Porcine gammadelta T cells: possible roles on the innate and adaptive immune responses following virus infection

gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.     

Biology of porcine T lymphocytes

The present review concentrates on the biological aspects of porcine T lymphocytes. Their ontogeny, subpopulations, localization and trafficking, and responses to pathogens are reviewed. The development of porcine T cells begins in the liver during the first trimester of fetal life and continues in the thymus from the second trimester until after birth. Porcine T cells are divided into two lineages, based on their possession of the alphabeta or gammadelta T-cell receptor. Porcine alphabeta T cells recognize antigens in a major histocompatibility complex (MHC)-restricted manner, whereas the gammadelta T cells recognize antigens in a MHC non-restricted fashion. The CD4+CD8- and CD4+CD8lo T cell subsets of alphabeta T cells recognize antigens presented in MHC class II molecules, while the CD4-CD8+ T cell subset recognizes antigens presented in MHC class I molecules. Porcine alphabeta T cells localize mainly in lymphoid tissues, whereas gammadelta T cells predominate in the blood and intestinal epithelium of pigs. Porcine CD8+ alphabeta T cells are a prominent T-cell subset during antiviral responses, while porcine CD4+ alphabeta T cell responses predominantly occur in bacterial and parasitic infections. Porcine gammadelta T cell responses have been reported in only a few infections. Porcine T cell responses are suppressed by some viruses and bacteria. The mechanisms of T cell suppression are not entirely known but reportedly include the killing of T cells, the inhibition of T cell activation and proliferation, the inhibition of antiviral cytokine production, and the induction of immunosuppressive cytokines.     

Lymphocyte subsets in porcine tonsillar crypt epithelium

The palatine tonsils are part of the mucosa-associated lymphoid tissue (MALT), strategically located in the oropharynx at the entrance of respiratory and gastrointestinal tracts, and are recognized portals of entry and sites of multiplication and persistence of several pathogens in pigs. As the tonsillar crypt epithelium is in close contact with external environment and the underlying lymphoid tissue, the characterization of the intra-epithelial lymphocyte subpopulations is essential for the understanding of initial steps of pathogenesis of several diseases. In this work we investigated specific lymphocyte subsets in the tonsillar crypt epithelium of 10 adult healthy pigs, using monoclonal antibodies against lymphocyte markers CD3, CD4, CD8, gammadelta T cell receptor and immunoglobulin light-chain in an avidin-biotin immunoperoxidase technique. The crypt epithelium was usually extensively infiltrated by a diverse population of T cells and by B cells. The degree of infiltration of each subset was variable among animals and within individual animals. In the T cell population CD4 cells and gammadelta TCR cells predominated over CD8 cells. These data suggest that the crypt lymphoepithelium is capable of participating in both cellular and humoral immune responses and that gammadelta T cells may play an important role in the defense of this mucosa.     

A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells

A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.     

Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle

The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350-420kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days -21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNgamma) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and gammadelta TCR) and to detect the activation marker CD25 (alpha chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Delta%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Delta%CD25+ data for days 0-70 of all subsets, except CD4-CD8+gammadelta TCR- (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group (P<0.05). IFNgamma production by PBMC from the vaccinated group showed significantly higher results (P<0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly (P<0.0001) higher neutralizing antibody titers than the control group from day 28 onward.     

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

Lymphocyte development in fetal piglets: facts and surprises

The developing porcine fetus offers an excellent opportunity for the study of lymphocyte development. Studies on B cell, alphabeta T cells and gammadelta T cells in the last decade have expanded our knowledge of lymphocyte development in pigs. These studies have revealed several interesting differences between swine, mice and humans. For example, porcine peripheral lymphocytes include CD4+CD8+ alphabeta T cells and an abundance of gammadelta T cells that may even prevail over the alphabeta population. There are numerous CD2- gammadelta T cells in the blood and a large number of CD8alphaalpha-bearing cells that include NK cells, conventional gammadelta and alphabeta T cells. All porcine B lymphocytes are CD25(lo) and sIgM+ B cells may differ in the expression of CD2 antigen. Unlike mice, porcine B cells appear approximately 2 weeks before T cells and progenitors undergo VDJH rearrangement at 20th day of gestation (DG20) in the yolk sac and DG30 in the fetal liver before consummating high level lymphogenesis in the bone marrow after DG45. Early B cells show an unexpectedly high proportion of in-frame rearrangements, undergo switch recombination in thymus on DG60 and use N-region insertion from the time of the earliest VDJ rearrangement. The genomic repertoire of VH, DH and JH genes is small compared to mice and humans and swine appear to depend on junctional diversity for the majority of their repertoire. The limited VH repertoire of swine contrasts sharply with the porcine TCRbeta repertoire, which is extensive, extraordinarily conserved and nearly identical to that in humans. Therefore, swine present an example of two highly related receptor systems that have diverged in the same species.     

Ontogeny of T lymphocytes and intestinal morphological characteristics in neonatal pigs at different ages in the postnatal period

To evaluate morphological characteristics and development of the immune system at different ages in neonatal pigs, 4 piglets were euthanized at 7, 14, and 18 d of age for collection of blood, bile, and intestinal tissue for morphological measurements. Blood was collected for differential cell counts, lymphocyte blastogenesis, immunoglobulin (Ig) concentrations, cytokine concentrations, and flow cytometric analysis. Bile was collected for quantification of Ig-A and Ig-M. Villus width and crypt depth from duodenum sections, as well as ileum crypt depth, were reduced (P < or = 0.08) in 18-d-old pigs compared with 7-d-old pigs. No age-related differences (P > or = 0.11) were observed in the number of goblet cells with neutral and acidic mucins, serum or enteric Ig concentrations, IL-2, IL-4, spontaneous lymphocyte proliferation, or leukocyte concentrations. When measured as counts per minute (cpm) and as a stimulation index (SI), lymphocyte proliferation responses to phytohaemagglutinin increased (P = 0.05) between 7 and 14 d of age; no changes (P = 0.10) occurred at 18 d of age. No age-related changes (P = 0.39) were observed in response to pokeweed mitogen (PWM) when measured as cpm; however, the SI from PWM-induced lymphocytes decreased (P = 0.04) 4-fold between 7 and 18 d of age. The CD4+:CD8+ and populations of lymphocytes expressing CD2+CD4+CD8- (T helper cells) and CD25+CD4+CD8- (activated T helper cells) were greater (P > or = 0.04) at 7 d of age than at 14 and 18 d. Populations of T lymphocytes, cytotoxic T cells (CD2+CD4-CD8+), activated lymphocytes (CD25+), and activated cytotoxic T cells (CD25+CD4-CD8+) were greater (P > or = 0.02) in 18-d-old pigs compared with 7-d-old pigs, whereas CD2+CD4-CD8- [double negative cells] were lower (P = 0.08) in 18-d-old pigs compared with 14-d-old pigs. The percentage of CD2+ T cells was 8.4% at 7 d of age, and by the time the pigs reached 18 d of age, the percentage of CD2+ T cells was 33.8%. Moreover, the percentage of gammadelta T cells was greater (P = 0.02) in 18-d-old pigs than in 7-d-old pigs (74.8 vs. 46.1%, respectively). Results indicate that the porcine immune system and gut are continuously changing as the young pig matures. Changes occurred in lymphocyte phenotypic expression and functional capabilities, as well as morphology and mucin production, and their role may be to further protect the neonate from antigenic challenge as protection from passive immunity declines.     

Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets

A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restimulation of PBMC with EHV-1 induced IFN-gamma production by a significantly higher percentage of total (CD5(+)) T lymphocytes, and CD4(+) and CD8(+) T lymphocyte subsets among post-EHV-1 infection PBMC samples compared to pre-infection samples. This response was associated with an increase in virus-specific CTL activity, a critical immune effector for the control of EHV-1 infection and disease. No significant increase in IFN-gamma production by B lymphocytes was observed. These data demonstrate that EHV-1 challenge infection of ponies results in increased production of IFN-gamma by virus-specific T lymphocytes, and that this response can be quantitated using flow cytometry.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.     

Regional variations in the distribution of small intestinal intraepithelial lymphocytes in three inbred strains of mice

The regional variation in the intraepithelial lymphocytes (IELs) in the small intestine was examined in BALB/c male and female mice and C3H/He and C57BL/6 male mice. The small intestines were taken from 11 to 12-week-old mice and divided equally into 3 parts (the proximal, middle and distal parts). IELs were isolated from each part of the intestine and analyzed with flow cytometer. The number of IELs was highest in the proximal part and lowest in the distal part. The distribution of IEL subsets was markedly different between the proximal and the distal parts, and that in the middle part showed the intermediate pattern. The percentage of alphabeta T cells were higher in the distal part. In alphabeta T cell subset, the percentage of CD8alphaalpha T cells was higher in the proximal part, whereas those of CD4 and CD4CD8alphaalpha double positive T cells were higher in the distal part. In gammadelta T cell subset, no regional variations were found. The regional variations in the number and subsets of IELs showed almost the same patterns between male and female BALB/c mice and similar patterns among three strains of mice. This strongly suggests that the regional variations in the small intestinal IELs are common to mouse species.     

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains

Poultry represents an important source of Salmonella infection in man. Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars. In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S. typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry. Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically. Blood samples and tissues were examined between days 1 and 12 of age.Chicks inoculated with S. typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals. The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age. In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen. Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5. As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen.In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level. The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...