Porcine circovirus type 2 (PCV2) is the primary component of porcine circovirus-associated disease (PCVAD), a systemic infection that can result in mortality rates of 70 to 80% in clinically affected pigs (Opriessnig et al., 2007). The virus was first associated with disease in the late 1990s (Allan et al., 1998), and between 2004 and 2005 an increase in the number and severity of PCVAD cases was reported in Canada (Carman et al., 2006), Kansas, Iowa, and North Carolina (Cheung et al., 2007). A strain of PCV2, PCV2b, not previously isolated in North America, was identified (Cheung et al., 2007). The source and route of transmission of PCV2b in North America is unknown. Potential routes of PCV2 transmission include direct contact with infected animals and contaminated fomites or vertical transmission. Horizontal transmission of PCV2 through spray-dried plasma (SDP) products is another hypothesis.
Incorporating SDP protein into the diet of weanling pigs to improve feed intake and growth performance, especially after disease challenge has been well documented (Hansen et al., 1993; Coffey and Cromwell, 2001; Lawrence et al., 2004; Zhao et al., 2007; Torrallardona, 2010). Porcine circovirus type 2 is a small circular DNA virus and has been shown to be extremely resistant to inactivation as evidenced by a 1-log reduction after dry-heat treatment of freeze-dried PCV2 at 120°C for 30 min (Welch et al., 2006) and resistance to a 15-min 70°C heat treatment in cell culture (O’Dea et al., 2008).
Commercially manufactured SDP protein containing 2.47 × 105 PCV2 genomic copies/mL fed to weaning pigs did not result in seroconversion or viremia during a 45-d observation period (Pujols et al., 2008). However, pigs had a small amount of maternal PCV2 antibodies before initiation of the trial (Pujols et al., 2008).
To investigate the infectivity of PCV2 after the spray-drying process, SDP derived from a pig experimentally infected with PCV2b was tested in a swine bioassay using PCV2-naïve animals.
MATERIALS AND METHODS
The experimental protocol in this study was approved by the Iowa State University Institutional Animal Care and Use Committee.
Animals and Housing
Colostrum-fed, crossbred, specific-pathogen-free (SPF) pigs were purchased from a herd that is routinely tested for major swine pathogens and known to be free of PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, and swine influenza virus (SIV). Twelve pigs obtained from 2 litters were weaned at 3 wk of age and transported to the Livestock Infectious Disease Isolation Facility at Iowa State University, Ames. Upon arrival, the pigs were ear-tagged, randomly divided into groups of 3 pigs using a random permutation generator (freeware available online at http://www.randomization.com), and housed in 4 separate rooms; there were no partitions separating the 3 animals within each room. Each room had 18 m2 of solid concrete floor space, separate ventilation systems, and 1 nipple drinker. All groups were fed a balanced, pelleted, complete feed ration free of animal proteins (with the exception of whey), and antibiotics (Nature’s Made, Heartland Coop, Alleman, IA) once per day.
Pigs were left un-inoculated (NEG), inoculated with an experimentally produced reconstituted PCV2-infected SDP product intraperitoneally (SDP-IP) or by oral gavage (SDP-OG), or inoculated intraperitoneally with plasma from a PCV2-infected pig (POS). Blood samples [8.5-mL serum separator tube (Fisher Scientific, Pittsburgh, PA), immediately centrifuged at 2,000 × g for 10 min at 4°C] were collected weekly after inoculation for 7 wk and tested for the presence of anti-PCV2-IgG antibodies and PCV2 DNA.
PCV2-Contaminated Plasma Source
The source of plasma inoculum was blood from a colostrum-fed, crossbred, SPF pig experimentally infected with 5 × 104.0 50% tissue culture infectious dose (TCID50) of PCV2b (GenBank EU340258) at 3 wk of age as part of a separate study (A. R. Patterson, unpublished data). At 35 d postinoculation (DPI), the pig was killed with an overdose of pentobarbital (Fatal Plus, Vortech Pharmaceutical, Dearborn, MI) due to development of clinical signs consistent with PCVAD including severe dyspnea, diarrhea, and loss of condition. After euthanasia, 2 L of blood were collected in jars containing 14,300 USP Heparin Units (Hospira Inc., Lake Forest, IL)/L of blood. The plasma was immediately centrifuged at 2,000 × g for 10 min at 4°C in 50-mL centrifuge tubes and stored at 4°C until use. The plasma was tested for the presence of PCV2 DNA by quantitative PCV2 PCR; 7.75 log10 PCV2 genomic copies/mL of plasma were detected. The diagnosis of PCVAD in the donor pig was further confirmed by the presence of intense PCV2 antigen staining by immunohistochemistry (IHC; Sorden et al., 1999) in the lungs, intestine, and lymphoid tissues. Transmissible gastroenteritis virus, rotavirus, and Lawsonia intracellularis were not detected by IHC. Evaluation for PRRSV (Prickett et al., 2008), SIV (Richt et al., 2004), and Mycoplasma hyopneumoniae (Calsamiglia et al., 1999) by PCR were negative. The IHC assays and PCR tests were performed according to the standard operating procedures for the Iowa State University Veterinary Diagnostic Laboratory.
Three hundred milliliters of collected plasma was spray-dried using a bench-top spray dryer (Yamato Model ADL310, Yamato Scientific Co. Ltd., Tokyo, Japan). To ensure that the spray dryer was not contaminated with PCV2 before the initiation of the run, it was disinfected with an oxidizing disinfectant (Virkon S, Dupont, Pharmacal Research Laboratories Inc., Naugatuck, CT) according to the manufacturers’ recommendations. Ten swabs were then taken from various components of the apparatus, placed in sterile saline, and confirmed to be PCV2-negative by real-time PCR (Opriessnig et al., 2003).
For drying of the plasma, the bench-top spray-drier manufacturers’ recommendations were followed. A 0.4-mm nozzle was used with the following parameters: Tinlet (inlet air temperature) of 166°C, aspiration rate of 0.6 m3/min, Toutlet (outlet temperature) of 67°C, and an 820 mL/h sample flow rate under 0.1 MPa of pressure. The resulting SDP product was stored at 4°C until use, at which time it was reconstituted in sterile saline to a concentration of 0.33 g/mL. This concentration was based on the amount of available product, the number of animals in the study and the amount of saline needed to reconstitute the SDP product. Some of the differences between the experimentally produced SDP used in this study and commercially produced SDP include, but are not limited to, the source animal(s), disease status, pooling effects, processing temperatures, product retention time, and postdrying conditions (Table 1).
Pigs in the SDP-IP and SDP-OG groups were inoculated with 3 mL of the reconstituted SDP product as described above. Pigs in the POS group were inoculated intraperitoneally with 3 mL of the untreated plasma. Pigs in the NEG group were sham-inoculated intraperitoneally with 3 mL of sterile saline.
Blood samples were collected on the day of inoculation, and weekly thereafter until 49 DPI. The blood was collected in 8.5-mL serum separator tubes (Fisher Scientific), immediately centrifuged at 2,000 × g for 10 min at 4°C and stored at −80°C until use. Serum samples were tested by an open-reading frame (ORF) 2-based PCV2 IgG ELISA as described previously and were considered positive if the calculated sample-to-positive (S/P) ratio was 0.2 or greater (Nawagitgul et al., 2002).
PCV2 DNA Quantification
Extraction of DNA from serum samples was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Extracts of DNA were used for quantification of the PCV2 genomic DNA copy numbers by real-time PCR. Described previously primers for ORF1 of PCV2 (Opriessnig et al., 2003) were used for quantification of the PCV2 genomic DNA copy numbers by real-time PCR. The PCR reaction consisted of 25-µL PCR mixtures that contained 12.5 µL of commercially available master mix (TaqMan Universal PCR Master Mix, Applied Biosystems, Carlsbad, CA), 2.5 µL of DNA extract, 1 µL of forward and reverse primers, and 0.5 µL of detection probe with concentrations of 10 µM. On each plate 5 progressive 1:10 dilutions of a known copy number of PCV2 genomic DNA excised from a purified PCV2 DNA clone were included to generate a standard curve. Each plate was run in the sequence detection system (7500 Sequence Detection System, Applied Biosystems) under the following conditions: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C.
Polymerase chain reaction products that were amplified from a virus recovered from a 49 DPI serum sample from an animal in the SDP-IP, SDP-OG, and POS groups was sequenced and compared with the inoculum. A nested PCR was used to amplify the entire ORF2 gene for sequencing (Opriessnig et al., 2006b). Polymerase chain reaction products were purified using the QIAquick PCR-purification kit (Qiagen) per the manufacturers’ directions and sequenced at the Iowa State University DNA facility. Sequences were analyzed with Sequence Scanner 1.0 (Applied Biosystems) and compared with the sequence of the inoculum using the BLAST (basic local alignment search tool; Zhang et al., 2000).
All pigs were necropsied at 49 DPI. Severity of macroscopic lung lesions (scored from 0 to 100% of the lung affected) and the size of lymph nodes [score ranged from 0 to 3; 0 (normal), 1 (2 times the normal size), 2 (3 times the normal size), 3 (4 times the normal size)] were estimated in a blinded fashion as described previously (Opriessnig et al., 2006a). Sections of lymph nodes (superficial inguinal, mediastinal, tracheobronchial, mesenteric), tonsil, thymus, ileum, kidney, colon, spleen, and liver were collected at necropsy and fixed in 10% neutral-buffered formalin and routinely processed for histological examination.
Histopathology and IHC
Microscopic lesions were evaluated by a pathologist that was blinded to treatment groups. Lung sections were scored for the presence and severity of interstitial pneumonia ranging from 0 (normal) to 4 (severe interstitial pneumonia) as described previously (Halbur et al., 1995). Sections of heart, liver, kidney, ileum, and colon were evaluated for the presence of lymphohistiocytic inflammation and scored from 0 (none) to 3 (severe). Lymphoid tissues including lymph nodes, tonsil, and spleen were evaluated for the presence of lymphoid depletion ranging from 0 (normal) to 3 (severe) and histiocytic inflammation and replacement of follicles ranging from 0 (normal) to 3 (severe; Opriessnig et al., 2004).
Detection of PCV2-specific antigen in selected formalin-fixed and paraffin-embedded sections of lymph nodes (superficial inguinal, mediastinal, tracheobronchial, and mesenteric), tonsil, spleen, Peyer’s patches, and thymus was performed by using IHC and a rabbit polyclonal antiserum as described previously (Sorden et al., 1999). Antigen scoring of PCV2 was done by a pathologist blinded to treatment groups. Scores ranged from 0 (no signal) to 3 (more than 50% of the lymphoid follicles contain cells with PCV2-antigen staining; Opriessnig et al., 2004).
An overall microscopic lymphoid lesions score that accounts for lymphoid depletion, histiocytic inflammation, and PCV2 antigen present in lymphoid tissues was calculated for each pig as described previously (Opriessnig et al., 2004) and ranged from 0 (normal) to 9 (severe).
To test the null hypothesis that there was no effect of time on differences between the groups, a repeated-measures ANOVA was used (James and McCulloch, 1990). The experimental unit was the individual pig. For the model, group (NEG, SDP-IP, SDP-OG, and POS) was the fixed, independent variable and the continuous data (log-transformed genomic copies/mL or anti-PCV2-IgG S/P ratios) were dependent variables. If a significant effect (P < 0.05) was noted in the repeated measures analysis, a 1-way ANOVA was performed at each DPI using the described previously model to assess significant differences between groups. For statistical analysis of lymphoid lesions, a 1-way ANOVA was performed using the overall microscopic lymphoid lesions score as a dependent variable and group (NEG, SDP-IP, SDP-OG, and POS) as the fixed, independent variable in the ANOVA. If an ANOVA was significant, pairwise comparisons using the Tukey-Kramer adjustment were done to determine which groups were different. Statistical analysis was performed using JMP 7 (SAS Inst. Inc., Cary, NC).
Clinical Presentation and Macroscopic Lesions
Clinical disease was not observed in any of the pigs for the duration of the study. No remarkable gross lesions were observed. Individual pigs in the POS, SDP-IP, and SDP-OG groups had slightly enlarged (P > 0.05) mediastinal lymph nodes compared with the NEG group.
Anti-PCV2-IgG Antibody Concentrations
Group mean and SE for anti-PCV2-IgG antibody S/P ratios are presented in Figure 1. Pigs within the NEG group remained seronegative throughout the duration of the trial. By 21 DPI, 2/3 of the pigs in the POS group seroconverted and by 28 DPI all 3 pigs had seroconverted. In the SDP-IP group, 2/3 of the pigs seroconverted on 35 DPI and by 49 DPI all of the pigs had seroconverted. In the SDP-OG group, 2/3 of the pigs seroconverted by 35 DPI and 1 animal remained negative through 49 DPI. Throughout the trial, there were no significant (P > 0.05) differences between anti-PCV2-IgG antibody S/P ratios among pigs in the POS, SDP-OG, or SDP-IP groups. At 49 DPI, POS, SDP-OG, and SDP-IG pigs had significantly (P < 0.05) greater group mean S/P ratios compared with the NEG group.
PCV2 DNA Quantification and Sequencing
Group means and SE for log10-transformed PCV2 genomic copies/mL are presented in Figure 2. Pigs within the NEG group remained negative throughout the duration of the trial. In the POS group, all pigs were viremic by 14 DPI. In the SDP-IP group, 1 of the 3 pigs was viremic on 14 DPI, 2 of the 3 pigs were viremic on 21 DPI, and by 35 DPI all 3 pigs were viremic. In the SDP-OG groups, 1 of the 3 pigs became viremic at 14 DPI, 2 of the 3 pigs were viremic by 28 DPI, and all 3 pigs were viremic at 35 DPI. A difference (P < 0.05) in amount of PCV2 DNA was observed between the POS and the SDP-IP at 14 DPI and between the SDP-IP and SDP-OG groups at 49 DPI (Figure 2). No other differences were noted between the POS, SDP-IP, and SDP-OG groups. By 35 DPI, all groups had significantly (P < 0.05) greater amounts of PCV2 DNA compared with the NEG group. Sequence analysis of the PCV2 recovered from 1 pig from each group that became viremic had 100% similarity with the inoculum.
Histopathology and IHC
Pigs within the NEG control group had no microscopic lesions associated with PCVAD (overall microscopic lymphoid lesions score of 0) and no lung lesions. Pigs within the POS control group had a mean overall microscopic lymphoid lesions score (±SE) of 3.3 ± 0.2. Specifically, there was mild-to-moderate lymphoid depletion and histiocytic replacement of lymphoid follicles in 3 of 3 pigs that was associated with small amounts of PCV2 antigen in 2 of the 3 pigs in the POS control group. Pigs within the SDP-IP and SDP-OG groups had a mean overall microscopic lymphoid lesions score of 3.7 ± 2.8 and 2.7 ± 1.2, respectively. Specifically, there was moderate lymphoid depletion and histiocytic replacement of follicles in lymphoid tissues in 1 of 3 animals and small-to-moderate amounts of PCV2 antigen were demonstrated in 3 of the 3 animals in the SDP-IP group. In the SDP-OG group, there was mild lymphoid depletion in lymphoid tissues in 3 of 3 animals and small-to-moderate amounts of PCV2 antigen were present in 2 of the 3 animals. There was no significant (P > 0.05) difference in overall microscopic lymphoid lesions scores among the POS, SDP-IP, and SDP-OG groups. Lesion scores in all groups were numerically greater in comparison to NEG pigs; however, the difference was significant (P < 0.05) only for the SDP-IP group. Significant differences in lung lesions were not observed (P > 0.05) among treatment groups.
Porcine circovirus type 2 is known to be shed in feces, urine, nasal, and oral secretions (Segalés et al., 2005) and remain in tissues for extended periods of time (Bolin et al., 2001). Vertical transmission of the virus has also been documented (Madson et al., 2009). The combination of multiple shedding routes with the ability of the viruses to withstand environmental stress leads to the possibility of horizontal transmission. Potential routes for horizontal transmission include direct contact with infected animals or indirect contact with contaminated fomites, aerosols, or mechanical vectors. Based on previous work, direct contact with infected animals is likely more efficient than other sources of transmission (Andraud et al., 2008).
A potential mechanism for the spread of PCV2b that has not been thoroughly investigated is transmission through SDP products. However, several studies have reported lack of viability of viruses in SDP. Pseudorabies virus and PRRSV were not detected in a study where bovine plasma was spiked with the aforementioned viruses, spray-dried, and tested in cell culture for presence of infectious virus (Polo et al., 2005). In addition, commercially produced SDP product incorporated into the diet of weanling pigs did not result in seroconversion to any of the tested viruses (Polo et al., 2005). Similarly, when porcine plasma was spiked with 106 TCID50 swine vesicular disease virus (SVDV)/mL, virus was not detected in spray-dried samples by virus isolation (Pujols et al., 2007). In contrast to PRRSV, PRV and even other stable, nonenveloped viruses such as SVDV (Turner and Williams, 1999), PCV2 has been shown to be extremely resistant and to maintain viability at temperatures of 60°C for 24 h and 75°C for 15 min (Welch et al., 2006; O’Dea et al., 2008).
The bench-top model of a spray-dryer used in this experiment uses a cocurrent flow of heated air and atomized spray in an open-cycle system to evaporate moisture from an aqueous or organic solution (Buchi training papers, spray drying; Buchi Labortechnik AG, Flawil, Switzerland, 1997–2002). Although inlet temperatures of 240°C can be achieved during this process, the time in which the product is exposed to this temperature is short. This design enables drying of plasma products without the destruction of various proteins including antibodies to PCV2, Mycoplasma sp., PRRSV, transmissible gastroenteritis virus, and SIV (Borg et al., 2002). There are differences between a bench-top model and commercial units; the main difference being size, which affects the retention time of the product within the chamber. The retention time is balanced through control of various parameters (inlet temperature, liquid feed rate, and so on) to minimize damage to the proteins and maximize the efficiency of the drying process (Maa et al., 1998).
It has been demonstrated that when SDP products are incorporated into the diets of weanling pigs, dietary plasma proteins promote immune modulation of pro- and anti-inflammatory cytokines resulting in improvements in growth variables (Moretó and Perez-Bosque, 2009). This same process, which allows proteins to remain stable, may allow extremely resistant viruses such as PCV2 to retain infectivity. For example, the outlet temperature can be generalized as the maximum product temperature (Buchi training papers, spray drying; Buchi Labortechnik AG, 1997–2002). Using this generalization, the temperature of the experimentally produced SDP in this experiment was between 67 to 71°C, similar to previously reported temperatures where PCV2 remained viable for 15 min (75°C) or 24 h (60°C; Welch et al., 2006; O’Dea et al., 2008). Therefore, the ability of PCV2 to cause seroconversion and viremia in naïve animals in this study was not unexpected. The parameters used in this study were based on recommendations of the manufacturer of the bench-top spray-dry unit used. Further determination of the necessary inactivation temperature for PCV2 by this method is warranted.
Based on the development of viremia and time of seroconversion, it is highly probable that within the SDP-IP and SDP-OG group intrapen transmission occurred. Previous work has shown that the mean time for a newly infected animal to infect a susceptible animal is approximately 18 d when naïve animals are placed into the same pen as experimentally infected animals (Andraud et al., 2008). Although ideally each pig would have been housed individually to perform replicates of the experiment, the conditions of the trial answer the question of whether a group of animals can be infected with PCV2 through exposure to an experimentally generated SDP product containing PCV2. However, this study does not indicate the relative magnitude and importance of this source of infection under field conditions.
Transmission of PCV2 from external sources into the experimental rooms was controlled in this study by utilizing multiple sampling teams whenever possible. If entering multiple rooms on the same day was absolutely necessary, a shower was taken between rooms and different face masks, gloves and coveralls were worn in each room. Throughout the study, negative control animals housed under the same conditions as experimental groups failed to seroconvert or become viremic. Additionally, sequence analysis of the PCV2 recovered from 1 viremic pig from each group confirmed 100% similarity with the inoculum.
In contrast to the results of this experiment, in a study in which SDP was incorporated into the diet of 3- to 4-wk-old SPF pigs, neither seroconversion nor viremia was detected (Pujols et al., 2008). In contrast to the present study, in which PCV2-naïve pigs were utilized, pigs with decreased concentrations of passively derived anti-PCV2-IgG as determined by immunoperoxidase monolayer assay (all pigs were negative based on a capture ELISA) were used by Pujols et al. (2008), which is more representative of commercial pigs. The increased sensitivity of the immunoperoxidase monolayer assay in comparison with the capture ELISA likely indicates that the amount of passively acquired antibody were small (Pujols et al., 2008). Possible passively acquired antibodies that may have remained undetected by the ORF-2 ELISA in the animals used in the current study can be ruled out as the PCV2-naïve status of the source herd was monitored over time and also confirmed by additional serological assays on the entire sow population (data not shown). Previously, in animals with large amounts of passively acquired antibodies, decreased viremia and seroconversion after experimental challenge with PCV2 was observed; however, small amounts of passively acquired antibody titers were not found to be generally protective (McKeown et al., 2005). As anti-PCV2 antibodies can mask or delay infection we decided to use a bioassay model based on naïve pigs.
Additional differences to the study by Pujols et al. (2008) include the use of different sources of SDP and a different route of inoculation. In the current study, plasma containing 5.63 × 107 PCV2 genomic copies/mL from an experimentally inoculated pig with clinical and histological evidence of PCVAD was used to directly inoculate the animals in the POS control group and to generate the SDP product used to inoculate the animals in the SDP-IP and SDP-OG groups. In the study by Pujols et al. (2008), commercially processed SDP containing 2.47 × 105 PCV2 DNA copies/mL was used. Although information is not available on the minimum infectious dose for PCV2, in previous work by the authors, oral administration of meat containing 104 PCV2 DNA copies/mL of homogenized tissue resulted in seroconversion and viremia in naïve animals (Opriessnig et al., 2009). In published experimental studies, the most common route of infection is the intra- or oronasal route using doses ranging from 102 TCID50 to 106 TCID50/mL (Tomás et al., 2008). In the current study, the main objective was to determine if SDP was infectious. Therefore, pigs were inoculated via intraperitoneal injection and by oral gavage in a tightly controlled bioassay model. In contrast, Pujols et al. (2008) incorporated the SDP in the diet of pigs for 45 d, which more closely simulates field conditions.
This work provides direct evidence that experimentally produced porcine SDP collected from a PCV2b-experimentally infected pig is infectious to naïve animals through the intraperitoneal and oral gavage routes. However, commercially produced products utilize pooled plasma from clinically healthy pigs, and it is not appropriate to fully extrapolate results from this experimentally produced SDP process to the commercially produced spray dried porcine plasma process and product used in the swine industry today.