South African Territories Research Articles

Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs

Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South African Territories (SAT) type viruses, which exist as distinct genetic and antigenic variants in different geographical regions. A cross-serotype chimeric virus, vKNP/SAT2, was engineered by replacing the external capsid-encoding region (1B-1D/2A) of an infectious cDNA clone of the SAT2 vaccine strain, ZIM/7/83, with that of SAT1 virus KNP/196/91. The vKNP/SAT2 virus exhibited comparable infection kinetics, virion stability and antigenic profiles to the KNP/196/91 parental virus, thus indicating that the functions provided by the capsid can be readily exchanged between serotypes. As these qualities are necessary for vaccine manufacturing, high titres of stable chimeric virus were obtained. Chemically inactivated vaccines, formulated as double-oil-in-water emulsions, were produced from intact 146S virion particles of both the chimeric and parental viruses. Inoculation of guinea pigs with the respective vaccines induced similar antibody responses. In order to show compliance with commercial vaccine requirements, the vaccines were evaluated in a full potency test. Pigs vaccinated with the chimeric vaccine produced neutralizing antibodies and showed protection against homologous FMDV challenge, albeit not to the same extent as for the vaccine prepared from the parental virus. These results provide support that chimeric vaccines containing the external capsid of field isolates can be successfully produced and that they induce protective immune responses in FMD host species.

A qualitative risk assessment of factors contributing to foot and mouth disease outbreaks in cattle along the western boundary of the Kruger National Park

Between November 2000 and the end of 2007, five outbreaks of foot and mouth disease (FMD) occurred in cattle in the area adjacentto the Kruger National Park (KNP) in the north-eastern corner of South Africa. To help understand the factors behind these outbreaks a qualitative risk assessment based on the World Organisation for Animal Health (OIE) assessment framework was adopted, using available data from published sources and various unpublished South African sources. Risk was assessed on the basis of the following factors: data on South African Territories (SAT) type infections of buffalo and impala in the KNP, permeability of the fence along the western boundary of the KNP, the potential for contact between livestock and wildlife susceptible to FMD in areas adjacent to the KNP, and the level of herd immunity in cattle generated by prophylactic vaccination. Scenario pathways for FMD occurrence outside the KNP are presented as a conceptual framework to qualitatively assess the risk of FMD outbreaks. Factors that are likely to have most influence on the risk were identified: fence permeability, vaccination coverage, or the efficiency of animal movement control measures. The method and results are provided as an approach that may be used as a basis to evaluate the risk of FMD outbreaks occurring in other wildlife/livestock interface areas of southern Africa.

Evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the SAT serotypes

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals that is primarily controlled by vaccination of susceptible animals and movement restrictions for animals and animal-derived products in South Africa. Vaccination using aluminium hydroxide gel-saponin (AS) adjuvanted vaccines containing the South African Territories (SAT) serotypes has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone and in controlling outbreaks in the free zone. Various vaccine formulations containing antigens derived from the SAT serotypes were tested in cattle that were challenged 1 year later. Both the AS and ISA 206B vaccines adjuvanted with saponin protected cattle against virulent virus challenge. The oil-based ISA 206B-adjuvanted vaccine with and without stimulators was evaluated in a field trial and both elicited antibody responses that lasted for 1 year. Furthermore, the ISA 206 adjuvanted FMD vaccine protected groups of cattle against homologous virus challenge at very low payloads, while pigs vaccinated with an emergency ISA 206B-based FMD vaccine containing the SAT 1 vaccine strains were protected against the heterologous SAT 1 outbreak strain.

Molecular characterization of SAT-2 foot-and-mouth disease virus isolates obtained from cattle during a four-month period in 2001 in Limpopo Province, South Africa

Foot-and-mouth disease (FMD) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. The virus is a single-stranded RNA virus that has a high rate of nucleotide mutation and amino acid substitution. In southern Africa the South African Territories (SAT) 1-3 serotypes of FMD virus are maintained by large numbers of African buffaloes (Syncerus caffer), which provide a potential source of infection for domestic livestock and wild animals. During February 2001, an outbreak of SAT-2 was recorded in cattle in the FMD control zone of South Africa, adjacent to the Kruger National Park (KNP). They had not been vaccinated against the disease since they form the buffer between the vaccination and free zones but in the face of the outbreak, they were vaccinated as part of the control measures to contain the disease. The virus was, however, isolated from some of them on several occasions up to May 2001. These isolates were characterized to determine the rate of genetic change in the main antigenic determinant, the 1 D/2A gene. Nucleotide substitutions at 12 different sites were identified of which five led to amino acid changes. Three of these occurred in known antigenic sites, viz. the GH-loop and C-terminal part of the protein, and two of these have previously been shown to be subject to positive selection. Likelihood models indicated that the ratio of non-synonymous to synonymous changes among the outbreak sequences recovered from cattle was four times higher than among comparable sequences isolated from wildlife, suggesting that the virus may be under greater selective pressure during rapid transmission events.

Evaluation of laboratory tests for sat serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (fmd) vaccination and infection appeared to be...

A serological survey for antibodies to foot-and-mouth disease virus in indigenous Tswana goats and sheep in Kasane, Maun and Shakawe districts in northwestern Botswana

A serological survey was conducted in apparently healthy, unvaccinated indigenous Tswana goats and sheep in Kasane, Maun and Shakawe districts in northwestern Botswana in order to determine in these animals, the levels of exposure to the South African Territories (SAT) serotypes: SAT 1, SAT 2 and SAT 3 of foot-and-mouth disease virus (FMDV). A total of 250, 142 and 134 goat sera originating respectively from Kasane, Maun and Shakawe districts were tested for FMDV antibodies against the three SAT serotypes by the liquid phase blocking enzyme-linked immunosorbent assay and 26 of 250 (10.4%), 5 of 142 (3.5%) and 18 of 134 (13.4%) were positive either to SAT 1 or SAT 3, or to both serotypes. None of the goats' sera was positive to SAT 2 serotype. All sheep sera (n = 9) tested negative against all three serotypes of the virus. The findings are discussed in relation to results of other serological surveys carried out elsewhere.

Studies of genetically defined chimeras of a European type A virus and a South African Territories type 2 virus reveal growth determinants for foot-and-mouth disease virus.

The three South African Territories (SAT) types of foot-and-mouth disease virus (FMDV) display great genetic and antigenic diversity, resulting from the independent evolution of these viruses in different geographical localities. For effective control of the disease in such areas, the use of custom-made vaccines is required. To circumvent the tedious process of vaccine strain selection, an alternative in the control process is being investigated. Specifically, it is proposed to replace the antigenic determinants of an infectious genome-length cDNA copy of a good SAT vaccine strain with those of appropriate field strains, producing custom-made FMDV chimeras for use in vaccine production. Here the construction of an infectious genome-length cDNA copy of the SAT2 vaccine strain, ZIM/7/83, is described, created utilizing an exchange-cassette strategy with an existing A(12) genome-length cDNA clone. The virus derived from this cDNA (designated vSAT2) displayed excellent growth properties in cell culture, indicating its potential usefulness in the production of custom-made vaccine strains. Evaluation of the growth of various SAT2/A12 chimeras created during the derivation of SAT2 infectious cDNA suggested incompatibilities between the non-structural proteins of ZIM/7/83 and the 5' UTR of A(12).

Retrospective genetic analysis of SAT-1 type foot-and-mouth disease outbreaks in West Africa (1975–1981)

The complete 1D genome region encoding the immunogenic and phylogenetically informative VP1 gene was genetically characterized for 23 South African Territories (SAT)-1 viruses causing foot-and-mouth (FMD) disease outbreaks in the West African region between 1975 and 1981. The results indicate that two independent outbreaks occurred, the first involved two West African countries, namely Niger and Nigeria, whilst the second affected Nigeria alone. In the former epizootic, virus circulation spanned a period of 2 years, whilst in the latter virus was recovered from the field over a 3 year period. Comparison of the West African viruses with SAT-1 viruses from other regions on the continent revealed that the two West African lineages identified in this study are regionally distinct. Furthermore, variation in VP1 gene length was identified in SAT-1 viruses for the first time, further emphasizing the uniqueness of these pathogens in West Africa. This first retrospective analysis in which the molecular epidemiology of SAT-1 viruses in West Africa is reported, provides a useful measure of the regional variation of these viruses and is an essential first step in the establishment of a West African sequence database that will be a useful reference for future outbreak eventualities.

Review of the status and control of foot and mouth disease in sub-Saharan Africa

Six of the seven serotypes of foot and mouth disease (FMD) virus (i.e. all but Asia 1) are prevalent in Africa although there are marked regional differences in distribution. Three of these serotypes are unique to Africa, namely the three South African Territories (SAT) serotypes. Serotype C may also now be confined to Africa because it has not been reported elsewhere recently. In southern Africa at least, the SAT serotypes have an intimate and probably ancient association with African buffalo (Syncerus caffer) that is instrumental in their maintenance. Within each of the six prevalent serotypes, with the possible exception of C, there are a number of different lineages with more or less defined distributions (i.e. topotypes) that in some cases are sufficiently immunologically different from one another to require specific vaccines to ensure efficient control. This immunological diversity in prevalent serotypes and topotypes, in addition to uncontrolled animal movement in most parts of the continent, render FMD difficult to control in present circumstances. This fact, together with poorly developed intercontinental trade in animals and animal products has resulted in the control of FMD being afforded a low priority in most parts of the continent, although the northern and southern regions of the continent are an exception. As a consequence, eradication of FMD from Africa as a whole is not a prospect within the foreseeable future. In southern Africa, the use of fencing and other means to strictly control the movement of wildlife and livestock as well as judicious application of vaccine has resulted in countries of the region being able to access beef and other livestock markets in Europe and elsewhere in the developed world. Significant marketing of livestock and livestock products from Africa outside the continent is unlikely to be achieved unless similar approaches can be developed for other regions of Africa. This will result in continuing under-exploitation of a valuable resource in the arid and semi-arid regions of Africa, with increasing marginalisation of human populations living there.

Foot and mouth disease: the experience of South Africa.

Foot and mouth disease (FMD) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP) and surrounding game parks in South Africa. The last outbreak of the disease in domestic stock outside the FMD control zone occurred in 1957. Due to the success in containing the disease, the country was accorded zone freedom from FMD without vaccination by the Office International des Epizooties (OIE: World organisation for animal health) in 1995. This status was lost in September 2000 when the first-ever recorded case of serotype O in South Africa was diagnosed in a piggery in KwaZulu-Natal after the illegal feeding of untreated swill. In November 2000, an outbreak of FMD caused by serotype South African Territories (SAT) 1 was diagnosed in a feedlot within the free zone of Mpumalanga Province. The SAT 1 outbreak was traced to cattle in the FMD control zone south of the KNP after the game-proof fence surrounding the KNP was severely damaged by floods. This enabled buffalo to come into direct contact with cattle outside the KNP. A further outbreak caused by SAT 2 was diagnosed within the FMD control zone in February 2001, also as a result of buffalo having escaped from the KNP. All these outbreaks were successfully contained, with the re-instatement of zone freedom from FMD without vaccination by the OIE in May 2002. These outbreaks made it necessary to re-examine the methods of control and containment of FMD that have been practised for many years and which are in line with accepted international practices. The authors describe the rationale for the different control strategies that were followed, the need for a multidisciplinary approach to disease control, the interface between control and technological and diagnostic support and the lessons learned. Some suggestions for future control strategies are also offered.

Genetic heterogeneity in the foot-and-mouth disease virus Leader and 3C proteinases

The Leader and 3C proteinases of foot-and-mouth disease virus (FMDV) are responsible for almost all the proteolytic processing events of the viral polyprotein precursor. Investigation into the genetic heterogeneity of the regions encoding these proteins from isolates of six FMDV serotypes revealed the 3C proteinase to be more conserved than the Leader proteinase. Maximum likelihood analysis indicated similar phylogenetic groupings for the non-structural protein coding regions of both the Leader and 3C. These groupings were different from the structural VP1 protein coding region which, as shown previously, grouped according to serotype. Two distinct clades were apparent for both the Leader and 3C coding regions: one comprising of serotypes A, O and C together with SAT (South African Territories) isolates from eastern Africa. The other clade consisted of SAT isolates originating from southern Africa. Only one virus isolate, obtained from a buffalo in Uganda, did not conform to this phylogenetic pattern. This SAT 1 virus grouped with types A, O and C in the Leader analysis, but with the southern African SAT types in the 3C analysis, implicating intertypic recombination. The leader proteinases of southern African SAT type isolates differed from those present in European type isolates, particularly in the self-processing region. A three-dimensional structure was modeled for the Leader proteinase of one of the SAT type viruses, ZIM/7/83/2, and compared with the previously elucidated crystal structure of O1Kaufbeuren Leader proteinase. The active sites of the two leaders were found to superimpose closely, despite the observed sequence variation between the two molecules. Comparison of the 3C proteinase P1 cleavage sites suggested that the FMDV 3C proteinase may possess a broader substrate specificity, as observed in hepatitis A virus 3C proteinase.

Foot and mouth disease in Zambia: a review of the aetiology and epidemiology and recommendations for possible control

In Zambia, foot and mouth disease (FMD) has been caused by all three of the South African Territories serotypes (SAT 1, 2 and 3) and by European types O and A. Three areas of the country which have experienced repeated occurrences of the disease are considered high-risk areas. The three areas are as follows: the southern border area between Zambia and Zimbabwe, Botswana and Namibia, the Kafue Flats and the northern border with Tanzania in the Nakonde and Mbala districts. The transfer mechanism of the virus is poorly understood but the African buffalo (Syncerus caffer) is considered to be the natural host, acting as a reservoir of infection for the SAT types of the virus. Cattle are known to be carriers of the virus for up to two and a half years and individual semi-domesticated buffalo have been reported to act as carriers for up to five years. In wild herds of buffalo, the virus has been recorded for periods of up to twenty-five years. Current control measures include mass vaccination of cattle in high-risk areas and restrictions on the movement of cattle from areas in which contact exists with buffalo. New protocols should be developed for the prevention and control of FMD, including the enforcement of livestock movement control, improved disease surveillance and reporting, and the monitoring of FMD virus in carrier cattle and buffalo. These measures will contribute towards building the confidence of the regulatory bodies of importing countries in the region.

Characterization of the structural-protein-coding region of SAT 2 type foot-and-mouth disease virus.

The South African Territories (SAT) types of foot-and-mouth disease (FMD) virus show marked genomic and antigenic variation in sub-Saharan Africa that is to a large extent geographically determined. This has implications for selection of appropriate vaccine strains as well as the accuracy of laboratory diagnosis. However, adaptation of field isolates as vaccine strains is cumbersome, time consuming and expensive. We propose the construction of recombinant viruses in which specific antigenic determinants can be manipulated. To achieve this goal, the structural-protein-coding region of a SAT 2 vaccine strain, ZIM 7/83/2, was determined and compared with two other known SAT 2 P1 regions. Five hypervariable regions were identified of which four are situated within VP1. The cleavage sites for proteolytic processing differs from serotype A, while the junction between P1/2A is variable within the SAT 2 serotype. These differences could influence the construction of recombinant vaccines.

Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A, C, O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

Characterization of monoclonal antibodies against a type SAT 2 foot-and-mouth disease virus

This paper is the first to describe characterization of monoclonal antibodies (MAbs) against a South African Territories 2 (SAT 2) foot-and-mouth disease virus (isolate Rho 1/48). Twelve MAbs which neutralized homologous virus were characterized in indirect and sandwich ELISA using purified Rho 1/48 virus particles, subunits, trypsin-treated, and chemically denatured virus. All the MAbs inhibited haemagglutination by parental virus. Binding of the MAbs to 73 SAT 2 field isolates was measured in a sandwich ELISA and defined four distinct antigenic regions. Preliminary characterization of escape mutants selected with some of the MAbs using virus neutralization tests, ELISA, and amino acid sequencing is included. MAbs 2, 25, 40, 48 and 64, reacted with a linear epitope on the VP1 loop region. An amino acid change at position 149 (valine to glutamic acid) was detected in mutants selected by MAb 2 and 40 and this eliminated binding and neutralization by all the other MAb. This epitope was conformation-dependent and was conserved in all 73 isolates of SAT 2 examined. Escape mutants isolated with MAb 41 and 44, had changes at positions 156 (glycine to aspartic acid), or 158 (serine to leucine) respectively. These MAbs bound with Rho 1/48 only out of 73 field strain viruses studies and the reactions of MAbs from the other groups was unaltered. MAb 27, 28 and 37 reacted with a conformation-dependent epitope on VP1 which was not conserved in field isolates. All mutants selected by these MAbs had a single amino acid substitution at position 149 (valine to alanine). The same change was always found in field isolates which did not bind MAbs from this group. MAb 11 reacted with a linear epitope associated with amino acids 147 or 148 on VP1 and showed similar binding characteristics to a conformation dependent MAb 7, no amino acid residue changes were found within VP1 for monoclonal antibody 7 mutants.

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.

The nucleotide sequence of the structural-protein-coding region of foot-and-mouth disease virus serotype SAT3

The nucleotide sequence coding for the structural proteins and nonstructural protein P2A has been determined for a foot-and-mouth disease virus (FMDV) isolated in Africa. This virus, serotypically designated SAT3 (South African Territories type 3), shows about 60% homology at the nucleotide level to prototype viruses from the O, A and C serotypes of FMDV. The highest region of variability was shown in structural protein VP1, presumably a consequence of its position on the surface of the virus and its exposure to selection pressure by neutralising antibody. Within this region amino acids (aa) 141–160, which have been shown to represent an immunodominant region in other FMDV serotypes, showed hypervariability as well as the insertion of 5 or 9 additional aa relative to the O 1, and C 1 serotypes, respectively. In contrast, the sequence of nonstructural protein P2A was completely conserved indicating a probable important role in virus replication.

Antigenic comparison of the polypeptides of foot-and-mouth disease virus serotypes and other picornaviruses

The cross-reactivity of proteins coded for by the seven serotypes of foot-and-mouth disease virus (FMDV) was assessed by reaction of infected cell lysates with polyclonal and monospecific antisera against the structural and nonstructural proteins of FMDV type Al 2 strain 119ab. It was shown that the homologous polypeptides from most serotypes are antigenically related. The least cross-reactivity occurred between VP1, VP3, and the protease (3C) of type Al 2 and South African Territories types 1 and 3. There was also a reduced degree of reactivity of Al 2 VP1 serum with VP1 from some A subtypes and the other serotypes. Comparison of FMDV proteins with polypeptides from other picornaviruses by a radioimmune binding assay revealed a low level of reactivity of antisera against some Al 2 poly-peptides with encephalomyocarditis virus (EMCV) infected cell lysates but no reactivity with bovine enterovirus type 1 and swine vesicular disease virus infected cells. The same EMCV proteins were immunoprecipitated by the various reactive Al 2 antisera, but the reaction was abolished if the lysate from EMCV infected cells was denatured prior to immunoprecipitation.

Research in Central Africa : Dr. J. E. Keyston

SUPPORT was forthcoming from all branches of scientific opinion for proposals put forward in 1946 at the Royal Society Empire Scientific Conference that research in Africa, south of the equator, should be planned and organised on a regional basis. Even before the Conference, the three Central African territories of Southern Rhodesia, Northern Rhodesia and Nyasaland were contemplating arrangements for a joint attack upon the research problems of their own sub-region of Africa, and for collaboration with the research organisations in, the neighbouring territories of East Africa and the Union of South Africa. The Central African Council, which is a consultative assembly of representatives of the three Central African countries, has given effect to proposals made by a Special Committee on Joint Research set up at the end of 1945, by appointing a ‘research ‘secretary whose first duty will be to survey the research facilities available in the Central African, East African and South African territories, to report on any additional facilities required in Central Africa and on the relations to be established between research work and workers in Central Africa and in other countries. This survey is expected to lead to recommendations regarding the nature, priority and future organisation of research in Central Airica.