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 , r_s) between SNT and LPBE were highly significant (r_s > 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.     

Presence of antibodies to non-structural proteins of foot-and-mouth disease virus in repeatedly vaccinated cattle

For the purpose of removing infected animals by detecting humoral immune responses to non-structural proteins of the foot-and-mouth disease (FMD) virus, antibodies induced by contaminated residual non-structural proteins contained in less pure FMD vaccine can be problematic for serological screening. The aim of the present study was to measure the possible presence of antibodies against these non-structural proteins in repeatedly vaccinated calves and beef cattle. Five imported FMD vaccines were examined using two commercial ELISA kits, UBI FMDV NS EIA and Ceditest FMDV-NS, for serological testing. After five doses of vaccination, the serum of one calf tested positive, and two vaccines induced a significant increase in anti-3ABC antibodies in calves. This finding demonstrated that a positive reaction to non-structural proteins due to impurities in the FMD vaccine was detectable using commercial tests. A low percentage of field sera sampled from beef cattle in Kinmen also tested positive, but the key factor resulting in the positive reactions could not be positively identified based on our data.     

Serological evidence of FMD subclinical infection in sheep population during the 1999 epidemic in Morocco.

During 1999, 11 outbreaks of foot and mouth disease (FMD) were declared in the east and central part of Morocco. All the FMD clinical cases reported were cattle. In order to analyse the serological status of sheep from the FMD outbreak areas, 598 sheep sera were tested using a liquid-phase blocking ELISA (LPBE) to detect antibodies against FMDV structural proteins. The study confirmed the presence of FMDV specific antibodies in 77 clinically normal sheep, indicating that unrecognised FMDV-infected sheep could represent a potential risk of FMD dissemination in Morocco.Subsequently, sera from flocks of sheep that had been exposed to FMD outbreaks were assayed by an indirect ELISA using the recombinant FMDV non-structural protein 3ABC expressed in E. coli to evaluate the potential use of this serological test in future epidemiological studies and the development of FMD control strategies. The results indicated that the 3ABC-ELISA was able to detect antibodies indicative of infection with FMDV in asymptomatic sheep in field conditions.     

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.     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

Colostral Transfer of Rinderpest Neutralizing Antibody to Offspring of Våccinated Dams

The transfer of maternal antibodies to Friesian and buffalo calves born of dams vaccinated against rinderpest was through colostrum only. Colostral antibody titers at the time of parturition were higher than the serum titer. Two hours after suckling, a high level of rinderpest neutralizing antibodies was detected in the sera of newborn animals. The half-life of maternal antibodies in buffalo and Friesian calves was found to be approximately 33 and 29 days respectively. By the age of 7-8 months, 60 per cent of buffalo calves and 80 per cent of Friesian calves had no detectable levels of rinderpest neutralizing antibody.     

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.     

Non-structural protein 3A for differentiation of foot-and-mouth disease infected and vaccinated animals in Haryana (India)

There are severe international trade restrictions on foot-and-mouth disease (FMD) affected areas. Because of endemic nature of FMD, India started FMD control programme (FMD-CP) using mass vaccination in selected states including Haryana (year 2003). Although no significant incidence of the disease was reported after launching FMD-CP in the state but in order to participate in international trade of animal and animal products, veterinary authorities have to prove that there is no FMD virus (FMDV) circulation in the animal population, for which it is necessary to differentiate the FMD infected and vaccinated animals. For this purpose, an in-house indirect ELISA utilizing baculovirus-expressed FMDV non-structural protein (NSP) 3A was used to find evidence for virus circulation (prevalence of anti-NSP 3A-specific antibodies) by examining serum samples that were collected either before start of FMD-CP or after completion of third phase (Pre-4th) of vaccination in Haryana (India). A significant reduction (P < 0.01) in prevalence of anti-NSP 3A-specific antibodies (possibly carriers) was observed 2 years after launching FMD-CP in Haryana. However, in cattle the percentage of animals with anti-NSP 3A-specific antibodies was found to be significantly higher (P < 0.01) than buffalo, both before (P < 0.01) and after (P < 0.01) launching FMD-CP in the state. The findings of this study suggest that use of FMDV vaccine in cattle and buffaloes in endemic areas reduces virus circulation (carriers) in the vaccinated herds and that the current 3ANSP-ELISA can be successfully used to monitor the FMDV circulation in endemic areas.     

Infectious bovine keratoconjunctivitis: evidence for genetic modulation of resistance in purebred Hereford cattle

A vaccination study was conducted in a herd of purebred Hereford cattle representing 4 selection (genetic) lines. For each of 2 years, half of the cattle were vaccinated with a pilus-enriched Moraxella bovis bacterin. Cows were vaccinated before parturition, and calves were vaccinated at 2 to 3 months of age. None of the cattle was vaccinated for 1 year preceding and 1 year after the 2 years in which cattle were vaccinated. There was a significantly (P less than 0.05) lower percentage of infectious bovine keratoconjunctivitis (IBK) in calves during years cattle were vaccinated than during years cattle were not vaccinated. During years cattle were vaccinated, there were lower percentages of IBK in vaccinated calves when compared with the percentages of IBK in nonvaccinated calves. When calves were compared on the basis of selection lines, regardless of the vaccination group, there were consistent differences in the percentages that developed IBK. Although calves with pigmented and nonpigmented eyes (representing all 4 genetic lines) developed IBK, the genetic line of calves with the most pigmented eyes had the lowest (P less than 0.05) percentage of IBK. Also, across all genetic lines, there was less IBK in pigmented eyes than in nonpigmented eyes. Seemingly, vaccination of dams, before parturition, and young calves reduced the occurrence of severe IBK in a herd situation under natural exposure conditions. The resistance or susceptibility in cattle under good management may be influenced by genetic factors.     

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.     

Differentiating infection from vaccination in foot-and-mouth-disease: evaluation of an ELISA based on recombinant 3ABC

Recent devastating outbreaks of foot-and-mouth disease (FMD) in Europe have reopened the discussion about the adequacy of the non-vaccination strategy implemented by the EU in 1991. Here we describe the evaluation of a new commercially available test kit for the discrimination between vaccination and infection. The test is based on the detection of antibodies against the recombinant non-structural (NS) protein 3ABC. In contrast to immunization with vaccines free of 3ABC, these antibodies are elicited as a consequence of infection. Testing more than 3600 negative sera from several countries revealed a specificity of > 99% for bovine, ovine, and porcine samples. Antibodies specific for 3ABC can be detected as soon as 10 days post-infection. As compared with the occurrence of antibodies against structural proteins of FMDV, anti-3ABC antibodies can be detected 5-10 days later, depending on the species. No anti-3ABC antibodies were detected in sera from vaccination experiments or in field sera from vaccinated animals. However, anti-3ABC antibodies can be detected in vaccinated animals upon challenge. These results provide evidence that this test can facilitate the use of vaccines in new strategies against FMD.     

A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C. II. Application

The complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was evaluated to detect antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. Log10 serum titres of uninfected, unvaccinated cattle (n = 100) were less than 1.80 in the CTB-ELISA. Sera from cattle vaccinated with either monovalent or trivalent vaccines were tested in both the CTB-ELISA and the serum neutralisation test (SNT); titres in both tests correlated positively (P less than 0.001). Titres of sera from cattle, sheep, and pigs vaccinated twice with FMDV A10 Holland also correlated positively in both tests. In another experiment, cattle vaccinated with FMDV strain C1 Detmold were intradermolingually challenged 3 weeks after primary vaccination; at the same time two controls were challenged. At 8 days after challenge, serum titres of the controls were distinctly higher in the CTB-ELISA than in the SNT, whereas serum titres of the vaccinated cattle were equally high in both tests. In potency tests for monovalent vaccines against FMDV strains A10 Holland, O1 BFS or C1 Detmold, serum titres correlated strongly in both tests with protection against the homologous FMDV strain. We concluded that the CTB-ELISA is not only sensitive, but easier to perform and more rapid and reproducible than the SNT. The CTB-ELISA may be useful in evaluating the immune response in cattle during FMD vaccine potency tests.     

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 competition enzyme immunoassay for brucellosis diagnosis

Two monoclonal antibodies (MAbs) conjugated with horseradish peroxidase were used independently in a competitive enzyme immunoassay (cEIA) to detect Brucella specific antibodies in 1120 sera from Brucella-free cattle, 61 from cattle known to be infected with B. abortus, and 207 sera from vaccinated calves. The results were compared to those obtained in the complement fixation test (CFT). The cEIA with both MAbs proved to be more sensitive than the CFT because no false-negative results were obtained. In addition, discrimination between sera from infected and vaccinated animals was more evident.     

Passive transfer of antibodies to Shiga toxin-producing Escherichia coli O26, O111 and O157 antigens in neonatal calves by feeding colostrum

To study whether or not passive immunity of neonatal calves against Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157 was obtained by colostrum administration, serum antibodies in calves after the feeding were determined by enzyme-linked immunosorbent assay (ELISA) in comparison with antibodies in colostrum and sera from donor dams. The highest antibody titers to STEC in colostrum from dams were detected soon after parturition. The antibody titers were found to be elevated in sera of neonatal calves (4-9 hr after birth) orally administered with colostrum with high antibody titers, suggesting that passive immunity of neonatal calves to STEC infection may be obtained by feeding colostrum. These results suggest that colostrum administration to neonatal calves may play an important role in elevating serum antibodies against STEC in neonatal calves.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.     

The immunization of calves against heartwater: subsequent immunity both in the absence and presence of natural tick challenge

Cattle, vaccinated as calves with Cowdria ruminantium-infected tick stabilate, were challenged 6, 12 and 24 months later. In the absence of tick challenge, vaccination of calves induced a partial immunity against subsequent challenge at 12 and 24 months. In animals exposed to ticks, the resistance was no better than that of control, unvaccinated cattle. When they were challenged at 6 months of age there was no difference between vaccinated and unvaccinated calves, either in the absence or presence of tick challenge, and all the animals manifested a high degree of natural resistance. This study therefore suggests that the value of vaccinating Afrikander-cross calves in heartwater endemic areas should be further investigated. The indirect fluorescent antibody (IFA) test proved to be a valuable means of monitoring the serological response of vaccinated animals and detecting the sero-conversion of animals exposed to tick infection. On one hand, there was good correlation between the febrile reaction and the results of the IFA test on the sera of vaccinated and control cattle challenged with the heartwater agent, in that all sero-positive animals were resistant to challenge. On the other hand, though, a considerable percentage of the animals that were serologically negative were also resistant to challenge.     

Functional analysis of antibody responses of feedlot cattle to bovine respiratory syncytial virus following vaccination with mixed vaccines.

The antibody response of cattle to bovine respiratory syncytial virus (BRSV) immunization was investigated using 4 different commercially available mixed vaccines. Forty, 5-6 month old, beef calves, randomly assigned to groups of 10, were vaccinated on day 0 and 21 with 1 of 3 inactivated vaccines, (3 groups), or a modified live virus (MLV) vaccine. BRSV-specific antibody responses were measured prior to vaccination and on day 35 by using an enzyme linked immunosorbent assay (ELISA), virus neutralization assay (VN), a fusion inhibition assay (FI); and responses were also measured for their ability to facilitate antibody dependent, complement mediated cytotoxicity (ADCMC) of BRSV infected cells. Sera from day 35 were, in addition, analyzed by use of an IgG1, IgG2 isotype specific ELISA. All vaccines induced significant increases in BRSV specific IgG antibody as measured by ELISA, but only one inactivated and the MLV vaccine induced significant increases in VN titers. Fusion inhibiting antibody titers were low or undetected in calves vaccinated with the inactivated vaccines. Vaccination with modified live virus induced significantly higher titers of fusion inhibiting antibodies, which are considered to be most highly correlated with protection. The VN to ELISA and FI to ELISA ratio of the calves that received MLV vaccine were significantly greater than the calves receiving the 3 inactivated vaccines. Vaccination with MLV induced the highest IgG2/IgG1 ratio. This difference was small, and only significant relative to 2 of the inactivated vaccine groups, which were not significantly different from each other. The higher proportion of IgG2 isotype in the MLV sera was not associated with lower ADCMC, a function not attributed to this isotype. The VN and FI titers, but not the ELISA value of the sera, were most predictive of ADCMC. The inactivation processes apparently alter epitopes and affect the induction of functional antibodies.     

Vaccine immune response and interference of colostral antibodies in calves vaccinated against rabies at 2, 4 and 6 months of age born from antirabies revaccinated females

Considering the high prevalence of rabies in cattle, we aimed to evaluate the interference of colostral antibodies transferred to calves after birth and the benefit of administering an antirabies vaccination in two-month-old calves compared to vaccinating at 4 and 6 months of age. Calves born from females revaccinated against rabies during the third trimester of pregnancy were studied. Forty-eight hours after parturition, blood samples from dams and offspring were collected, and antirabies neutralizing antibody titers were analyzed using the Rapid Focus Fluorescent Inhibition Test. We found that all calves had similar titers of antibodies transferred through the colostrum. Furthermore, none of the calves presented a satisfactory serological response after the first vaccination, but all had an appropriate response after revaccination. This study demonstrates that antirabies vaccination should be recommended for calves at two months of age in endemic and epizootic situations.