Foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods

Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10 vesicular epithelia, which were derived from submissions from suspect cases of FMD or swine vesicular disease (SVD). Sixteen samples were derived from six FMD virus positive epithelia representing four different serotypes (two each of types O and A and one each of types Asia 1 and SAT 2), two from samples which had been found to be negative by antigen ELISA and virus isolation (VI) in cell culture and two from SVD virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of three of the initial suspensions. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The best of the RT-PCR assays used in each laboratory gave comparable results while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. This prototype panel of samples would appear suitable for external quality assurance of these tests but would benefit from the inclusion of more negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.     

口蹄疫诊断技术研究进展

口蹄疫的有效控制关键在于早期检测 ,然而有很多疾病症状与口蹄疫相似 ,仅靠临床症状难以确诊 ,因此必须进行实验室诊断。实验室诊断包括病毒学诊断和血清学诊断。病毒学诊断方法有病毒分离、补体结合试验、酶联免疫吸附试验 ( EL ISA)、RT-PCR以及乳胶凝集试验 ( L AT)。 RT-PCR有待进一步完善 ,而用于野外检测的现场诊断方法已取得可喜进展。血清学诊断包括中和试验和 EL ISA,中和试验已经被 EL ISA方法取代 ,并且通过检测非结构蛋白的抗体可以区分感染动物和免疫动物。更加快速、敏感、可靠以及用于检测潜伏感染的诊断技术将是今后研究的热点。     

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9-10th-day and 7-8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003-2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.     

Application of non-structural protein ELISA kits in nationwide FMD surveillance in pigs to demonstrate virus circulation in Taiwan

Large scale surveillance of FMD non-structural protein (NSP) antibody in pigs was conducted to monitor for FMD virus circulation in Taiwan using Ceditest and UBI NSP ELISA kits after recurrence of FMD in 2009. A total of 53,759 serum samples were collected from pigs in the auction markets in 2009. There were 43 farms with positive FMD NSP reactors to both NSP ELISA tests in the nationwide surveillance. After tracing back, clinical examination and the NSP ELISA testing using both Ceditest and UBI on 14 follow-up serum samples from all the herds with confirmed NSP reactors in 2009, there were 4 farms classified as positive on follow-up testing criteria. In this surveillance, we have demonstrated that the NSP ELISA tests of outbreak farms followed by clinical and serological investigation could be used to detect FMD circulation in the pig population in Taiwan even while the national compulsory vaccination program is ongoing.     

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.     

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.     

应用实时荧光定量TaqMan RT-PCR检测口蹄疫病毒

按照口蹄疫病毒(FMDV)聚合酶3D基因序列,设计合成了引物和探针,经各反应务件的优化,建立了实时荧光定量RT-PcR技术,对细胞培养物、水泡液、水泡皮及分泌物、血液中的FMDV进行了特异性检测和敏感性试验。结果,用300nmol／L的引物浓度和200nmol／L探针浓度,获得的CT值较小,而△Rn最大;可检测到相当于9．1TCID50的病毒RNA;与VSV和其他水泡性病毒不发生交叉反应;制作的标准曲线中各浓度范围内有极好的线性关系,且线性范围宽,相关系数为0．984;组内和组间试验重复性的变异系数(CV)分别为5．4％和6．7％;与常规RT-PCR相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。     

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.     

Serosurveillance of foot-and-mouth disease in sheep and goat population of India

Serological investigation to detect foot-and-mouth disease (FMD) virus circulation in the domestic small ruminant population of India was conducted. A total of 4407 and 4035 serum samples from sheep and goats, respectively were collected at random covering majority of the states across the country during 2010–2012. These samples were analyzed for antibodies against the non-structural proteins (NSP) of FMD virus in an indirect 3AB NSP ELISA and against the structural proteins (SP) in a liquid phase blocking (LPB) ELISA. A total of 20.35% sheep and 13.60% goats were found to be positive for 3AB NSP antibodies providing a serological evidence of extensive viral activity. In LPB ELISA, only 4.54% sheep and 6.27% goats were found to have protective antibody (log₁₀ titre of ≥1.8) against all three serotype strains in the vaccine, which correlates with “no or sparse vaccination” scenario in these species in the country. Hence, to check silent amplification and dissemination of virus in a mixed farming set up, small ruminants may be brought under the ambit of routine vaccination and surveillance programmes.     

Relationship between the anti-FMD virus antibody reaction as measured by different assays, and protection in vivo against challenge infection.

The antibody response of cattle after vaccination against foot-and-mouth disease (FMD) virus was monitored using the serum neutralization test (SNT), the sandwich ELISA, liquid-phase ELISA, sandwich competition ELISA, liquid-phase competition ELISA, and the liquid-phase sandwich competition (blocking) ELISA. The competition ELISAs (in particular the "blocking" ELISA) were the most effective at detecting reactivity in these cattle sera. However, 95% of negative sera also competed in the most sensitive ELISA (the "blocking" ELISA) to titres of 1:32 (4% of the sera competed to a titre of 1:128). Comparisons between the different ELISAs, and between these ELISAs and the SNT, demonstrated that the tests were not measuring exactly the same reaction of antibody with FMD virus. With respect to the capacity of animals to resist FMD virus challenge, neither the SNT nor the competition ELISAs were consistently able to identify such animals. The anti-FMD virus antibody titres obtained could be classified into three zones; the "white zone" wherein antibody titres were high and donor animals likely to be protected; the "black zone" wherein antibody titres were low and donor animals likely to be susceptible to infection; the "grey zone" wherein the antibody titres were intermediary and no interpretation could be made with respect to protection. Assays such as ELISA and SNT cannot and do not measure immunological protection; they are a measure of antibody responses and nothing more, and should be interpreted in terms of the "three zone" phenomenon.     

Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody

For the rapid and accurate evaluation of the IgA antibody level of porcine epidemic diarrhea virus (PEDV) in pig serum and milk,a specific PEDV IgG monoclonal antibody (MAb) 8A3 was screened from four strains of PEDV MAb,which could capture all virus particles (inactivated virus cell culture medium) of PEDV efficiently.In this method,the coating concentration of 6.0 μg/mL showed the optimal performance of MAb 8A3,the cut-off value (D_{450 nm}) was settled as 0.34,it had no cross-reactivity with the positive serums of common porcine viruses.Compared with immune-peroxidase monolayer assay (IPMA),the concordance rates of established ELISA for positive and negative serum detection were 98.7% (152/154) and 98.0% (145/148),respectively.For positive and negative samples of colostrum and milk,the concordance rates of the established ELISA compared with IPMA were 100% (60/60) and 95.8% (23/24),respectively.IgA levels in colostrum and milk samples during lactation detected by established ELISA were highly correlated with trends in neutralizing titers (kappa=0.835).Collectively,the indirect ELISA in this study had high sensitivity and specificity,it was a rapid and objective method suitable for large-scale detection of PEDV IgA in clinical samples.     

Evaluation of foot and mouth vaccination for yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>) in Pakistan

In northern Pakistan, many farming communities rely on domestic yak (Bos grunniens) as a principle source of income. A 2006 participatory disease surveillance report from this region indicated that foot-and-mouth disease (FMD) is the most prevalent annual disease of yak. Our objectives of this study were to determine exposure levels of yak to FMD virus; implement a vaccination program based on current, regional FMD virus serotypes and subtypes; and quantify immune responses following vaccination. Blood samples were used to determine pre-vaccination exposure of animals to FMD virus by antibody presence to non-structural proteins of FMD virus using a 3-ABC trapping indirect ELISA. Vaccine used consisted of FMD serotypes ‘O’ (PanAsia-2), ‘A’ (Iran-05), and ‘Asia-1’ (Shamir), but changed later during the study to match newly circulating viruses in the country (‘O’-PanAsia-2; ‘A’-Turk-06 and Asia-1-Sindh-08). Three hundred sixty-three blood samples were tested from selected villages to determine pre-vaccination FMD virus exposure in yak with an average of 37.7%. Immune responses from initial vaccination and booster dose 30 days later showed clear protective levels (as mean percent inhibition) of antibodies against structural proteins of serotypes ‘O,’ ‘A,’ and ‘Asia-1.’ These responses remained above threshold positive level even at day 210 following initial vaccination. Results of sero-surveillance and anecdotal information of repeated FMD outbreaks demonstrate the persistence of FMD virus of yak in northern Pakistan. Laboratory results and field observations clearly indicated that yak can be protected against FMD with a good quality vaccine with FMD serotype(s) matching current, regionally circulating FMD virus.     

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.     

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (ELISA) and complement fixation test

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.     

A rapid enzyme-linked immunosorbent assay for the detection of foot-and-mouth disease virus in epithelial tissues

A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.