization of novel porcine astroviruses (PAstVs) with high prevalence and frequent co-infection of individual pigs with multiple PAstV types

Many astrovirus (AstV) species are associated with enteric disease, although extraintestinal manifestations in mammalian and avian hosts have also been described. In this study, the prevalence rates of porcine AstV types 1-5 (PAstV1-PAstV5) were investigated using faecal samples from 509 pigs of which 488 (95.9%) came from farms with a history of diarrhoea. All of the five known PAstV types were found to circulate in pigs in the USA, and co-infection of a single pig with two or more PAstV types was frequently observed. A high overall prevalence of 64.0% (326/509) of PAstV RNA-positive samples was detected, with 97.2% (317/326) of the PAstV RNA-positive pigs infected with PAstV4. Further genomic sequencing and characterization of the selected isolates revealed low sequence identities (49.2-89.0%) with known PAstV strains, indicating novel types or genotypes of PAstV2, PAstV4 and PAstV5. Some new features of the genomes of the PAstVs were also discovered. The first complete genome of a PAstV3 isolate was obtained and showed identities of 50.5-55.3% with mink AstV and the novel human AstVs compared with 38.4-42.7% with other PAstV types. Phylogenetic analysis revealed that PAstV1, PAstV2 and PAstV3 were more closely related to AstVs from humans and other animals than to each other, indicating past cross-species transmission and the zoonotic potential of these PAstVs.

The spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma.

Abstract Porcine epidemic diarrhea virus (PEDV) is considered an emergent pathogen associated with high economic losses in many pig rearing areas. Recently it has been suggested that PEDV could be transmitted to naïve pig populations through inclusion of spray-dried porcine plasma (SDPP) into the nursery diet which led to a ban of SDPP in several areas in North America and Europe. To determine the effect of spray-drying on PEDV infectivity, 3-week-old pigs were intragastrically inoculated with (1) raw porcine plasma spiked with PEDV (RAW-PEDV-CONTROL), (2) porcine plasma spiked with PEDV and then spray dried (SD-PEDV-CONTROL), (3) raw plasma from PEDV infected pigs (RAW-SICK), (4) spray-dried plasma from PEDV infected pigs (SD-SICK), or (5) spray-dried plasma from PEDV negative pigs (SD-NEG-CONTROL). For the spray-drying process, a tabletop spray-dryer with industry-like settings for inlet and outlet temperatures was used. In the RAW-PEDV-CONTROL group, PEDV RNA was present in feces at day post infection (dpi) 3 and the pigs seroconverted by dpi 14. In contrast, PEDV RNA in feces was not detected in any of the pigs in the other groups including the SD-PEDV-CONTROL group and none of the pigs had seroconverted by termination of the project at dpi 28. This work provides direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious PEDV in the plasma. Additionally, plasma collected from PEDV infected pigs at peak disease did not contain infectious PEDV. These findings suggest that the risk for PEDV transmission through commercially produced SDPP is minimal.

Serum antibodies and shedding of infectious porcine circovirus 2 into colostrum and milk of vaccinated and unvaccinated naturally infected sows.

Serum antibodies and shedding of porcine circovirus type 2 (PCV2) into lacteal secretions were examined in naturally infected sows. Total (TA) and neutralising (NA) antibodies against PCV2 were evaluated in serum and colostrum from 20 vaccinated (Vac) and 21 unvaccinated (N-vac) sows. Anti-PCV2 IgA titres and PCV2 infectious titres were determined in colostrum and milk. All sows had high TA and NA levels in serum and colostrum. Infectious PCV2 was detected in 22/41 colostrum samples (7/20 Vac and 15/21N-Vac sows) and 5/20 milk samples (1/5 Vac and 4/15N-Vac sows). Anti-PCV2 IgA was found in high levels in colostrum and varying levels in milk. Infectious PCV2 may be present in milk and colostrum of naturally infected sows, even in the presence of NA.

Identification of recently described porcine parvoviruses in archived North American samples from 1996 and association with porcine circovirus associated disease.

The association of porcine circovirus (PCV) type 2 and porcine parvovirus (PPV) type 1 as a cause of porcine circovirus associated disease (PCVAD) is well established. The objective of this study was to investigate the prevalence rates of classical PPV1 and recently recognized PPV2-5 in serum and lung samples from pigs and farms with known PCV2 status. A total of 586 serum samples and 164 lung homogenates collected from 1996 to 2013 in the USA and Canada were utilized. All samples were tested for PPV1-5 and PCV2. PCV2 was detected in 27.7% (162/586) and PPV in 48.8% (286/586) of the serum samples, whereas 78.7% (129/164) of the lung tissues were positive for PCV2 and 56.7% (93/164) were positive for PPV. Overall, PPV2 had the highest prevalence rates in sera (35.2%) and tissues (42.7%). Concurrent infection of PCV2 and PPV occurred in 14.3% (84/586) of the serum samples and in 49.4% (81/164) of the tissue samples. Moreover, the prevalence of PPV1 or PPV2 DNA was significantly higher in tissues containing high amounts of PCV2 DNA compared to non-PCVAD cases. The frequency of concurrent PPV/PCV2 infection was higher for PCVAD herds compared to negative or subclinically infected herds. PPV2, PPV3 and PPV4 were all identified in samples collected in 1998 and PPV5 was first identified in 2006. The obtained findings indicate that similar to PCV2, PPVs are widespread in North American pigs. Nevertheless, diagnostic investigations into PCVAD cases should give more consideration to the role of PPV1 and PPV2 as contributing cofactors.

Commercial PCV2a-based vaccines are effective in protecting naturally PCV2b-infected finisher pigs against experimental challenge with a 2012 mutant PCV2

Current commercial PCV2 vaccines are all based on PCV2a and have been shown to be effective in reducing PCV2a and PCV2b viremia and PCV2-associated lesions and disease. The recent emergence of novel mutant PCV2 (mPCV2) strains and linkage of mPCV2 with cases of porcine circovirus associated disease (PCVAD) in vaccinated herds have raised concerns over emergence of vaccine-escape mutants and reduced efficacy of PCV2a-based vaccines. The aim of this study was to determine the ability of three commercial PCV2a-based vaccines administered in the presence of an ongoing PCV2b infection and passively-acquired anti-PCV2 antibodies to protect conventional pigs against experimental challenge with mPCV2 at 11 weeks of age. Fifty naturally PCV2b-infected 2-week-old pigs were divided into five treatment groups with 10 pigs each. Pigs were unvaccinated (positive and negative controls) or vaccinated at 3 (VAC-A, VAC-B, VAC-C) and at 5 weeks of age (VAC-C). At 11 weeks of age, all pigs except the negative controls were challenged with a 2012 U.S. strain of mPCV2. The experiment was terminated 21 days after challenge. Under the conditions of this study, vaccinated pigs were protected against PCV2 viremia and lesions whereas non-vaccinated pigs were not. Moreover, concurrent PCV2b and mPCV2 infection was demonstrated in all positive controls and 3/10 had microscopic lesions consistent with PCVAD while negative controls infected with PCV2b alone did not develop PCVAD. The results indicate that concurrent PCV2b/mPCV2 infection can trigger PCVAD development and that commercial vaccines are effective in protecting conventional pigs against emerging mPCV2 strains.

A commercial vaccine based on PCV2a and an experimental vaccine based on a variant mPCV2b are both effective in protecting pigs against challenge with a 2013 U.S. variant mPCV2b strain

During 2012 and 2013, an apparent increase in porcine circovirus associated disease occurred in the USA. A variant PCV2b strain designated mPCV2b was recovered from many of these cases. This raised concerns of a decrease in efficacy of commercially available PCV2 vaccines. The objective of this study was to compare the ability of a commercial PCV2a-based vaccine and an experimental mPCV2b-based vaccine to control mPCV2b-associated disease, lesions, and viremia in a challenge model. Twenty-six caesarian-derived, colostrum-deprived pigs were randomly assigned to one of four groups: (1) vaccinated with a commercial PCV2a-based vaccine and challenged (PCV2a-VAC; n=7), (2) vaccinated with an experimental mPCV2b-based vaccine and challenged (mPCV2b; n=7), (3) sham-vaccinated with saline and challenged (positive controls; n=7), and (4) sham-vaccinated with saline without challenge (negative controls; n=5). Vaccination was done on D0 and D14, challenge was done on D28 using a tissue homogenate containing PRRSV and mPCV2b and the experiment was terminated on D49. Among the challenged pigs, 47.6% (10/21) developed severe clinical disease and either died or had to be humanely euthanized between D39 and D48 (11-20 days after challenge). PCV2 viremia was almost completely absent in the vaccinated groups regardless of vaccine type except for two PCV2a-vaccinated pigs which had detectable PCV2 DNA levels on individual days after challenge. Microscopic lesions typical of PCV2 infection were limited to the positive control group which developed mild-to-severe lesions associated with low-to-abundant PCV2 antigen. Under the conditions of this study, PCV2 vaccines regardless of PCV2 type were effective against mPCV2b challenge.