Immunogenic and protective properties of GP5 and M structural proteins of porcine reproductive and respiratory syndrome virus expressed from replicating but nondisseminating adenovectors

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for significant economic losses in the porcine industry. Currently available commercial vaccines do not allow optimal and safe protection. In this study, replicating but nondisseminating adenovectors (rAdV) were used for the first time in pigs for vaccinal purposes. They were expressing the PRRSV matrix M protein in fusion with either the envelope GP5 wild-type protein (M-GP5) which carries the major neutralizing antibody (NAb)-inducing epitope or a mutant form of GP5 (M-GP5m) developed to theoretically increase the NAb immune response. Three groups of fourteen piglets were immunized both intramuscularly and intranasally at 3-week intervals with rAdV expressing the green fluorescent protein (GFP, used as a negative control), M-GP5 or M-GP5m. Two additional groups of pigs were primed with M-GP5m-expressing rAdV followed by a boost with bacterially-expressed recombinant wild-type GP5 or were immunized twice with a PRRSV inactivated commercial vaccine. The results show that the rAdV expressing the fusion proteins of interest induced systemic and mucosal PRRSV GP5-specific antibody response as determined in an ELISA. Moreover the prime with M-GP5m-expressing rAdV and boost with recombinant GP5 showed the highest antibody response against GP5. Following PRRSV experimental challenge, pigs immunized twice with rAdV expressing either M-GP5 or M-GP5m developed partial protection as shown by a decrease in viremia overtime. The lowest viremia levels and/or percentages of macroscopic lung lesions were obtained in pigs immunized twice with either the rAdV expressing M-GP5m or the PRRSV inactivated commercial vaccine.     

Mannan oligosaccharide increases serum concentrations of antibodies and inflammatory mediators in weanling pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Mannan-containing products are capable of modulating immune responses in animals. However, different products may have diverse immunomodulation. The experiment was conducted to examine effects of mannan oligosaccharide (Actigen; ACT) on growth performance and serum concentrations of antibodies and inflammatory mediators in weanling pigs (Sus scrofa) experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 32 PRRSV-negative pigs (3 wk old) were randomly assigned from within blocks to 1 of 4 treatments in a 2 by 2 factorial arrangement [2 types of diet: control (0%) and ACT addition (0.04%); and with and without PRRSV] in a randomized complete block design. Pigs were blocked by initial BW within sex. Ancestry was equalized across treatments. Pigs (8/treatment) were kept individually in each pen. After 2 wk of an 8-wk period of feeding the treatments, pigs received an intranasal inoculation of PRRSV or sham medium at 5 wk of age. Infection by PRRSV decreased ADG, ADFI, and G:F throughout the experiment (P < 0.01). Actigen did not affect ADG (P = 0.450), but decreased (P = 0.047) ADFI from 28 to 42 days postinoculation (DPI). During that time, ACT improved G:F in infected pigs but not in sham controls (interaction, P = 0.009). Dietary ACT did not affect viremia in infected pigs (P > 0.05), but increased PRRSV-specific antibody titer at 35 DPI (P = 0.042). Infection with PRRSV induced the febrile responses of pigs from 3 to 10 DPI (P < 0.001) with return to normal at 14 DPI. During the experimental period, the rectal temperature of pigs was found slightly elevated by ACT (P = 0.045). Infected pigs had greater serum concentrations of IL-1β, tumor necrosis factor (TNF)-α, IL-12, interferon (IFN)-γ, IL-10, and haptoglobin (Hp) than sham controls (P < 0.001). These results indicate that PRRSV stimulated secretion of cytokines involved in innate, T-helper 1, and T-regulatory immune responses. Actigen tended to decrease the serum TNF-α concentration regardless of PRRSV (P = 0.058). The ACT × PRRSV interaction was significant for IL-1β (P = 0.016), IL-12 (P = 0.026), and Hp (P = 0.047), suggesting that infected pigs fed ACT had greater serum concentrations of these mediators than those fed the control. The increases in IL-1β and IL-12 may favorably promote innate and T-cell immune functions in infected pigs fed ACT. Feeding ACT may be useful as ACT is related to increased PRRSV antibody titers and G:F in infected pigs at certain times during infection.     

Identification and characterization of auto-anti-idiotypic antibodies specific for antibodies against porcine reproductive and respiratory syndrome virus envelope glycoprotein (GP5)

Auto-anti-idiotypic antibodies (Aab-2s) were detected from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The Aab-2s were specific against the idiotypic antibodies (Ab-1s) to the envelope glycoprotein GP5 of PRRSV and were detected from serum samples collected between 21 and 98 days post-infection (DPI). Serological characterization indicated that the Aab-2s recognized the idiotype located within or near the antigen-combining sites of the anti-GP5 antibodies, which was shared by both mouse MAb anti-GP5 and swine polyclonal antibodies. The fact that the Aab-2 inhibited the anti-GP5 antibodies from binding to PRRSV and that they were detected at different time periods in pigs that cleared the infection prior to 98 DPI versus pigs in which virus was detected at 98 DPI suggests that Aab-2 antibodies may play a role in immunity to PRRSV infection.     

猪圆环病毒2型感染对猪繁殖与呼吸综合征灭活疫苗体液免疫的影响

本试验采用ELISA方法对单独接种猪繁殖与呼吸综合征病毒(PRRSV)变异株灭活苗组(HP组,n=3)和PCV2感染且出现病毒血症后接种PRRSV变异株灭活苗组(PCV2/HP组,n=3)不同时相血清中的PRRSV抗体进行检测;并对HP和PCV2/HP组血清中PCV2特异的抗体和核酸分别进行ELISA和荧光定量PCR检测。结果表明,在首免后70 d HP组血清平均抗体效价极显著高于PCV2/HP组(P<0.01);首免后56、63和77 d HP组平均抗体效价明显高于PCV2/HP组。其中在首免后56和63 d HP组抗体阳性率均达67%(2/3),PCV2/HP组在相应时相抗体阳性率为0;在首免后70和77 d HP组抗体阳性率均达100%(3/3),PCV2/HP组在相应时相抗体阳性率仅为0和33%(1/3)。结果提示PCV2感染可在一定程度上抑制PRRSV变异株(JXA1)特异性的抗体反应。     

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log₂) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.     

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((R)) 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.     

Immune responses in pigs induced by recombinant canine adenovirus 2 expressing the glycoprotein 5 of porcine reproductive and respiratory syndrome virus

To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine™ 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection.     

Comparative evaluation of PRRS virus infection in vaccinated and naïve pigs

The purpose of this study was to evaluate the time-course of the immune response to a field Porcine Respiratory and Reproductive Syndrome virus (PRRSV) strain in PRRS-naïve, untreated pigs, as well as in four groups of age and breed-matched pigs injected with a live attenuated PRRS vaccine, its adjuvant, an inactivated PRRS vaccine and an irrelevant, inactivated Porcine Circovirus type 2 (PCV2) vaccine, respectively. PRRSV infection was confirmed in all groups by PCR and antibody assays. The antibody response measured by ELISA took place earlier in pigs injected with the live attenuated vaccine, which also developed a much stronger serum-neutralizing antibody response to the vaccine strain. Yet, no clear protection was evidenced in terms of viremia against the field virus strain, which showed 11.1% nucleotide divergence in ORF7 from the vaccine strain. In vitro, the interferon (IFN)-γ response to PRRSV was almost absent on PVD 60 in all groups under study, whereas the prevalence of interleukin (IL)-10 responses to PRRSV was fairly high in PCV2-vaccinated animals, only. Results indicate that distinct patterns of immune response to a field PRRSV strain can be recognized in PRRS-vaccinated and naïve pigs, which probably underlies fundamental differences in the development and differentiation of PRRSV-specific immune effector cells.     

Comparison of two swine Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays for detection of antibodies from vaccinated pigs and field serum samples.

Mycoplasma hyopneumoniae (Mhyo) causes mycoplasmal pneumonia, an economically important disease of swine. Serodiagnosis of Mhyo is based on the current available commercial enzyme immunoassays for detection of swine antibodies against Mhyo, which are the indirect enzyme-linked immunosorbent assay (ELISA) and the blocking ELISA (B-ELISA). Because of the limited information available for these ELISAs, these 2 assays were compared by testing 347 serum samples collected from vaccinated pigs at 0, 13, 28, 43, and 62 days postimmunization (DPI), 50 samples from nonvaccinated pigs, and 1,013 field serum samples. The results of comparison study showed that the specificity for both ELISAs was 99.2% generated from 139 non-vaccinated negative samples. The sensitivities for indirect ELISA generated from samples collected from animals that received the vaccine at DPI 13, 28, 43, and 62 were 0%, 95.7%, 88.4%, and 92.6%, respectively, whereas the sensitivities for B-ELISA were 0%, 98%, 100%, and 97%, respectively. The overall agreement of 96.7% and 80.3% was generated between 2 ELISAs from negative and vaccinated pigs and from field samples, respectively.     

猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响

采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应.     

Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n=8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n=9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n=9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n=9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n=9), pigs infected with VR-2385 (n=9), and pigs infected with NC16845b (n=9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.     

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.     

Characterization of antibody response to porcine reproductive and respiratory syndrome virus ORF5 product following infection and evaluation of its diagnostic use in pigs.

The sensitivity and specificity of recombinant open reading frame 5 products used in the Western blotting assay for confirmation of porcine reproductive and respiratory syndrome virus (PRRSV) serologic status were evaluated. The recombinant antigen-based assays were specifically compared with a commercial enzyme-linked immunosorbent assay (ELISA) for PRRSV antibodies using 1) PRRSV antibody-negative reference sera (n = 30), 2) naturally infected pig sera (n = 40), 3) sequential sera obtained from 24 experimentally infected pigs, and 4) sera submitted to 3 state diagnostic laboratories (n = 200). The recombinant antigen assay yielded an average increased sensitivity of 10% over the commercial PRRSV ELISA. The negative controls (group 1 sera) showed no difference between the 2 assays. This comparison confirmed that the recombinant antigen-specific assay was more sensitive than the commercial ELISA and is well suited for routine confirmation of the presence of PRRSV antibodies.     

Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets

Pseudorabies virus (PRV) has been developed as a vaccine vector for expressing foreign immunogens. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), continues to be a major problem to the pork industry worldwide. Many vaccine strategies have been developed to control the disease but most of them turn out to be unsuccessful. The objective of this research was to explore the feasibility of PRV-based vector vaccine in protection against PRRSV. A live attenuated vaccine-based PRV recombinant expressing the envelope protein GP5 of PRRSV was generated using recombinant DNA techniques. The Bartha-K61-derived recombinant virus, named rPRV-GP5, was shown to express PRRSV GP5 efficiently. Sixteen healthy piglets were assigned to one of four groups (one to four, four pigs per group). Animals in Groups 1 and 2 were each inoculated intramuscularly and intranasally with 10(7.0) PFU of rPRV-GP5 and its parent Bartha-K61, respectively; Group 3 were vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 4 was served as non-vaccinated control. One month later, all animals were all challenged with 10(6.5) TCID(50) of virulent PRRSV CH-1a. All animals in Groups 1 and 3 remained clinically healthy before and after challenge, with only a short period of fever (no more than 41 degrees C and 3 days), mild and gradually improving lung and kidney lesions, and short-term viremia (2 and 3 week, respectively) in spite of no detectable anti-PRRSV antibody before challenge. On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 41 degrees C) after challenge, and severe lung, kidney and spleen lesions and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PRRSV challenge in vaccinated pigs.     

Effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs

OBJECTIVE: To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. ANIMALS: Four hundred forty 6- to 8-week-old PRRSV-na?ve pigs. PROCEDURES: Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). RESULTS: Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.     

Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.     

猪繁殖与呼吸综合征病毒重组M蛋白间接ELISA抗体检测方法的建立及应用

为建立一种敏感、特异、快速、高通量的猪繁殖与呼吸综合征病毒(PRRSV)抗体血清学检测方法,本研究利用原核表达技术表达了PRRSV M蛋白,将纯化后的重组M蛋白作为包被抗原建立了检测PRRSV抗体的间接ELISA方法。参照已发表的PRRSV基因组M基因序列,设计合成1对特异性引物,RT-PCR扩增了长约435 bp的M基因片段,将目的片段亚克隆至pET32a(+)表达载体中,经IPTG诱导获得了以包涵体形式表达的重组M蛋白,重组蛋白纯化后,免疫印迹检测结果表明具有良好的抗原性和特异性。以重组M纯化蛋白为包被抗原,经间接ELISA反应条件的优化,建立了检测PRRSV抗体的间接ELISA方法,该方法检测猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病病毒(PRV)、猪传染性胃肠炎病毒(TGEV)、猪圆环病毒2型(PCV2)其他6种常见猪病病原的阳性血清均为阴性;该方法批内与批间重复性试验的变异系数分别小于5%和10%;该方法与商品化ELISA试剂盒的符合率为95.3%。本研究建立的M-ELISA检测方法将为猪群免疫PRRS疫苗后抗体水平监测及PRRSV野毒感染的快速诊断与流行病学调查等提供了一种简便易行、快速、高通量的血清学抗体检测方法。     

In vivo and in vitro effects of moderately virulent African swine fever virus on mitogenesis of pig lymphocytes

Six pigs were infected oro-nasally with a moderately virulent African swine fever (ASF) virus from the Dominican Republic (DR II). The effect of virus infection on the pig's immune system was tested by measuring peripheral leucocyte numbers and the ability of mononuclear leucocytes (MNL) to respond by lymphocyte proliferation (LP) to the mitogens phytohemagglutinin-P (PHA-P), concanavalin-A (Con-A), and pokeweed mitogen (PWM). All 6 pigs developed high viremias between 4 and 18 days post-inoculation (DPI) which became undetectable by 32 to 46 DPI. Virus was found in erythrocytes, plasma, and mononuclear leucocytes from peripheral blood. Overall, virus infection had only minor effects on the number of circulating leucocytes, lymphocytes, monocytes and granulocytes. At the early acute phase of infection slight neutrophilia and lymphocytopenia were observed with mildly elevated monocyte numbers and slightly depressed neutrophil numbers that continued from the time of evident reduction in viremia to beyond the period of viral clearance. The infected pigs readily produced high titers of ASF virus antibody shortly after the onset of viremia. No significant differences in LP responses of MNL from the 6 pigs to PHA-P, Con-A and PWM were observed after infection when compared to those obtained with MNL from normal pigs. The in vitro addition of infectious ASF virus to MNL from normal pigs did not affect LP responses to any of the three mitogens. These results do not support the hypothesis that immunosuppression is a consequence of ASFV infection of pigs.     

Evaluation of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody (IFA) test for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibody in pigs from conventional farms

To evaluate the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for detecting the porcine reproductive and respiratory syndrome virus (PRRSV) antibody, conventional pigs in PRRSV-positive and -negative commercial farms were examined. Antibody development patterns in ELISA and IFA tests were compared in 3 week old piglets experimentally infected with the PRRSV. The virus was detected from 2 days post infection (PI) and then the antibody titers and S/P ratios rose by both methods. A total of 208 serum samples were collected from 4 PRRSV-negative farms and 210 samples from PRRSV-positive farms, and were tested for the PRRSV antibody by IFA and ELISA. The titer of 64 should be set as the cut-off point in IFA for field sera. Similarly, the cut-off S/P ratio should be set at 0.4 in ELISA. A high degree of correlation was observed between antibody titers by the two methods in these 418 samples, with a correlation coefficient of 0.84. The coincidence rate between the two tests was 84.7% (354/418). In non-coincident cases, ELISA was able to detect the antibody with a low titer in the serum samples which were negative in IFA but from PRRSV positive farms. ELISA was more sensitive than IFA to detect PRRSV infected animals or farms.