Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV.     

Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies

Monoclonal antibodies (MAB) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared, using their virus-neutralizing (VN) activity in the presence or absence of guinea pig, rabbit, or swine complement. The MAB against the peplomer protein had similar VN titers for TGEV in the presence or absence of complement from any source. The MAB against the matrix protein had VN activity for TGEV only in the presence of complement, whereas MAB specific for the nucleocapsid protein did not possess VN activity in the presence or absence of complement. Serum from sows containing antibodies against TGEV peplomer and matrix proteins had slightly higher VN titers in the presence of complement than in the absence of complement. High concentrations of complement from swine serum (128 U) had little effect on the infectivity of TGEV for swine testes cells, whereas 32 U of complement from rabbit serum and 64 U of complement from guinea pig serum were able to neutralize virulent and attenuated TGEV in the absence of known antibodies for TGEV. Complement (less than or equal to 8 U) from any source did not decrease the infectivity of TGEV by greater than 0.5 log10 units.     

Virus isolation and immune responses in susceptible swine exposed with pseudorabies virus (Shope strain).

Eighteen seronegative swine weighing from 9 to 11 kg were exposed intranasally with the Shope strain of pseudorabies virus (PRV) and were observed for 21 days in an experiment to detect virus shedding and immune responses. All swine had PRV in their nasal passages at 7 days after exposure; they also had precipitating antibodies to PRV as determined by the microimmunodiffusion test (MIDT) and very low levels of virus-neutralizing (VN) antibodies. The PRV was isolated from only 2 swine at postexposure day 14; all swine were MIDT positive, and VN titers ranged from 4 to 128. Virus was not isolated from the swine at 21 days after exposure, but all were MIDT positive; VN titers ranged between 8 and greater than or equal to 256.     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

Development and evaluation of a microimmunodiffusion test for detection of antibodies to pseudorabies virus in swine serum.

A microimmunodiffusion test (MIDT) was developed for the detection of pseudorabies virus (PRV) antibodies in swine serum. The optimal medium for the MIDT was determined to contain 0.69% agarose in 0.05 M tris buffer (pH 7.2) with 0.025% sodium azide and no NaCl. The PRV antigen prepared by (NH4)2SO4 precipitation of viral fluids (42.5 g/100 ml), dialyzed against distilled water, and concentrated to approximately 100-fold of the original volume with polyethylene glycol (mol wt 20,000) provided a good reproducible antigen. The sensitivity of the MIDT was compared with the microtitration procedure of the virus-neutralization (VN) test by assaying 2,203 swine serums for PRV antibodies. An equal percentage of serums was positive in both tests; 419 had VN titers of greater than or equal to 4, and 421 were MIDT positive. Serums (314) that had VN titers of greater than or equal to 16 were all positive by the MIDT. Of serum samples with a VN titer of 8 (53), 50 were MIDT positive, a 94% correlation, and of 52 serums that had VN titers of 4, 36 were MIDT positive, a 69% correlation. In addition, 8 serums that had titer of less than 4 by VN test were positive by MIDT. Seventy-one serum samples were too cytotoxic, markedly hemolyzed, or contaminated to evaluate properly in the VN test; of these serums, 13 were MIDT positive. The MIDT is an accurate, rapid, economical, and sensitive diagnostic test for the detection of PRV antibodies in swine serums.     

Colostral IgA, IgG, and IgM-IgA fractions as fluorescent antibody for the detection of the coronavirus of transmissible gastroenteritis.

Colostrum from sows and gilts inoculated with virulent transmissible gastroenteritis virus was fractionated into the 3 major immunoglobulin classes, IgA, IgG, and IgM-IgA fractions, by chromatographic and gel-filtration procedures. Each fraction was assayed for purity with rabbit anti-porcine serum and rabbit monospecific anti-porcine IgG, anti-porcine IgA, and anti-porcine IgM. These analyses showed that the IgG and IgA fractions were pure. The IgM fraction contained some IgA in the polymeric form and was designated the IgM-IgA fraction. Each Ig was assayed for virus-neutralizing activity on swine testes cells by the plaque-reduction method before and after conjugation with fluorescein isothiocyanate. On the basis of activity per milligram of protein, the virus-neutralizing titers were 1:641, 1:44, and 1:6.8 for the IgA, IgG, and IgM-IgA fractions respectively; the fluorescent antibody titers were 1:31.3, 1:0.1, and 1:15.6, respectively, for the same Ig.     

猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位区基因的克隆及原核表达

参照GenBank发表的猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位的编码区基因序列,设计一对引物,通过PCR扩增后,将约为600bp的目的片段克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32(a)的T7启动子下游,构建的重组质粒pET-gB经IPTG诱导,在大肠杆菌BL21(DE3)中获得了高效表达。SDS-PAGE结果显示,表达产物分子量约为42.4KDa,主要以包涵体形式存在。BandScan分析表明,表达量约占菌体蛋白的60.5%。利用His亲和层析方法得到了纯化的表达产物。Western blotting结果显示,重组蛋白能与阳性血清发生特异性反应,具有较好的抗原反应原性,可以作为检测用抗原。     

Immune responses in swine given lipid-conjugated pseudorabies viral antigens.

The immune response was compared in pigs given inactivated pseudorabies virus (PRV) antigens (with or without adjuvant) or PRV antigens covalently conjugated with a fatty acid (lauric acid) to enhance delayed-type hypersensitivity. The pigs were given 2 inoculations, 14 days apart, and were challenge exposed 28 days after the 1st inoculation. Pibs inoculated with PRV antigens, with or without adjuvant, had significant virus-neutralizing (VN) antibodies before challenge exposure, but the pigs inoculated with lipid-conjugated PRV antigens had no detectable VN antibodies, with the exception of 1 pig. All inoculated pigs were positive by the microimmunodiffusion test at postinoculation day 14 and remained positive throughout the experiment. The inoculated pigs had delayed-type hypersensitivity reactions when skin tested a postinoculation day 25; the pigs inoculated with lipid-conjugated PRV antigens had a more pronounced reaction. Inoculated pigs had mild respiratory signs on the 3rd through the 6th days after challange exposure, with no observable difference in severity between the inoculated groups. The control pigs had acute signs of PRV, and 3 or 4 pigs died 5 to 8 days after challenge exposure. The average VN titers of the different inoculated groups of pigs were nearly equal 2 weeks after challenge exposure. Results indicated that both humoral antibodies and cell-mediated immunity have a role in PRV infections in swine.     

猪伪狂犬病病毒多克隆抗体的制备及免疫学活性研究

本研究旨在获得抗猪伪狂犬病病毒(PRV)闽A株的多克隆抗体,为PRV的治疗与检测提供理论基础.本研究在PK-15细胞上进行PRV的增殖,测定其TCID₅₀为10^-7.372,粗提蛋白后,测定PRV蛋白浓度为3.6 mg/mL.试验选用25只健康、雄性、体重为2.5 kg±0.2 kg的新西兰大白兔为试验动物,用获得的PRV为抗原免疫后,获得抗PRV多克隆抗体.测定其抗血清效价为1:32 000,抗原包被稀释度为1:40,最佳包被条件为4 ℃ 12 h,最佳封闭时间为1 h,酶标二抗最佳工作稀释度为1:8 000.细胞病变中和试验结果表明,本研究制备的PRV抗血清在1:16的稀释情况下能保护50%的PK-15细胞免受PRV的攻击,而阴性血清不能保护PK-15细胞免受PRV的感染.结果表明本研究成功制备了PRV多克隆抗体.     

Preparation of Polyclonal Antibodies against Swine Pseudorabies Virus and Study on the Immunological Activity

This study was aimed to obtain polyclonal antibody against swine pseudorabies virus (PRV) Min A strain,and provide a theoretical basis for the study of the treatment and detection of PRV.This study was performed on PK-15 cell and proliferation of PRV was measured as TCID₅₀ 10^-7.372,the protein concentration of PRV was measured as 3.6 mg/mL.Choosing five healthy male rabbits (2.5 kg±0.2 kg) as experimental animals and using PRV obtained as the antigen,we got polyclonal antibody against PRV.Antiserum titer was 1:32 000,antigen coating dilution was 1:40,the best coating conditions was 4 ℃ 12 h,the best blocking time was 1 h,the best working dilution of enzyme labled antibody was 1:8 000,the result of cell lesions neutralization test showed that PRV antiserum prepared in this assay at 1:16 dilution could protect 50% of PK-15 cells from being infected by PRV,and negative serum couldn't protect PK-15 cells from being infected by PRV.The study successfully prepared polyclonal antibodies against PRV.     

Immunohistologic study of pulmonary and lymphatic tissues from gnotobiotic pigs inoculated with ara-T-resistant strain of pseudorabies virus

Ara-T-resistant strain of pseudorabies virus (PRV) was inoculated intranasally into six 2-week-old gnotobiotic pigs. Five inoculated pigs were sneezing and coughing. In pigs 1 to 4 killed on postinoculation days (PID) 3, 5, 7, and 9, respectively, PRV antigen was detected in respiratory epithelial cells, and pigs had severe pneumonitis. In pigs 5 and 6 killed on PID 11 and 13, respectively, PRV antigen was localized in macrophages in alveoli and necrotizing nodules. Immunoglobulin-containing cells (IgG, IgM, and IgA) were detected first in pneumonic lesions in pig 4 killed on PID 9. Detection of immunoglobulin-containing cells was coincident with pulmonary inflammation and regeneration of pneumonic lesions. The number of IgG-containing cells was greater than that of IgM- and IgA-containing cells. Corresponding to transient viral multiplication, IgG-, IgM-, and IgA-containing cells were demonstrated first in lymphatic tissues in pig 1 killed on PID 3 and their number was 5 to 10 times more than those in control pigs 7 and 8. Seemingly, PRV replication in lymphatic tissues stimulated the proliferative response of specific immunoglobulin-producing cells, and the appearance of immunoglobulin-containing cells in the lungs was associated with clearance of PRV and regeneration of pneumonic lesions.     

检测猪伪狂犬病病毒gE抗体红细胞凝集试验的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)—猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为抗原,建立了检测猪伪狂犬病毒gE抗体的红细胞凝集试验。利用方阵滴定试验筛选出最佳抗原工作浓度为55μg/mL,血清最佳稀释度为1∶20,作用时间15 min,与猪瘟(CSF)、猪细小病毒(PPV)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪布氏杆菌病(Brucellosis)阳性血清和PRV gE缺失疫苗接种的猪免疫血清均不出现红细胞凝集现象,与PRV标准阳性血清反应出现肉眼可见的凝集圈。与美国进口的PRV抗体检测gE-ELISA诊断试剂盒检测结果比较,50份猪血清的阴、阳性检出符合率均为100%。红细胞凝集试验检测方法具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

A fusion protein of IgG fc and mouse-derived antigen on the surface of pseudorabies virus particles does not accelerate production of harmful auto-reactive antibodies

Previously we reported that immunization with pseudorabies virus (PRV), harboring chimeric Fc on the surface of the virus particles (PRV/Fc), induced higher immune responses than normal PRV particles. The chimeric Fc was fused with mouse transferrin receptor of transmembrane domain (mTR) and the Fc region of immunoglobulin G1. Since it has been reported that some chimeric protein of Fc and self-antigen induce auto-reactive antibodies, in this present study, we examined whether PRV/Fc induces auto-reactive antibodies that react with mTR. PRV/Fc immunized mice produced higher levels of anti-PRV antibodies and antibodies that reacted with mouse-derived 3T3/A31 cells (A31 cell), compared to normal PRV immunized mice. However, antibodies that reacted with mTR in A31 cells were not detected in both Western blot analyses and indirect immunofluorescence assay. The antibodies reacted with an antigen of approximately 16 kDa in A31 cells, but this antigen has a different molecular mass from that of mTR. The antibody also reacted with the antigen of approximately 16 kDa in RK13 cells in which the virus had been propagated. In addition, antibodies induced by immunization with normal PRV also reacted with the same antigen in A31 and RK13 cells. Moreover, neither kidney disorders, in which high levels of mTR were expressed, nor clinical symptoms of autoimmune diseases were observed in mice immunized with either PRV or PRV/Fc. These results indicated that the antibodies were not induced by mTR-Fc, but were instead induced by trace amounts of RK13 derived antigens contained in PRV or PRV/Fc preparations, and cross-reacted with equivalent molecules in mouse derived A31 cells. Therefore, this study confirmed that immunization with PRV/Fc did not induce harmful auto-reactive antibodies.     

Characteristics of cytotoxic cells induced by Toxoplasma lysate antigen in mouse spleen.

Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL male-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen cells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive, and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK) cells induced by IL-2.     

Attenuated properties of thymidine kinase-negative deletion mutant of pseudorabies virus

A thymidine kinase (TK)-negative (TK-) deletion mutant of the Bucharest (BUK) strain of pseudorabies virus (PRV) was isolated. The mutant, designated as PRV (BUK d13), did not revert to TK-positive (TK+), even when propagated in medium that selected for TK+ viruses. The mutant also replicated equally well at 39.1 C and 34.5 C, and was easily distinguished from other PRV strains by molecular hybridization experiments, restriction nuclease fingerprints, and plaque autoradiography or other assays for the TK phenotype. The PRV (BUK d13) had greatly reduced virulence for mice and rabbits, compared with parental TK+ strains, PRV (BUK-5) and PRV (BUK-5A-R1), and provided mice with solid protection against the TK+ BUK and Aujeszky strains of PRV. Experiments were done in 5- to 6-week-old pigs to assess the safety and efficacy of PRV (BUK d13) in the natural host. In one experiment, pigs were vaccinated IM with 7.5 X 10(8) plaque-forming units of TK- PRV (BUK d13), and were then challenge exposed intranasally (IN) with 4.3 X 10(8) TCID50 of virulent PRV [Indiana-Funkhauser (IND-F)]. Vaccinated pigs did not have clinical signs of illness after vaccination or after challenge exposure. One nonvaccinated control pig died on postchallenge day 4; a 2nd nonvaccinated control pig became moribund, but eventually recovered. Pigs developed virus-neutralizing antibodies after vaccination, and had a secondary immunologic response after challenge exposure; however, PRV was not isolated from the tonsils or trigeminal ganglia of vaccinated pigs at postchallenge exposure day 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection from pseudorabies virus challenge in mice by a combination of purified gII, gIII and gVI antigens.

The antigens gII, gIII and gVI were purified from the lysates of the pseudorabies virus (PRV) -infected HmLu-1 cells using Sepharose 4 B coupled with MAbs against these antigens. Mice immunized with either gII, gIII, gVI antigen or a mixture of them were challenged intraperitoneally with 8.5 x 10(3) plaque forming units of PRV. All the mice immunized with 1.5 and 4.5 micrograms of the mixture and 4.5 micrograms of the gIII antigen survived. The sera of mice immunized with the mixture had virus neutralizing activity which was independent of complement, hemagglutination inhibition activity, and recognized major (93 kilodaltons) and minor (129, 74, 68 and 50 kilodaltons) PRV proteins under reducing conditions in western blotting. The serological activity levels in the lower survival group were not different from those in the complete survival. These results indicate that the mixture of each glycoprotein is more effective for eliciting of protective immunities in mice and that serological activity do not always correlate with protection.     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.     

近期部分规模化猪场猪伪狂犬病野毒抗体监测情况调查

猪伪狂犬病仍然是我国猪群的重要威胁性传染病，通常造成母猪繁殖障碍以及仔猪的神经症状和高死亡率，同时伪狂犬病病毒也是猪呼吸系统疾病综合征的重要原发性病原。用gE-ELISA野毒鉴别诊断方法共检测了来自8个省、市46个猪场1940份血清样品，其中阳性样品657份，样品总阳性率33.87%，阳性猪场25个。在检测的各场中，阳性率最高猪场达到75.76%(125/165)。而部分进行了科学的疫苗免疫和实施了严格的生物安全措施的猪场始终保持猪伪狂犬病阴性(0/135)。利用基因缺失疫苗科学免疫配合伪狂犬抗体鉴别诊断技术是控制和净化伪狂犬病的重要手段。     

Second-generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes

A modified-live pseudorabies virus (PRV) vaccine, designated PRV(dlg92/d1tk), with deletions in the thymidine kinase (tk) and glycoprotein-gIII (g92) genes, was derived from the PRV (Bucharest [BUK]-d13) vaccine strain. The vaccine virus also contained a deletion in glycoprotein gI. Despite 3 deletions, PRV(dlg92/d1tk) replicated to high titers in cell culture from 30 C to 39.1 C. Enzyme assays and autoradiography revealed that PRV(dlg92/d1tk) did not induce a functional tk activity in infected tk- RAB(BU) cells (rabbit skin). Rabbit skin cells were infected with PRV(dlg92/d1tk), with vaccine strains derived from BUK or Bartha K strains of PRV or with the virulent Illinois (ILL), Indiana-Funkhauser (IND-F), and Aujeszky (Auj) strains of PRV and were labeled with [3H]mannose from 4 or 5 to 24 hours after infection to investigate whether these viruses induced the synthesis of glycoprotein gIII. Nonionic detergent extracts were prepared and immunoprecipitated with antisera from pigs vaccinated with tk(-)-PRV(BUK-d13) or tk+-Bartha K, pigs vaccinated with tk+-PRV(BUK) strains and then challenge exposed to tk+-PRV(IND-F), naturally infected domestic or feral pigs, and pigs vaccinated with tk-)-PRV(dlg92/d1tk). Mouse monoclonal antibodies against PRV glycoproteins gIII, gp50, and gII were also studied. After immunoprecipitation, labeled PRV-specific proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. The PRV glycoprotein-gII complex, but not glycoprotein gIII, was synthesized in PRV(dlg92/d1tk)-infected cells. Glycoprotein gII and gIII were made in cells infected with PRV vaccine strains BUK, Bartha K, and BUK-d13 and with virulent PRV strains ILL, IND-F, and Auj. Cells infected with PRV(dlg92/d1tk) and with PRV strains ILL, IND-F, Auj, Bartha K, BUK, and BUK-d13, excreted into the cell culture medium a highly sulfated glycoprotein gX of about 90 kilodaltons. Antibodies to glycoprotein gIII were not detected in the sera of pigs inoculated with PRV(dlg92/d1tk), but were found in all other swine sera.     

Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of na?ve PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.