Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.     

Kinetics of immune response to foot-and-mouth disease virus (type Asia 1) in experimental cattle

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle. An erratum to this article can be found at     

Interplay of foot-and-mouth disease virus, antibodies and plasmacytoid dendritic cells: virus opsonization under non-neutralizing conditions results in enhanced interferon-alpha responses

ABSTRACT: Foot-and-mouth disease virus (FMDV) is a highly infectious member of the Picornaviridae inducing an acute disease of cloven-hoofed species. Vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-I interferon (IFN) by plasmacytoid dendritic cells (pDC). The present study demonstrates that the opsonising antibody titres mediating enhanced IFN-α responses in pDC were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. However, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. Both uncomplexed virus and immune complexed virus stimulated pDC via Toll-like receptor 7. An additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pDC activation by FMDV strongly differed between viral isolates. Altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses.     

Reconstitution of immunosuppression mice with mononuclear cells from donors sensitized to foot-and-mouth disease virus (FMDV)

Passive transfer experiments were performed to serve as a basis for analyzing the immune response of adult mice to FMDV infection. Animals were irradiated (750 rad: 1 lethal dose 50%) and reconstituted with allogeneic mononuclear cells from blood, spleen, thymus and peritoneal cavity from donors 2 and 8 days post-inoculation (p.i.). Donors were primed with 10 000 suckling mouse 50% lethal doses of FMDV strain O1 Campos. The following parameters were studied in recipient mice challenged with 10 000 suckling mouse 50% lethal doses of the same virus: (1) viremia; (b) FMDV neutralizing antibody titres; (c) sheep red blood cell (SRBC) hemagglutinating antibody titres. Viremia was substantially prolonged in irradiated control mice, which did not produce detectable antibodies to FMDV or SRBC. In contrast, the span of viremia was markedly shorter in animals reconstituted with cells obtained 8 days p.i. and its eclipse coincided with the onset of neutralizing antibody production. An equally efficient antibody response to the inoculation of SRBC was observed in these animals. No effect was detected after the transfer of cells obtained 2 days p.i. It is concluded that the humoral immune response plays a predominant role in the recovery from FMDV experimental infection in adult mice.     

Immune responses of swine inoculated with a recombinant fowlpox virus co-expressing P12A and 3C of FMDV and swine IL-18

Two recombinant fowlpox viruses (rFPV-P1 and rFPV-IL18-2AP12A) containing foot-and-mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 were evaluated to determine their abilities to induce humoral and cellular responses in the presence or absence of swine IL-18 as genetic adjuvant. The ability to protect swine against homologous virus challenge was examined. All swine were given booster vaccinations at 21 days after the initial inoculation and were challenged 10 days after the booster vaccination. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-P12A and rFPV-IL18-P12A developed specific anti-FMDV ELISA antibody and neutralizing antibody and T-lymphocyte proliferation was observed. Cellular immune function was evaluated via examination of IFN-gamma production in swine peripheral blood serum. The results demonstrate the potential viability of a fowlpox virus-based recombinant vaccine in the control and prevention of FMDV infections.     

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs,swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD₅₀) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.     

Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals

Non-structural protein (NSP) 3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals. An indirect ELISA was established, using purified His-tagged 3ABC fusion protein as antigen, for detection of the antibody response to FMDV NSP 3ABC in different animal species. The method was validated by simultaneous detection of the early antibody responses to NSP and structural protein (SP) in FMDV Asia 1 infected animals. The performance of the method was also validated by detection of antibody in reference sera from the FMD World Reference Laboratory (WRL) in Pirbright, UK, and comparison with two commercial NSP ELISA kits. The results showed that the antibody response to SP developed more quickly than that to NSP 3ABC in FMDV infected animals. In contact-infected cattle, the antibody response to NSP 3ABC was significantly delayed compared with that to SP antibody. The early antibody responses to SP and NSP 3ABC in FMDV inoculated cattle and contact-infected or inoculated sheep and pigs were generally consistent. In pigs, 3ABC antibody was linked to the presence of clinical signs; however, in sheep, subclinical infection was detected by the development of 3ABC antibodies. Therefore, the antibody responses to 3ABC varied between host species. Eight out of 10 positive serum samples from FMD WRL were tested to be positive at cutoff value of 0.2. The rate of agreement with the ceditest FMDV-NS and the UBI NSP ELISA were 98.05% (302/308) and 93.2% (287/308), respectively. The prevalence of 3ABC antibodies reached 71.4% in some diseased cattle herds. The further work is required to evaluation the performance of this method in different animal species and different field situations.     

猪免疫口蹄疫灭活疫苗后外周血细胞亚群变化的研究

为了解动物在注射口蹄疫灭活疫苗后机体免疫状态的变化情况,本试验选取口蹄疫阴性猪进行疫苗免疫,利用流式细胞术检测方法来测定外周血中免疫细胞亚群的变化。结果显示,树突状细胞在免疫后能够快速产生应答,在24 h内其含量出现明显升高;B细胞和Th细胞含量在免疫后第10天都出现了明显升高,且两者之间的变化存在着高度相关性,而抗体水平的变化情况与这两种细胞含量的变化也高度相关;细胞毒性T细胞含量在免疫后也出现升高。这说明灭活疫苗免疫动物后不仅可以激活机体的体液免疫应答反应,产生高水平抗体,发挥免疫保护作用,更重要的是能够在短时间内激发机体的固有免疫应答,同时能够激活细胞免疫应答,全方位发挥免疫保护作用。     

Study on Change of Lymphocyte Subpopulations in Swine after Immunization with Inactivated Vaccine

In order to investigate the change of lymphocyte subpopulations in the peripheral blood after injection of foot-and-mouth disease inactivated vaccine, blood samples were collected after vaccination and assayed with flow cytometry.The results showed that the proportion of dendritic cells (DCs) increased significantly in a short time after vaccination, after which the number of B cells, helper T (Th) cells and cytotoxic lymphocytes (CTLs) also raised with significance.The changing trend of B cells proportion was highly relevant with Th cells and a same correlation also appeared in the antibody titers.This indicated that the innate immune system could be activated rapidly after vaccination with the inactivated vaccine, and the adaptive immune response including cellular immune response and humoral immune response could be invoked subsequently.The high level of neutralizing antibodies was of great importance in evading FMDV and the cellular immune response also exerted a crucial role.     

Effectiveness of vaccines and vaccination programs for the control of foot-and-mouth disease in Uganda, 2001–2010

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. In Uganda, FMD outbreaks are mainly controlled by ring vaccination and restriction of animal movements. Vaccination stimulates immunity and prevents animals from developing clinical signs which include lameness, inappetence, and decreased production. Ring vaccination and restriction of animal movements have, however, not successfully controlled FMD in Uganda and outbreaks reoccur annually. The objective of this study was to review the use of FMD virus (FMDV) vaccines and assess the effectiveness of vaccination programs for controlling FMD in Uganda (2001–2010), using retrospective data. FMD vaccine distribution patterns in Uganda (2001–2010) matched occurrence of outbreaks with districts reporting the highest number of outbreaks also receiving the largest quantity of vaccines. This was possibly due to “fire brigade” response of vaccinating animals after outbreaks have been reported. On average, only 10.3 % of cattle within districts that reported outbreaks during the study period were vaccinated. The average minimum time between onset of outbreaks and vaccination was 7.5 weeks, while the annual cost of FMDV vaccines used ranged from US $58,000 to 1,088,820. Between 2001 and 2010, serotyping of FMD virus was done in only 9/121 FMD outbreaks, and there is no evidence that vaccine matching or vaccine potency tests have been done in Uganda. The probability of FMDV vaccine and outbreak mismatch, the delayed response to outbreaks through vaccination, and the high costs associated with importation of FMDV vaccines could be reduced if virus serotyping and subtyping as well as vaccine matching were regularly done, and the results were considered for vaccine manufacture.     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

Bovine serum panel for evaluating foot-and-mouth disease virus nonstructural protein antibody tests.

A panel of 36 sera has been assembled from experimental cattle that had been infected by inoculation or contact exposure with 4 serotypes of foot-and-mouth disease virus (FMDV) with or without prior vaccination. Virus replication and persistence had been characterized in all of the animals. The proportion of the sera scored positive by 5 tests for antibodies to the nonstructural proteins of FMDV varied, suggesting that the panel can discriminate between the sensitivity with which such tests are able to identify infected cattle. Use of this panel will help in assessment of new tests and quality control of existing methods.     

Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant.

The epidemiological analysis of the cattle population during the eradication plan of foot and mouth disease (FMD) in Argentina clearly indicated a higher incidence of the disease in animals within their first year of age. It is important to improve the efficacy of the vaccination in those animals. In a previous report, we have shown the effect of an immunomodulator, Avridine (Avr), in the enhancement of the immune response elicited by FMD virus (FMDV) vaccines in experimental hosts [Berinstein, A., Pérez Filgueira, M., Schudel, A., Zamorano, P., Borca, M., Sadir, A.M., 1993. Avridine and LPS from Brucella ovis: effect on the memory induced by foot-and-mouth disease virus vaccination in mice. Vaccine 11, 1295-1301]. In this report, we analyze the effect of Avr in the improvement of the anti-FMDV immune response elicited in young animals immunized with a tetravalent vaccine. The anti-FMDV antibody response was evaluated using a liquid-phase blocking sandwich ELISA (LPBE) [Smitsaart, E.N., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V., Schudel, A.A., 1998. Assessment using ELISA of the herd immunity levels induced in cattle by foot and mouth disease oil vaccines. Prev. Vet. Med 33, 283-296] while the cellular response was detected using an antigen specific lymphoproliferative test [Zamorano, P., Wigdorovitz, A., Chaher, M., Fernández, F., Sadir, A., Borca, M., 1994. Localization of B and T cell epitopes on a synthetic peptide containing the major immunogenic site of FMDV O1 Campos. Virology 201, 383-387]. The results show that, while no differences were detected in the cellular response, the anti-FMDV antibody reaction was significantly (<0.05) higher in animals immunized with the immunogen containing Avr. At 90 days post vaccination, 89-100% of the animals immunized with Avr presented predicted protection (PP) higher than 82% while just 50-61% of the animals immunized with vaccine without immunomodulator presented that characteristic. Also, it is shown that the increase in the anti-FMDV antibody titre in animals immunized with the vaccine containing Avr was mediated by an increase in the levels of both IgG1 and IgG2 which presented a significative correlation with LPELISA antibodies titres. It is concluded that the addition of Avr in the FMDV vaccines improve the immune status of the calves, the cattle population that suffers the highest epidemiological risk.     

A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.     

Immunization of cattle against Aujeszky's disease with inactivated adjuvant vaccine

The experiments with sheep and young cattle were carried out to test the immunizing efficacy of inactivated adjuvant vaccine against Aujeszky's disease. The vaccine application at doses of 1 ml and 2 ml to lambs at the age of eight to ten months caused the neutralizing antibody production with a significant rise of titres after revaccination. A survival of infection induced with a dose of 10(5.5) TKID50 of virulent virus was recorded in 62.5% of once vaccinated animals and in 87.5% of twice vaccinated animals. When applying different doses of vaccines (from 1 to 10 ml) to young cattle, the antibody reaction level was directly dependent on the inoculum quantity. The double inoculation of animals with vaccines of 2 ml and 5 ml caused the neutralizing antibody production at titres of 1:35, or 1:46. The animals, immunized with the live or inactivated IBR-vaccine possessing high antibody titres against IBR-virus, reacted upon the vaccination with inactivated Aujeszky's vaccine anamnestically, by early production of antibodies in high titres. Metaphylactic vaccination (2 ml of vaccine) of cattle in herds with an acute course days, however earlier during five days from the revaccination when it was carried out in seven days following the first vaccination.     

Foot-and-mouth disease: past,present and future

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals including cattle, pigs, sheep and many wildlife species. It can cause enormous economic losses when incursions occur into countries which are normally disease free. In addition, it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal products. The disease is caused by infection with foot-and-mouth disease virus (FMDV), a picornavirus. Seven different serotypes (and numerous variants) of FMDV have been identified. Some serotypes have a restricted geographical distribution, e.g. Asia-1, whereas others, notably serotype O, occur in many different regions. There is no cross-protection between serotypes and sometimes protection conferred by vaccines even of the same serotype can be limited. Thus it is important to characterize the viruses that are circulating if vaccination is being used for disease control. This review describes current methods for the detection and characterization of FMDVs. Sequence information is increasingly being used for identifying the source of outbreaks. In addition such information can be used to understand antigenic change within virus strains. The challenges and opportunities for improving the control of the disease within endemic settings, with a focus on Eurasia, are discussed, including the role of the FAO/EuFMD/OIE Progressive Control Pathway. Better control of the disease in endemic areas reduces the risk of incursions into disease-free regions.     

Immunogenicity of foot-and-mouth disease virus type O1 replicated in either monolayer or suspended BHK cell system

The efficacy of vaccines formulated from the 10th passage of foot-and-mouth disease virus (FMDV) type O1 in monolayer baby hamster kidney (BHK) cells and the 8th passage in suspension BHK cells was compared in steers. The vaccines were inactivated with ethylenimine, contained an equal amount of antigen and were emulsified in oil-adjuvant. Six animals were vaccinated with each vaccine. During the challenge of immunity (91 days post-vaccination, DPV), one out of the six steers from the monolayer vaccine group became infected with the challenge virus while none of the six steers from the suspension vaccine group contracted the disease during the test period. The neutralizing antibody titers (means) of the serum samples taken at different DPV also did not suggest significant variation between these vaccines. In addition exposure to FMDV infected animals demonstrated that both vaccines elicited an immune state in the vaccinates.     

Enhancing immune responses to inactivated foot-and-mouth virus vaccine by a polysaccharide adjuvant of aqueous extracts from Artemisia rupestris L.

BackgroundNew-generation adjuvants for foot-and-mouth disease virus (FMDV) vaccines can improve the efficacy of existing vaccines. Chinese medicinal herb polysaccharide possesses better promoting effects.ObjectivesIn this study, the aqueous extract from Artemisia rupestris L. (AEAR), an immunoregulatory crude polysaccharide, was utilized as the adjuvant of inactivated FMDV vaccine to explore their immune regulation roles.MethodsThe mice in each group were subcutaneously injected with different vaccine formulations containing inactivated FMDV antigen adjuvanted with three doses (low, medium, and high) of AEAR or AEAR with ISA-206 adjuvant for 2 times respectively in 1 and 14 days. The variations of antibody level, lymphocyte count, and cytokine secretion in 14 to 42 days after first vaccination were monitored. Then cytotoxic T lymphocyte (CTL) response and antibody duration were measured after the second vaccination.ResultsAEAR significantly induced FMDV-specific antibody titers and lymphocyte activation. AEAR at a medium dose stimulated Th1/Th2-type response through interleukin-4 and interferon-γ secreted by CD4⁺ T cells. Effective T lymphocyte counts were significantly elevated by AEAR. Importantly, the efficient CTL response was remarkably provoked by AEAR. Furthermore, AEAR at a low dose and ISA-206 adjuvant also synergistically promoted immune responses more significantly in immunized mice than those injected with only ISA-206 adjuvant and the stable antibody duration without body weight loss was 6 months.ConclusionsThese findings suggested that AEAR had potential utility as a polysaccharide adjuvant for FMDV vaccines.     

Detection of foot-and-mouth disease virus infection in vaccinated cattle

The aim of this study was to evaluate the value of commercially available kits for the detection of foot-and-mouth disease (FMD) virus infection in vaccinated cattle. The cattle were vaccinated with a commercial aqueous FMD vaccine type A24 and subsequently challenged 28 days post vaccination with homologous FMD virus. Seven of eight animals were protected from clinical disease and all became carriers. They were bled sequentially for up to 130 days post infection and samples of sera were tested with three ELISA kits: CHEKIT FMD-3ABC, Ceditest FMDV-NS and SVANOIR FMDV 3ABC-Ab ELISA. The Ceditest kit appears to be relatively higher sensitive than the others. When examined with this ELISA, all cattle developed of FMDV nonstructural proteins (NSPs) antibodies and remained positive throughout the period of the experiment. The response of antibodies against 3ABC antigen delayed in two cattle challenged with FMDV A24 virus. One of the cattle reacted negatively in Svanoir ELISA kit and sera from two animals were found negative in CHEKIT ELISA. It can be concluded that all tested kits can be a promising tool for FMD control and eradication campaigns in situation where emergency vaccination was applied.     

Recombinant pseudorabies virus expressing P12A and 3C of FMDV can partially protect piglets against FMDV challenge

One of the crucial factors for evaluation of an effective genetically engineered vaccine is whether susceptible animals are protected from virus challenge after vaccination. In this study, a recombinant pseudorabies virus (PRV-P12A3C) that expressed capsid precursor polypeptide P12A and nonstructural protein 3C of foot-and-mouth disease virus (FMDV) was used as a vaccine. The expression of P12A3C and its immunogenicity and protective efficacy against FMDV challenge were measured. Humoral and cellular immune responses were evaluated after each immunization. Subsequently, each piglet was challenged with 1000 ID₅₀ (50% infection dose) FMDV serotype O, named OR/80, which is used to produce vaccine in China. PRV-P12A3C induced a high level of neutralizing antibody and FMDV-specific lymphocytes. Inactivated vaccine provided 100% protection, and the vector strain (TK⁻/gE⁻/gI⁻) showed no protection. PRV-P12A3C induced 60% protection, compared with piglets that were vaccinated with TK⁻/gE⁻/gI⁻. The severity of clinical signs for the remaining two piglets was lighter and the appearance of vesicles was delayed.