Detection of antibody to the foot-and-mouth disease virus (FMDV) non-structural polyprotein 3ABC in sheep by ELISA.

The specificity and sensitivity of an ELISA for detecting IgG to the 3ABC non-structural protein of foot-and-mouth disease (FMD) virus was evaluated in FMD naive, aerosol-infected, aerosol plus direct contact infected and field-exposed sheep. All 12 sheep that were experimentally infected without prior vaccination seroconverted in the test, although fewer field sera from FMD-exposed sheep were scored seropositive compared to test results for structural protein antibodies. The 3ABC test specificity was 98 or 100% according to whether sera reacting in the doubtful range were scored as positive or negative. The test was then used to investigate the antibody response of sheep vaccinated against FMD and exposed to the virus by an aerosol challenge 4-14 days later. The response of individual animals varied. Whether immunised with high or low doses of vaccine, the development of 3ABC antibody was most likely in sheep from which live virus was recovered at or beyond 9 days post-challenge. Non-structural responses were also more frequent in animals from which multiple incidences of live FMD virus isolation (perhaps more indicative of true virus replication) were demonstrated.

Development of tests for antibodies against foot-and-mouth disease virus in cattle milk.

The liquid-phase blocking ELISA (LPBE) and a specific isotype assay (SIA) for bovine IgG1 were modified to detect antibodies against FMDV isolate O1 Manisa in cattle milk. Samples from vaccinated animals were mostly indistinguishable from negative control cattle in the LPBE but 90% of milks from convalescent animals (which had also been vaccinated several times previously) gave positive results. The SIA was able to detect 95% of cattle vaccinated up to 12 months previously, and 100% of the recovered animals examined. Both tests could, therefore, be used for surveillance purposes following an outbreak of FMD to identify herds of cattle which had been infected, but the SIA would be required to show the presence of vaccinated animals. Samples from convalescent cattle showed the highest correlation between antibody levels in milk and in sera. Agreement was also high among recently vaccinated animals, but declined with increasing time since immunisation, though there was still a strong correlation of IgG1 levels after 6 months.

The detection of antibodies against foot-and-mouth disease virus in sheep milk.

The liquid-phase blocking ELISA (LPBE) and a specific isotype assay (SIA) modified for caprine/ovine IgG1 and IgG2 were used to detect antibodies against foot-and-mouth disease virus isolate O(1) Manisa in sheep milk samples. The majority of samples from animals vaccinated 14-23 weeks previously were indistinguishable from naive sheep when tested in the LPBE but 97% were positive using the SIA. All milk samples taken at 7 days after parturition from immunised animals were positive by LPBE. Thereafter, this proportion declined, although occasional reactors were detected at every stage up to 83 days. However, when mean titres were calculated for every sampling point after lambing, results were only positive for the first 18 days in this test. By contrast, the SIA was able to detect almost all (i.e. 97%) the vaccinated animals up to 83 days post parturition. Milk samples from convalescent animals all gave positive results in the assays. Sicilo-Sarde sheep gave higher titres on average than the Commisane breed at comparable stages after lambing. Both tests could be used for surveillance purposes following an outbreak of FMD to identify flocks of sheep which had been infected, but the SIA would be required to show the presence of vaccinated animals unless they were sampled soon after parturition.

Predicting herd protection against foot-and-mouth disease by testing individual and bulk tank milk samples.

Four groups of cattle were tested for antibodies against foot-and-mouth disease (FMD) virus type O(1) over three 70 day vaccination cycles using the liquid-phase-blocking-ELISA (LPBE). First lactation cows showed the lowest titres and group protection levels (GPLs) against FMD virus strains with 'r' values < or =0.5 while second lactation animals gave the highest results. When mean serum titres for each group and sampling date were plotted against GPL a strong correlation was found. Revaccination was indicated at a mean titre of approximately log10 2.88 (1:760; R=0.93; n=86) if the herd was threatened by field strains with an 'r' value of 0.25, or log10 2.62 (1:420; R=0.83; n=48) if this ratio was 0.5. Significant overall correlation (R=0.53; n=624) was obtained between serum titres and milk IgG(1) results derived from the modified specific isotype assay (SIA). Milk titres equivalent to 1:100, 1:200, 1:400 and 1:800 were 1:3.8, 1:6.3, 1:10.4 and 1:17.1, respectively, in first lactation cows. Bulk tank milk samples demonstrated a repeating pattern of results corresponding to the vaccination cycle with no titre lower than log10 1.05 (1:11). Colostrum from first lactation animals showed mean SIA results of log10 4.06 (1:11,480) and early milk titres only levelled off approximately 11 days post partum (dpp).

Validation of the specific isotype assay to detect antibodies against foot-and-mouth disease virus in bovine milk.

The specific isotype assay (SIA) detects IgG1 against foot-and-mouth disease (FMD) virus in bovine milk. A strong correlation was demonstrated between milk antibody titres, and those in serum as measured by the liquid phase blocking ELISA. Thus the SIA would be useful on a herd basis to monitor the milk of vaccinated cattle to determine when re-immunisation is advisable. The SIA titration ELISA was then simplified to a single dilution test and optimised to differentiate the reactions in the milk of FMD-naive cows from those in animals which had been infected with FMD or vaccinated against the disease. For milk from immunised cattle, the pH of the sample was important and borderline positive specimens with a pH of 6.0 or below gave negative results. For milk from naive animals, the optical density (OD) registered in the SIA varied according to the time of year that samples were collected which, in turn, influenced the OD above which milks might be considered positive. Studies showed that the pH of milk could be maintained within the range suitable for the SIA by either storing for up to 1 week at 4 degrees C or by freezing at -20 degrees C for an indefinite period.

The detection of antibodies against foot-and-mouth disease virus (FMDV) in filter paper eluates from pig sera or whole blood by ELISA.

The use of pig blood samples dried on paper discs for the detection of antibodies against FMDV by the liquid phase blocking ELISA has been evaluated. The average volume of whole heparinised blood required to fully saturate 6.0 mm discs was 7.65 microliters (range 7.2 to 8.1; variation = 0.24, P = 0.05). When 200 clinically healthy animals were assessed by virus neutralisation (VN) titres up to 1/22 were recorded against types O, A and C, 97% being 1/11 or less. Using ELISA, results were more skewed. Overall, 91% showed titres of 1/32 or less, and there were occasional high non-specific reactors with types O and A. Using small groups of sera from experimental animals, a VN titre of 1/16 was found to be equivalent to an ELISA titre of approximately 1/100 (log10 2.0 +/- 0.2) with type O and 1/56 (log10 1.75 +/- 0.1) for type A. Although some loss in sensitivity from disc dried sera was found in selected negatives on testing at a single dilution of 1/32, sera from vaccinated animals with VN titres of 1/16 to 1/708 all gave strong positive results. A monoclonal antibody (Mab) assay was successfully developed to detect different swine anti-FMDV antibody isotypes.

Freeze-drying foot-and-mouth disease virus antigens. II. For use in the ELISA.

Live and inactivated preparations of foot-and-mouth disease virus strains 01 BFS 1860 and A22 IRQ 24/64 were freeze-dried in the presence or absence of additive solutions and assessed for their reactivity by ELISA at intervals over a six month storage period at various temperatures and also after reconstitution and subsequent storage with or without glycerination. The type specificity of all antigen preparations was maintained throughout the study period and the potency of antigens, judged by titration in ELISA, remained constant during the freeze-drying procedure and throughout subsequent storage at -20 degrees C and 4 degrees C with or without additives having been made to virus suspensions prior to freeze-drying. This was also the case with antigens reconstituted and stored at either -20 degrees C with glycerol or at 4 degrees C without glycerol. Certain additive solutions were necessary, however, to preserve the activity of antigens stored at the elevated temperature of 37 degrees C. The reactivity of all freeze-dried antigens was not unduly affected in the liquid-phase blocking ELISA using bovine convalescent antisera of each of the seven serotypes of foot-and-mouth disease virus and known negative, non-immune bovine sera. The results suggest that shipment and long-term storage of freeze-dried foot-and-mouth disease virus antigens is possible for use in the ELISA in the absence of refrigeration. This has attractive advantages for reducing both shipment and storage costs of antigens and for the development of ELISA kits for the diagnosis of foot-and-mouth disease virus.

Freeze-drying foot-and-mouth disease virus antigens. I. Infectivity studies.

The ability of foot-and-mouth disease virus strains type O1 BFS 1860 and type A22 IRQ 24/64 to retain infectivity after freeze-drying with or without additives being made to virus suspensions was studied. The infectivity titres of freeze-dried antigens was assessed at intervals over a six month storage period at various temperatures and also after reconstitution to the liquid phase and storage with or without glycerination. Certain additive solutions were necessary to prevent degradation of virus during the freeze-drying procedure which reduced any loss of infectivity caused by storage of products at 4 degrees C and 20 degrees C. Additive solutions composed of 10% sucrose and 5% lactalbumin hydrolysate; 10% skimmed milk; 4% peptone and 1% gelatin; and 5% dextran, 1% sodium glutamate and 5% sucrose all prolonged the keeping qualities of virus at the elevated temperature of 37 degrees C. The results indicate that short-term storage and shipment of freeze-dried foot-and-mouth disease virus antigens is possible without the need for refrigeration, thereby reducing transportation and storage costs. Reconstituted antigens survived better after glycerination and storage at -20 degrees C than did non-glycerinated samples stored at 4 degrees C.

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.

A comparative study of serological and biochemical methods for strain differentiation of foot-and-mouth disease type A viruses.

Three serological and three biochemical methods were used to compare five field isolates of foot-and-mouth disease virus (FMDV) from Western India with nine reference vaccine strains and five field isolates from other countries. The serological tests (liquid-phase ELISA and virus neutralization) were able to distinguish between the three reference vaccine strains examined, but the five Indian field isolates reacted poorly with antisera produced against these vaccine strains. Analysis of monoclonal antibody (mAb) data was difficult to interpret although clearly the field isolate A/IND/5/87 reacted to a lesser extent with one of the mAb panels (A10/Holland/42) than the other four Indian isolates. The A22/Iraq/24/64 mAbs did not react with any of the Indian field isolates and only significantly with one of the reference vaccine strains, A/IND/57/79. Polyacrylamide gel electrophoresis distinguished the reference vaccine strains from each other and from the field isolates. Additionally, one of the Indian isolates (A/IND/5/87) could be differentiated from the other four. Electrofocusing showed similarities between the reference vaccine strain A22/Iraq/24/64 and three of the Indian field isolates (A/IND/1/87, A/IND/2/87 and A/IND/3/87), however, A/IND/4/87 and A/IND/5/87 were distinct. Nucleotide sequencing showed that the isolates A/IND/1/87, A/IND/2/87 and A/IND/3/87 were very closely related to each other and related to A/IND/4/87, however, A/IND/5/87 was different.

Experimental infection of eland (Taurotrages oryx), sable antelope (Ozanna grandicomis) and buffalo (Syncerus caffer) with foot-and-mouth disease virus.

The course of experimental infection of a type SAT 1 FMDV strain was studied in buffalo, sable antelope and eland following tongue inoculation and contact and has been compared with that in cattle. All species became infected, although disease was less severe in the game animals and larger amounts of virus were required to infect game animals than cattle. Neutralizing antibody titres were high and were maintained for an extended period in buffalo, sable antelope and eland. The carrier state was demonstrated in buffalo for the longest period. Cattle carried virus for up to 56 days. Virus persistence in sable antelope was transitory and did not occur in eland.

Field and laboratory analysis of an outbreak of foot and mouth disease in Bulgaria in 1991.

In July 1991, an outbreak of foot and mouth disease (FMD) occurred near Stefan Karadjovo village in Boliarovo (south-east Bulgaria, close to the Turkish border). The virus isolated was identified in Bulgaria as serotype O and this was subsequently confirmed by the World Reference Laboratory for Foot and Mouth Disease in Pirbright (United Kingdom). Serological studies using bovine sera and monoclonal antibody analysis were made. In addition, the sequence of approximately 170 nucleotides at the 3' end of the 1D gene was determined for the field isolate and for vaccine strains used in Bulgaria. These were compared with other sequences of type O FMD viruses from outbreaks in the Middle East. Serum samples were taken from domestic animals in the region close to the outbreak and examined for anti-FMD virus antibodies to assess the extent (if any) of spread of the virus before or after the outbreak. No evidence of infection was found in these animals. The virus involved in the Bulgarian outbreak was antigenically similar to the O1 vaccine strains but probably did not originate from these strains. The virus was closely related genetically to a group of viruses isolated in the Middle East since 1987, suggesting that it may have been introduced into Bulgaria from an area in the Middle East by unidentified means.

Experimental transmission of foot-and-mouth disease virus from carrier African buffalo (Syncerus caffer) to cattle in Zimbabwe.

Four female cattle and three male African buffalo (Syncerus caffer) which were free of foot-and-mouth disease (FMD) virus were held together on an island in Lake Kariba, Zimbabwe. The buffalo were experimentally infected with FMD virus type SAT2, developed generalised disease and became virus carriers. While the buffalo were in the acute phase of the disease the susceptible contact cattle did not show lesions, no virus was recovered from them and they did not develop serum antibodies. However, five months later the cattle developed severe foot-and-mouth disease. Direct nucleotide sequencing of the virus used to infect the buffalo and of the virus from the in-contact cattle showed that the two isolates were almost identical. The results suggest that in nature it is possible for the virus to be transmitted from buffalo to cattle under the influence of factors not yet defined, and that there was very little change in the nucleotide sequence of the virus during the carrier period of five months.

Characterisation of foot-and-mouth disease virus strains circulating in Turkey during 1996-2004.

Two genotypes of foot-and-mouth disease virus serotype A were identified as the cause of disease outbreaks in Turkey during 1996-2004, while serotype O strains, identified during the same period, seem to represent an evolutionary continuum, and Asia1 strains were only rarely identified. The data presented are concordant with the conclusion that serotype A strains are repeatedly introduced to Turkey from the east and circulate only transiently in farming communities, while type O strains persist and re-emerge from endemic areas of Turkey. The co-circulation of strains belonging to two A genotypes for 6 years, as observed in the present study, is a remarkable difference compared to previous decades in which only one A genotype was transiently circulating, successively being replaced by others. This co-circulation was observed in spite of enforcement countrywide of biannual vaccination of more than 50% of the cattle during the same period. Mean r(1) values of 0.70 +/- 0.19 and 0.39 +/- 0.04 found for A96 and A99 isolates, respectively, compared to the A96 vaccine component reveal antigenic differences but also imply that the vaccine in use in Turkey should provide protection against both genotypes. It is suggested that further studies to reveal the nature of the difference in epidemiological dynamics of type A and type O strains might lead to an understanding of the measures required to control foot-and-mouth disease in islands of persistent circulation.

Evidence that high potency foot-and-mouth disease vaccine inhibits local virus replication and prevents the "carrier" state in sheep.

The ability of a single administration of a high, medium and low potency foot-and-mouth disease (FMD) vaccine to decrease or inhibit local virus replication and excretion in the oropharynx of sheep following aerosol challenge with homologous live virus 14 days later was examined. Unvaccinated sheep showed signs of clinical FMD, whereas all of the vaccinated sheep, regardless of antigen payload, were protected against clinical disease and development of viraemia. Virological and serological results confirmed that there had been no local virus replication in the oropharynx of sheep from the high potency vaccine group in contrast to moderate or substantial virus replication in the oropharynx of the low potency vaccinated or unvaccinated sheep respectively. The vaccines showed no evidence of promoting a local mucosal antibody response at the time of virus challenge, but were capable of stimulating a systemic gamma interferon response, the level of which was related to the antigen payload. This suggests that the systemic gamma interferon response could be a useful indicator of the ability of a FMD vaccine to elicit a sterile immunity and indicates that further work is warranted to investigate the role of systemic gamma interferon in this immunity. This is the first experiment to clearly show that high potency, high payload, FMD vaccines are capable of inhibiting local virus replication and consequently persistence and the carrier state in this target species.