Efficacy of a high quality O1/Campos foot-and-mouth disease vaccine upon challenge with a heterologous Korean O Mya98 lineage virus in pigs.

In 2010 serotype O foot-and-mouth disease virus of the Mya98 lineage/SEA topotype spread into most East Asian countries. During 2010-2011 it was responsible for major outbreaks in the Republic of Korea where a monovalent O/Manisa vaccine (belonging to the ME-SA topotype) was applied to help control the outbreaks. Subsequently, all susceptible animals were vaccinated every 6 months with a vaccine containing the O/Manisa antigen. Despite vaccination, the disease re-occurred in 2014 and afterwards almost annually. This study focuses on the in vivo efficacy in pigs of a high quality monovalent commercial O1/Campos vaccine against heterologous challenge with a representative 2015 isolate from the Jincheon Province of the Republic of Korea. Initially, viral characterizations and r1 determinations were performed on six viruses recovered in that region during 2014-2015, centering on their relationship with the well characterized and widely available O1/Campos vaccine strain. Genetic and antigenic analysis indicated a close similarity among 2014-2015 Korean isolates and with the previous 2010 virus, with distinct differences with the O1/Campos strain. Virus neutralisation tests using O1/Campos cattle and pig post vaccination sera and recent Korean outbreak viruses predicted acceptable cross-protection after a single vaccination, as indicated by r1 values, and in pigs, by expectancy of protection. In agreement with the in vitro estimates, in vivo challenge with a selected field isolate indicated that O1/Campos primo vaccinated pigs were protected, resulting in a PD50 value of nearly 10. The results indicated that good quality oil vaccines containing the O1/Campos strain can successfully be used against isolates belonging to the O Mya98/SEA topotype.

Antigenic and immunogenic spectrum of foot-and-mouth disease vaccine strain O1 Campos against representative viruses of topotypes that circulated in Asia over the past decade.

Identifying vaccine strains to control outbreaks of foot-and-mouth disease virus that could spread to new regions is essential for contingency plans. This is the first report on the antigenic/immunogenic relationships of the South American O1/Campos vaccine strain with representative isolates of the three currently active Asian type O topotypes. Virus neutralization tests using O1/Campos post-vaccination sera derived from cattle and pigs predicted for both species acceptable cross-protection, even after single vaccination, established by r1 values and by expectancy of protection using monovalent or polyvalent vaccines. The results indicate that effective oil vaccines containing the O1/Campos strain can be used against Asian isolates, expanding the scope of O1/Campos strain included in vaccine banks to control emergencies caused by Asian viruses, even on single-dose vaccination, and to cover the need of effective vaccines in Asia during systematic vaccination.

Importance of foot and mouth disease vaccine purity in interpreting serological surveys.

The aim of this study was to determine whether the degree of purity achieved in conventional vaccines against the foot and mouth disease virus in Argentina interferes with the interpretation of seroepidemiological surveys for confirming the absence of viral activity, which are performed to support the recognition of free zones practising vaccination. The evaluation of 168 vaccine series due to be marketed in Argentina (2006-2012) and subjected to official control testing in cattle, as well as repeated vaccination of cattle and other species using vaccines with high antigen concentrations, demonstrated that they did not induce antibodies to non-structural proteins (NSPs). The results show clearly that vaccines with satisfactory potency do not induce a response to NSPs, even by forcing the immune response through more concentrated doses with multiple valences and revaccination protocols at shorter irtervals than in vaccination campaigns. These results confirm that the vaccines used in routine vaccination programmes have a degree of antigen purification consistent with the needs observed on the basis of sampling for serological surveillance. Moreover, serological surveys conducted in 2006-2011 by Argentina's official Veterinary Services--the National Health and Agrifood Quality Service (SENASA)--on more than 23,000 sera per year from cattle included in the vaccination programme, in order to confirm the absence of virus circulation, revealed an average 0.05% of reactive results, consistent with the specificity of the tests. In conclusion, the vaccines produced by conventional methods and with proven potencythat are available in Argentina are sufficiently purified to ensure thatthey do not interfere with the interpretation of sampling for serological surveillance performed to support the recognition of FMD-free zones practising vaccination.

Early protection against foot-and-mouth disease virus in cattle using an inactivated vaccine formulated with Montanide ESSAI IMS D 12802 VG PR adjuvant.

Foot and mouth disease is an acute disease of cattle with a broad distribution around the world. Due to the fast spread of FMDV infections, control measures must be applied immediately after an outbreak, such as the use of vaccines that induce fast protection. Previously, it was shown that mice vaccinated with FMD inactivated virus (iFMDV) formulated with Montanide™ ESSAI IMS D 12802 VG PR adjuvant (802-iFMDV) were protected when they were challenged 4 and 7 days post-vaccination (dpv) with homologous virus. In this work, we describe the successful use of this formulation in cattle. In addition, adjuvant Montanide™ IMS 1313 VG NPR was also tested. 802-iFMDV vaccine was able to confer 100% protection against viral challenge at 4 and 7 dpv, while eliciting low antibody levels, at 7 dpv. 1313-iFMDV vaccine induced protection in 60% of cattle. At 4 dpv, 1313-iFMDV vaccinated animals presented increased levels of IFNγ but not of macrophages. At 4 and 7 dpv, macrophages, IFNγ, nasal IgA and IgG1 antibodies against FMDV, and opsonophagocytosis were increased in animals vaccinated with 802-iFMDV indicating that these phenomena could be involved in protection.It is the first time that total protection against FMDV at early stages post-vaccination is reported using a single dose of the formulation iFMDV plus Montanide™ ESSAI D IMS 12802 VG PR adjuvant.

Foot-and-mouth disease vaccine potency testing in cattle using homologous and heterologous challenge strains: precision of the "Protection against Podal Generalisation" test.

The level of protection conferred by foot-and-mouth disease (FMD) vaccines in primovaccinated animals primarily depends on the potency of the vaccine and the relatedness of the vaccine strain and circulating field isolate. The "Gold Standard" FMD vaccine potency test is the in vivo test performed in the target species. The objective of the study was to determine the precision of the in vivo "Protection against Podal Generalisation" (PPG) FMD vaccine potency test in cattle using homologous (vaccine quality control) and heterologous (vaccine matching) viral challenge. The overall level of protection induced by the A(24) Cruzeiro/Brazil/55 vaccine used in six homologous PPG tests was 88.5%. Vaccine accordance (VACC) and vaccine concordance (VCON) were estimated to be 75.9% and 73.7%, respectively. In four heterologous challenge PPG tests, the overall level of cross-protection induced by the A(24) Cruzeiro/Brazil/55 vaccine against A Argentina/2001 challenge was 26.6%, with VACC and VCON values of 65.7% and 59.2%, respectively. Results indicate that the homologous PPG test is more reliable than the European Pharmacopoeia potency test, but that a larger number of animals should be used in order to increase the test's statistical power. In this regard, indirect alternative tests for vaccine potency and vaccine matching merit consideration.

A comparison of methods for measuring the antibody response in mice and cattle following vaccination against foot and mouth disease.

We present a comparison of methods for evaluating the potency of foot and mouth disease vaccine in the laboratory. The anti-FMDV antibodies (Ab) in vaccinated mice were tested by liquid phase (lp) ELISA, solid phase (sp) ELISA and virus neutralization (VN), and were compared with the Ab titres detected by lpELISA, which is the official test in Argentina for testing the potency of FMD vaccines and protection against a virulent challenge in cattle. The results demonstrated that it is possible to relate the Ab levels induced in vaccinated mice with both the Ab and protective responses elicited in cattle. Furthermore, it was found that the anti-FMDV Ab titres in mice detected by lpELISA 14 days after vaccination should be an accurate parameter for predicting the results of the challenge test in cattle. Thus, this test in mice appears to be an inexpensive and rapid alternative for testing FMD vaccines in cattle.

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus.

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.

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.

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines.

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of naïve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.

[Seroepidemiologic indicators for the evaluation of campaigns for the control of foot-and-mouth disease]. / Indicadores seroepidemiológicos para la evaluación de las campañas de control de fiebre aftosa.

A sero-epidemiological survey was conducted in two districts in Argentina between 1993 and 1995, to provide additional information on the epidemiology of foot and mouth disease (FMD) in Argentina and to assess the level of immunity in cattle populations, and the circulation of FMD virus. As part of the final stage of this survey, a comparison was made of the results obtained by the enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion techniques. Levels of population immunity against the four types of virus included in the vaccine increased progressively during the period of the survey until, in 1995, at the end of the vaccination period, the percentage of animals possessing adequate levels of protection was approximately 77% in yearlings, and more than 94% in cattle over one year old. During the three-year study, there was a clear tendency for viral activity to diminish, until in 1995 when between 3% and 0.6% were positive to the agar gel immunodiffusion test for the antigen associated with viral infection. By contrast, the ELISA detected antibody in about five times as many animals. The authors show how the increase in the level of population immunity was accompanied by a fall in viral activity.

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease.

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.

Detection of antibodies against foot-and-mouth disease virus using a liquid-phase blocking sandwich ELISA (LPBE) with a bioengineered 3D protein.

A liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA-3D) was developed to detect specific antibodies to the 3D protein in sera from foot-and-mouth disease (FMD) virus (FMDV)-infected animals. The assay uses a nonstructural 3D recombinant protein and two polyclonal antisera, one for capture (bovine) and the other for detector (guinea pig). The specificity of the assay was demonstrated by negative results with 101 sera of cattle from the FMD-free zone in Argentina and with bovine and porcine sera raised against various RNA and DNA viruses. The ELISA-3D was able to detect antibodies in cattle after natural or experimental infection with FMDV of A, O, or C types as early as 5 days postinfection and at later stages in persistently infected animals. Comparison of the results with those obtained with the routinely used agar gel immunodiffusion test and a previously described ELISA, both employing a partially purified virus-infection-associated antigen, shows that the ELISA-3D is highly sensitive and specific and gives reproducible results. Its use as a tool for monitoring viral activity and for certification of FMDV-free animals is recommended.

Immune response of calves to foot-and-mouth disease virus vaccine emulsified with oil adjuvant. Strategies of vaccination.

Calves born to vaccinated cows under the regular annual vaccination programme were vaccinated at different ages using commercial quadrivalent (01, A79, A87 and C85 FMDV strains) vaccine emulsified in oil adjuvant. The antibody responses of vaccinated calves were evaluated using liquid-phase blocking sandwich ELISA. All calves 20, 30 and 40 days old having high maternal antibody titres responded well to vaccination. Moreover, 25-57% of vaccinated calves showed protective antibody titres both at 90 and 120 days post-vaccination (d.p.v.), whereas none of the non-vaccinated animals achieved these levels. Calves aged 3-4 months with non-protective levels of colostral-derived antibodies responded with high antibody titres to vaccination which persisted for at least 4 months. In both groups of calves a certain degree of suppression of postvaccinal response was observed which was related to colostral antibody titres. Our results suggest that in order to reduce the proportion of calves susceptible to infection it is advisable to immunize calves as young as 20 days old to induce acceptable antibody titres for the following 4 months. In addition, a second vaccination 60 d.p.v. ensures high antibody levels in high disease risk areas.

Isotype profiles induced in Balb/c mice during foot and mouth disease (FMD) virus infection or immunization with different FMD vaccine formulations.

The IgG isotype response in Balb/c mice infected with FMDV or immunized with different vaccine formulations using inactivated virus particles as antigen was analyzed at various times post-inoculation. For this purpose an ELISA based on polyclonal antibodies for detection and quantification of mouse IgG isotypes with FMD virus (FMDV) specificity was developed. Three immunomodulators, which have been shown to be very effective in inducing strong and long-lasting antibody responses (Bahnemann, Arch. Virol. 1975, 47, 47-56; Polatnik and Bachrach, Appl. Microbiol. 1964, 12, 368-376), were employed to formulate different vaccines using aqueous and oil vehicles: a water-soluble fraction of the cell wall of Mycobacterium sp., a purified extract of lipopolysacharide from Brucella ovis and a synthetic lipoamide, Avridine. Infected animals between 14 and 60 days post-inoculation (d.p.i.) showed responses dominated by IgG2b, followed by IgG1, IgG2a and IgG3, respectively. The IgG3 isotype was the first, together with IgG1, to be elicited during the first 7 days after infection, whereas no IgG3 activity was detected in vaccinated animals at any time. With formulations including immunomodulators, persisting high levels of IgG2b (similar to those of infected animals) were detected until 180 d.p.i., while with conventional vaccines IgG2b responses were detected up to 60 d.p.i. Animals vaccinated with formulations including these immunomodulators presented an augmented resistance to viral challenge at 210 d.p.i. in relation with those immunized with conventional vaccines. The possible relationship of these differences in the isotype response and protection is discussed.

Validation of an inhibition ELISA using a monoclonal antibody for foot-and-mouth disease (FMD) primary diagnosis.

An inhibition ELISA (IH-ELISA) test for foot-and-mouth disease virus (FMDV) was validated using 106 epithelial samples from suspected cases of FMD in Argentina submitted to the Argentine National Diagnostics Laboratory (GELAB) over a period of 12 months and examined in parallel with the complement fixation test (CFT). IH-ELISA was found to be more sensitive, detecting 25% (26 samples) more FMDV positives than the CFT in original suspensions of field samples. The effect of storage conditions on 12S stability was examined. Plates stored at 4 degrees C blocked with 1% ovalbumin and plates stored at -20 degrees C with or without blocking buffer could be used for at least 90 days. When various brands of polystyrene plates were compared for 12ps adsorption it was found that those microplates of higher binding capacity were more efficient.

Comparative studies of BHV-1 variants by in vivo--in vitro tests.

The new encephalitogenic BHV-1.3 and previously characterized BHV-1 strains were studied with reference to their immunogenic and protective potency and their antigenic relationships using "in vitro" and "in vivo" tests. The "in vitro" results obtained by neutralization kinetics showed that the Los Angeles (LA) strain (BHV-1.1) and a vaginal isolate L-114 strain (BHV-1.2) had antigenic similarities. Conversely, the behavior of the encephalitogenic strain A-663 (BHV-1.3), was significantly distinct. The "in vivo" protection test was carried out in calves using LA and A-663 strains. Post-vaccination antibodies and challenge with A-663 strain showed that the immunogenic behavior and protective capacities of both strains were similar. Neutralization kinetics differences between BHV-1.1 and BHV-1.3 did not alter the "in vivo" protection against BHV-1.3 challenge.

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence

La seroneutralización (SN) en cultivos celulares, hemoaglutinación pasiva (HA), la inmunofluorescencia indirecta (IFI) y las pruebas inmunoenzimáticas (ELISA) son de utilizadas para la detección de anticuerpos específicos contra Herpesvirus-bovino 1 (BHV-1). La prueba de SN es la determinación de referencia, sin embargo las dificuldades de implementación por la necessidad de contar con cultivos celulares, así como la celeridad en la obtención de resultados, determinan la optimización de otra prueba de alternativa y equivalente especificidad y sensibilidad. Trabajos previous describen una alta correlación entre las técnicas de SN vs IFI y de SN vs ELISA, destacando una mayor sensibilidad para el ELISA. El presente trabajo compara la sensibilidad y especificidad de IFI, ELISA y SN en la detección de anticuerpos a BHV-1 utilizando 105 sueros bovinos. La especificidad de las técnicas comparadas se demostró por la seroconversión obtenida en sueros de animales experimentalmente infectados (Cuadro 1). Se observó una alta asociación entre sueros reactivos y no reactivos entre SN e IFI, SN y ELISA y entre Ifi y ELISA destacándose una mayor sensibilidad para el ELISA (Figuras 2 y 3). El estudio estadístico de los resultados obtenidos con las tres técnicas diagnósticas determinó un alto coeficiente de correlación entre las mismas (Cuadro 2). Debido a la simplicidad y facilidad de ejecución se recomienda la técnica de ELISA para estudios epizootiológicos de larga escala

Detection of foot-and-mouth disease virus by competitive ELISA using a monoclonal antibody specific for the 12S protein subunit from six of the seven serotypes.

Foot-and-mouth disease (FMD) prevention and control programs are dependent upon rapid, reliable diagnostic procedures. The widely used FMD diagnostic complement fixation (CF) procedures require a specific antiserum for each of the seven FMDV serotypes making the tests both cumbersome and difficult to standardize. An FMD diagnostic, monoclonal antibody based inhibition-ELISA procedure was developed. The test uses a single monoclonal antibody (MAb) that reacts with all European and South American FMDV isolates examined. The procedure detects a highly conserved epitope on the 12S protein subunit of FMDV which appears to be common to all FMDV's with the exception of the South African Territories 2 serotype. The results indicate that the sensitivity of this test is greater than CF and approaches that of virus isolation.

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence.

Serum neutralization (SN), indirect immunofluorescence (IFI) and enzyme-linked immunosorbent assay (ELISA) were compared for their sensitivity and specificity to detect bovine serum antibodies to Bovine Herpesvirus type 1 on 105 bovine serum samples. High correlation coefficients among all three techniques were observed, showing a higher sensitivity for the ELISA test. Due to its simplicity and its feasibility to be adopted in less equipped laboratories, the ELISA test is recommended for large scale epizotiological studies, serological diagnosis and detection of reactive animals.