Development and validation of a foot-and-mouth disease virus SAT serotype-specific 3ABC assay to differentiate infected from vaccinated animals.

The effective control of foot-and-mouth disease (FMD) requires sensitive, specific and rapid diagnostic tools. However, the control and eradication of FMD in Africa is complicated by, among other factors, the existence of five of the seven FMD virus (FMDV) serotypes, including the SAT-serotypes 1, 2 and 3 that are genetically and antigenically the most variable FMDV serotypes. A key diagnostic assay to enable a country to re-gain its FMD-free status and for FMD surveillance, is the 3ABC or the non-structural protein (NSP) enzyme-linked immunosorbent assay (ELISA). Although many kits are available to detect 3ABC antibodies, none has been developed specifically for the variable SAT serotypes. This study designed a SAT-specific NSP ELISA and determined whether this assay could better detect NSP-specific antibodies from FMDV SAT-infected livestock. The assay's performance was compared to validated NSP assays (PrioCheck®-NSP and IZSLER-NSP), using panels of field and experimental sera, vaccinated and/or infected with FMDV SAT1, SAT2 or SAT3. The sensitivity () of the SAT-NSP was estimated as 76% (70%, 81%) whereas the specificity was 96% (95%, 98%) at a 95% confidence interval. The sensitivity and specificity were comparable to the commercial NSP assays, PrioCheck®-NSP (82% and 99%, respectively) and IZSLER-NSP (78% and 98%, respectively). Good correlations were observed for all three assays.

Predicting antigenic sites on the foot-and-mouth disease virus capsid of the South African Territories types using virus neutralization data.

Foot-and-mouth disease virus (FMDV) outer capsid proteins 1B, 1C and 1D contribute to the virus serotype distribution and antigenic variants that exist within each of the seven serotypes. This study presents phylogenetic, genetic and antigenic analyses of South African Territories (SAT) serotypes prevalent in sub-Saharan Africa. Here, we show that the high levels of genetic diversity in the P1-coding region within the SAT serotypes are reflected in the antigenic properties of these viruses and therefore have implications for the selection of vaccine strains that would provide the best vaccine match against emerging viruses. Interestingly, although SAT1 and SAT2 viruses displayed similar genetic variation within each serotype (32 % variable amino acids), antigenic disparity, as measured by r(1)-values, was less pronounced for SAT1 viruses compared with SAT2 viruses within our dataset, emphasizing the high antigenic variation within the SAT2 serotype. Furthermore, we combined amino acid variation and the r(1)-values with crystallographic structural data and were able to predict areas on the surface of the FMD virion as antigenically relevant. These sites were mostly consistent with antigenic sites previously determined for types A, O and C using mAbs and escape mutant studies. Our methodology offers a quick alternative to determine antigenic relevant sites for FMDV field strains.

Experimental infection of giraffe (Giraffa camelopardalis) with SAT-1 and SAT-2 foot-and-mouth disease virus.

The potential role of giraffe (Giraffa camelopardalis) in the epidemiology and spread of foot-and-mouth disease (FMD) SAT types was investigated by experimental infection and detection of virus in excretions using virus isolation on primary pig kidney cell cultures. In two experiments separated by a period of 24 months, groups of four animals were needle infected with a SAT-1 or SAT-2 virus, respectively and two in-contact controls were kept with each group. Viraemia was detected 3-9 days post-infection and virus isolated from mouth washes and faeces only occasionally up to day 13. The SAT-1 virus was transmitted to only one in-contact control animal, probably via saliva that contained virus from vesicles in the mouth of a needle-infected animal. None of the animals infected with the SAT-2 virus had any vesicles in the mouth, and there was no evidence of transmission to the in-contact controls. No virus was detected in probang samples for the duration of the experiments (60 days post-infection), indicating that persistent infection probably did not establish with either of these isolates. Giraffe most likely do not play an important role in FMD dissemination. Transmission of infection would possibly occur only during close contact with other animals when mouth vesicles are evident.

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.

The efficacy of an experimental oil-adjuvanted encephalomyocarditis vaccine in elephants, mice and pigs.

An oil-adjuvanted inactivated encephalomyocarditis (EMC) vaccine was developed to protect a wild population of elephants against a natural outbreak of disease. The experimental vaccine was initially tested for efficacy by challenging mice and pigs. Mice showed protection against challenge and pigs developed high antibody levels. Since both vaccinated and control pigs failed to develop clinical disease, apparently due to the low virulence of the strain in this species, protection in pigs could not be evaluated. Vaccinated elephants developed high antibody titers which protected all vaccinates from a challenge roughly two months post-vaccination, whereas controls developed fatal or sub-clinical myocarditis. This is the first report of an inactivated EMC vaccine inducing high antibody titers in domestic and wild animal species. Due to the potency of this vaccine and the acceptability of the oil adjuvant used, it has potential for use in animals in zoological collections as well as in the pig industry.

Persistent infection of African buffalo (Syncerus caffer) with SAT-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission.

Transmission of a plaque-purified SAT-2 foot-and-mouth disease virus (FMDV) occurred erratically from artificially infected African buffaloes in captivity to susceptible buffaloes and cattle in the same enclosure; in some instances transmission occurred only after contact between persistently infected carriers and susceptible animals lasting a number of months. Because the rate at which FMDV mutations accumulated in persistently infected buffaloes was approximately linear (1.64 percent nucleotide substitutions per year over the region of the 1D gene sequenced), both buffaloes and cattle that became infected some months after the start of the experiment were infected with viruses that differed from the original clone. The nucleotide differences were reflected in significant antigenic change. A SAT-1 FMDV from a separate experiment inadvertently infected some of the buffalo in the SAT-2 experiment. The SAT-1 FMDV also accumulated mutations at a constant rate in individual buffaloes (1.54 percent nucleotide changes per year) but the resultant antigenic variation was less than for SAT-2. It is concluded that persistently infected buffaloes in the wild constantly generate variants of SAT-1 and SAT-2 which explains the wide range of genomic and antigenic variants which occur in SAT-1 and SAT-2 viruses in southern Africa.

A liquid-phase blocking ELISA for the detection of antibodies to rabies virus.

A liquid-phase blocking ELISA was adapted to the detection and titration of antibodies to principally the nucleoprotein of rabies virus. Sera from animals that had either been vaccinated against rabies or inoculated with street rabies viruses, as well as sera from animals that had no recorded contact with rabies, were tested. These included sera from people, cattle, sheep, goats, dogs, laboratory mice, rabbits, yellow mongooses, wild dogs and lions. Where possible, the results were compared with those obtained with a commercial kit incorporating an indirect ELISA that measures antibody to the rabies glycoprotein. There was a high correlation (r = 0.79) between the two tests. The blocking ELISA provides a single test suitable for the rapid detection of antibodies against rabies virus in the sera of any animal species and for that reason is particularly apt for epidemiological investigations in regions where species diversity is important, as in southern Africa.

Pseudorabies and transmissible gastroenteritis: a serological survey in South Africa.

Two specific and sensitive, indirect enzyme-linked immunosorbent assays (ELISAs) utilizing a protein G-peroxidase conjugate were developed to detect antibodies to the pseudorabies virus (PRV) and the transmissible gastroenteritis virus (TGEV) in pig sera. Sera from 5,337 pigs, obtained from various abattoirs in South Africa, were tested with both ELISAs. No serological evidence of infection with either PRV or TGEV was found in any of the pigs tested.

The role of the yellow mongoose (Cynictis penicillata) in the epidemiology of rabies in South Africa--preliminary results.

Ninety-seven yellow mongooses were captured in six different localities in South Africa and blood specimens for rabies antibody determination as well as brain and salivary glands for virus isolation were collected. No rabies virus or antibody to it was detected in any of the specimens. Parallel to the field study, two experimental infections were undertaken in which yellow mongooses were artificially infected with serial dilutions of two different rabies isolates (one from a dog and the other of mongoose origin) in order to determine the minimal lethal dose (MLD50), clinical signs, duration of illness, course of the disease, presence of virus in the saliva and salivary glands and development of antibodies to rabies virus. A significantly higher proportion of mongooses inoculated with mongoose virus died than did those inoculated with the dog isolate. However, the clinical signs, incubation period, duration of illness and development of antibodies were independent of the dose of the inoculum. The levels of rabies virus in the saliva and salivary glands were high in all clinically affected animals infected with the mongoose isolate but only one of the two mongooses which died following inoculation of the dog isolate contained detectable levels of virus in the salivary glands. Antibodies to rabies were detected only in the terminal stages of clinical disease.

Maintenance of foot and mouth disease viruses in buffalo (Syncerus caffer Sparrman, 1779) in southern Africa.

Using age-related infection rates derived from serological data in available deterministic and specially developed stochastic simulation models, it has been possible to establish that the basic reproductive rates for South African Territory (SAT) type foot and mouth disease virus in buffalo (Syncerus caffer) are high. The models predict that there is a periodicity of infection within herds and possibly the population as a whole. Thus, buffalo herds are likely to be more infectious at some times than at others. However, because most infections in buffalo are inapparent, such episodes are difficult to identify. There is wide intratypic variation within the SAT type virus populations circulating in buffalo. This was determined by sequencing part of the 1 D gene of buffalo isolates and establishing antigenic profiles with neutralising monoclonal antibodies and conventional antisera.

No serological evidence for the presence of swine vesicular disease virus in South Africa.

An indirect ELISA incorporating a protein A-peroxidase conjugate was developed for detecting antibodies to swine vesicular disease virus (SVDV) in pig sera. This test and a conventional virus neutralization test were found to be equally sensitive. A total of 2846 pig sera collected from various abattoirs in South Africa were tested using the indirect ELISA. No serological evidence of infection with SVDV in pigs in South Africa was found.

The suitability of a rolled BHK21 monolayer system for the production of vaccines against the SAT types of foot-and-mouth disease virus. I. Adaptation of virus isolates to the system, immunogen yields achieved and assessment of subtype cross reactivity.

In an examination of 34 southern African SAT-type foot-and-mouth disease viruses, all but 1 attained satisfactory levels of infectivity within 6 passages in rolled BHK21 monolayer cell cultures. However, there were marked differences between adapted viruses with respect to the mass of immunogen (146S material) produced. Several isolates which consistently produced levels greater than or equal to 2 micrograms/ml were identified. In cross neutralization tests using post-vaccinal sera, SAT-1 and SAT-2 isolates showed considerable diversity and none of the viruses tested would be expected to produce a broad-spectrum response if incorporated into a vaccine. On the other hand, when 2 of the SAT-2 isolates were incorporated into the same vaccine a distinctly broader response resulted.

Foot-and-mouth disease and the African buffalo (Syncerus caffer). II. Virus excretion and transmission during acute infection.

Three groups of young buffalo in captivity were infected by exposing them to similar buffalo in the acute stages of infection induced by needle inoculation with SAT 1 or 2 viruses. Clear foot lesions developed in most of the buffalo from which the relevant virus types were re-isolated. During the first week following infection virus was found in blood, nasal secretions, saliva, preputial secretions and faeces. Air samples collected in the immediate vicinity of acutely infected buffalo were also found to contain virus. However, the regularity of virus detection as well as the quantity of virus in buffalo specimens was generally lower than for cattle infected with viruses of the same type. Conversely, virus was detected in the nasal secretions or saliva of 3 buffalo up to 4 weeks after infection, a situation which has not been encountered in cattle. Susceptible cattle and impala (Aepyceros melampus) were penned together with or in the immediate vicinity of infected buffalo and shared feeding and watering facilities with the buffalo. The pattern of transmission which emerged indicated that transfer of these viruses from buffalo to other species probably occurs only in the acute stages of infection and where there is direct physical contact between the species.

Buffalo in the northern Natal game parks show no serological evidence of infection with foot-and-mouth disease virus.

A total of 594 sera collected from buffalo (Syncerus caffer) in the Hluhluwe/Umfolozi Game Reserve complex, Ndumu Game Reserve and the eastern shores of Lake St Lucia were examined for antibody to SAT 1, 2 and 3 types of foot-and-mouth disease (FMD) virus in neutralization tests. No neutralization of SAT 2 or 3 viruses was exhibited by any of the sera tested at final dilutions greater than 10. A small proportion (2,9%) of sera neutralized SAT 1 virus at dilutions up to 10, but these were considered to be due to non-specific reactions. This, together with the absence of clinical FMD in both cattle and game in this region over at least a 45-year period and the failure to isolate FMD virus from pharyngeal scrapings of buffalo sampled in the area, leads to the conclusion that FMD does not occur in these buffalo populations.