A nucleotide-specific polymerase chain reaction assay to differentiate rabies virus biotypes in South Africa

Antigenic and nucleotide sequence analyses have shown that two distinct biotypes of rabies virus are circulating in South Africa. One of these typically infects members of the family Canidae, while the other comprises a heterogeneous group of apparently indigenous viruses, infecting members of the Viverridae family. In recent times, it has become evident that a considerable amount of crossinfection may occur and the manifestation of viverrid rabies in non-viverrid animals in particular appears to have become more commonplace. Consequently, the need to rapidly distinguish between rabies virus biotypes has become increasingly important in efforts to monitor the epidemiology of rabies in the southern African region. In this study, a nested polymerase chain reaction (PCR) assay was developed to distinguish between these two groups of rabies viruses. Consensus oligonucleotides were used to amplify the cytoplasmic domain of the rabies virus glycoprotein and the adjacent intergenic region. The resultant amplicon was subsequently used as template in a second round heminested PCR in the presence of type-specific primers, thereby successfully generating amplicons of characteristic size for each biotype.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Understanding of the farmers' privilege concept by smallholder farmers in South Africa

Legislation on plant breeders’ rights – the Plant Breeders’ Rights Act, 1976 (Act No. 15 of 1976) – currently is being reviewed by the Department of Agriculture, Forestry and Fisheries. This legislation provides for farmers’ privilege, which is one of the exceptions to plant breeders’ rights. It allows farmers to save seed of protected varieties for their own use. Farmers’ privilege, and particularly its impact on smallholder farmers in developing countries, is a widely debated issue. During the public consultation process, several comments proposing amendments to the farmers’ privilege provision were received from various stakeholders. However, no comments were received from the smallholder farmers who may be directly impacted by this provision. This pilot study was undertaken to assess the understanding of the farmers’ privilege concept by smallholder farmers from the historically disadvantaged communities and their current practices with regard to seed saving. The results showed that the majority of the smallholder farmers were not aware of the existence of the legislation on plant breeders’ rights and therefore do not understand the farmers’ privilege concept and its implications. They also did not know whether the varieties they were using were protected by plant breeders’ rights or not. Little information has been published on the impact of plant breeders’ rights in South Africa in general. We hope that this study might inform policy decisions on matters related to plant breeders’ rights and the farmers’ privilege.http://www.sajs.co.za/hb201

Polyphasic taxonomy of rhizobia associated with legumes occuring in South Africa

The advantageous association between rhizobia and leguminous plants has motivated numerous studies into the diversity and identity of the associated bacterial symbionts. This, as well as developments in molecular microbiology, has led to major revisions of rhizobial taxonomy. Previous investigations of the rhizobia, associated with various leguminous plants from South Africa, concluded that most of the indigenous strains were related to the genus Bradyrhizobium (Dagutat, 1995; Kruger, 1998). The other rhizobial genera represented, albeit to a lesser extent, were Rhizobium, Sinorhizobium and Mesorhizobium. The major shortcoming of these investigations was the lack of sufficient genotypic characterisation. Since the completion of these initial investigations, a new rhizobial genus and several new species have been described. These developments and the additional isolation of rhizobia, from previously uninvestigated legumes, necessitated a more detailed analysis of the indigenous rhizobia. The aim of this study was therefore to study the diversity of the indigenous strains, focusing particularly on genotypic traits. A selection of indigenous rhizobia was characterised by partial 16S rDNA sequencing, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer (IGS) region, partial nifH sequencing and nodC RFLP. Based on 16S rDNA sequencing, most of the isolates could be assigned to a specific genus, most being related to the genus Bradyrhizobium. A group of isolates was also related to the genus Methylobacyterium. The IGS- RFLP analyses were sufficiently discriminatory to indicate additional variation among isolates which showed little or no 16S rDNA sequence variation. The nifH phylogenetic groupings correlated well with those obtained by 16S rDNA sequencing. However, nodC RFLP indicated that the indigenous rhizobia carry diverse range nodC genotypes, with only a few showing host-specific associations. In the absence of sequence data of these nodC genotypes, their origin and correspondence to known nodC genes of other rhizibial genera, remain uncertain. Considering the results obtained here and the phenotypic characteristics determined previously, several novel Bradyrhizobium and Mesorhizobium strains were identified, however, their specific status should be validated by DNA homology studies.Thesis (PhD (Microbiology))--University of Pretoria, 2002.Microbiology and Plant Pathologyunrestricte

Molecular genetic analysis of some enzootic rabies viruses of southern Africa

Rabies viruses are known to be able to infect a broad range of warm-blooded animals. In South Africa the disease is maintained in different animal species including dogs, jackals, bateared foxes and a variety of members of the viverridae family. These include the different mongoose species (principally the yellow mongoose), genets, suricates and a variety of small carmvores. The antigenic variation within the nucleoprotein gene has previously been investigated in efforts to characterise various isolates of rabies viruses in southern Africa. It was noted that two antigenically distinct groups are cocirculating in the country. In this study, the investigation into the epidemiology of rabies in South Africa was extended by molecular genetic analysis of a large number of isolates from wildlife and domestic animal hosts. Geographically and temporally distinct rabies viruses were previously studied by comparative nucleotide sequence analysis of DNA fragments encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region. Based on this analysis two main rabies virus groups were identified. Members of the first group infect canid host species and are closely related to the European rabies strains, while the viruses belonging to the second group circulate within different viverrid species. Although isolates of mainly mongoose origin were initially analysed, considerable heterogeneity within this group was evident. The current study was consequently undertaken to include rabies virus isolates from other viverrid host species. Following the same approach as the previous investigators (Nel et al., 1993 & von Teichman et al., 1995) four genetically distinct clusters were indicated within the viverrid lineage. These clusters corresponded closely to the geographic origin of the virus isolates independent of specific viverrid host. The results suggest genetic divergence and independent evolution of the viverrid viruses within geographically isolated regions. Spillover or cross-infection, where a viverrid virus is recovered from a canid host and vice versa, could not be attributed to a new rabies virus and were most likely initiated by interspecies transmission events. These results suggest little modification of the virus following infection of an atypical host. A phylogeny of the rabies virus variants in southern Africa was established based on sequence variation within the abovementioned genome regions. Although such an approach is most informative when compared to other methods, it is time-consuming and laborious. The use of strain-specific oligonucleotides was therefore investigated for rapid strain differentiation. Two oligonucleotides were designed based on the nucleotide sequences of the cytoplasmic domain of the glycoprotein and the G-L intergenic region. These oligonucleotides together with a common downstream primer were used to amplify DNA fragments of characteristic size, allowing for discrimination between the two rabies biotypes.Afrikaans: Dit is bekend dat die hondsdolheidsvirus verskeie warmbloedige diere kan infekteer. In suider Afrika word die siekte onderhou in gasheer spesies soos honde, jakkalse, bakoorvosse en lede van die Viverridae familie. Dit sluit verskeie meerkat spesies (hoofsaaklik die witkwasmuishond), kleinkolmuskejaatkat, stokstertmeerkat en 'n verskeidenheid klein karnivoorspesies in. Die antigeniese variasie binne die nukleoprotei"en geen is voorheen aangewend in 'n poging om hondsdolheidsvirus-isolate in suidelike Afrika te karakteriseer. Uit hierdie ondersoek het dit geblyk dat daar twee antigenies-onderskeibare virusgroepe in die land sirkuleer. In hierdie studie word die ondersoek na die epidemiologie van hondsdolheid in Suid-Afrika uitgebrei deur middel van genetiese analise van 'n groot aantal isolate afkomstig vanaf wilde- sowel as huishoudelike diere. Die genetiese variasie van geografies- en temporaal-verwyderde isolate is vantevore deur middel van vergelykende nukleotiedvolgorde-analise ondersoek. Hierdie vergelykings het die nuletensuurvolgordes van die sitoplasmiese gebied van die glikoprote1en en die G-L intergeniese gebied ingesluit. Hierdie analise het twee hoofgroepe hondsdolheidsvirusse aangedui. Lede van die eerste groep infekteer canid gasheer spesies en is nou verwant aan die Europese hondsdolheidsvirusse, terwyl die tweede groep binne verskillende viverrid spesies sirkuleer. Alhoewel daar aanvanklik virus isolate vanuit hoofsaaklik die witkwasmuishond ondersoek is, was daar heelwat heterogenisiteit binne die viverrid virusse gevind. Gevolglik is hierdie studie onderneem om hondsdolheidsvirusse vanaf antler viverrid gashere in te sluit. Deur gebruik te maak van dieselfde benadering as vorige navorsers (Nel et al., 1993; von Teichman et al., 1995) is vier geneties onderskeibare groepe binne die viverrid stamboom aangetoon. Hierdie groepe het nou ooreengestem met die geografiese oorsprong van die virus isolate, onafhanklik van 'n spesifieke gasheer. Hierdie resultate dui daarop dat onafhanklike ewolusie van die viverrid virusse binne ge1soleerde lokaliteite plaasvind. Kruis-infeksie, waar viverrid virusse canid gashere en omgekeerd infekteer, kon nie toegeskryf word aan 'n nuwe hondsdolheidsvirus me. Hierdie verskynsel word vermoedelik deur interspesie oordraag gebeure ge'inisieer. 'n Filogenetiese verwantskap van hondsdolheidsvirusse in suider Afrika is bepaal op grond van van die nuleotiedvolgorde variasie in bogenoemde genomiese gebiede. In vergeleke met antler metodes, is hierdie benadering baie informatief, maar dit kan tydrowend en moeisaam wees. Die gebruik van ras-spesifieke voorvoerders is daarom ondersoek as 'n alternatiewe metode vir differensiasie. Twee voorvoerders is ontwerp gebaseer op die volgordes van die sitoplasmiese gebied van die glikoprote1en en die G-L intergeniese gebied. Deur hierdie voorvoerders tesame met 'n gemeenskaplike stroom-af voorvoerder te gebruik word DNA fragmente van karakteristieke groottes geamplifiseer. Hierdie benadering voorsien dus 'n makliker manier vir onderskeiding tussen die twee virus groepe.Dissertation (MSc)--University of Pretoria, 1997.Microbiology and Plant PathologyMScUnrestricte