Gerrit J. Viljoen

Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of Lumpy skin disease virus

Summary. The genomic sequences of 3 strains of Lumpy skin disease virus (LSDV) (Neethling type) were compared to determine molecular differences, viz. the South African vaccine strain (LW), a virulent field-strain from a recent outbreak in South Africa (LD), and the virulent Kenyan 2490 strain (LK). A comparison between the virulent field isolates indicates that in 29 of the 156 putative genes, only 38 encoded amino acid differences were found, mostly in the variable terminal regions. When the attenuated vaccine strain (LW) was compared with field isolate LD, a total of 438 amino acid substitutions were observed. These were also mainly in the terminal regions, but with notably more frameshifts leading to truncated ORFs as well as deletions and insertions. These modified ORFs encode proteins involved in the regulation of host immune responses, gene expression, DNA repair, host-range specificity and proteins with unassigned functions. We suggest that these differences could lead to restricted immuno-evasive mechanisms and virulence factors present in attenuated LSDV strains. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this assumption. The molecular design of an improved LSDV vaccine is likely to be based on the strategic manipulation of such genes.

Advances in viral disease diagnostic and molecular epidemiological technologies

The early and rapid detection and characterization of specific nucleic acids of medico–veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications

Preface.- acknowledgements.- chapter 1: background.- 1. aims of this book, 2. what is pcr?, 3. what is the use of pcr?. 4. pcr and infectious diseases -the veterinary picture, 5. laboratory diagnostic technology, 6. references.- chapter two: traditional pcr, 1. traditional pcr, 2. pcr reaction, 3. pcr set up and optimization, 4. the pcr plateau effect, 5. radioisotope-pcr based methods, 6. references.- chapter three: real time pcr. the basic principles: 1. traditional pcr versus real time pcr, 2. optimising a real time pcr reaction, 3. references.- chapter four: new trends in the diagnosis and molecular epidemiology of viral diseases: 1. background, 2. pcr methods used in routine molecular diagnostics, 3. acknowledgements, 4. references.- chapter five: disease diagnosis using real-time pcr specific procedures for important veterinary pathogens.- chapter six: pcr laboratory set-up: 1. establishment of a pcr laboratory.- 2. quality assurance programme or accreditation.- 3. references.- chapter seven: analysis and troublesho0ting.- chapter eight: specifications for pcr machines.- glossary of terms.- index.

Developing a Progressive Control Pathway for African Animal Trypanosomosis

Progressive control pathways (PCPs) are stepwise approaches for the reduction, elimination, and eradication of human and animal diseases. They provide systematic frameworks for planning and evaluating interventions. Here we outline a PCP for tsetse-transmitted animal trypanosomosis, the scourge of poor livestock keepers in tropical Africa. Initial PCP stages focus on the establishment of national coordination structures, engagement of stakeholders, development of technical capacities, data collection and management, and pilot field interventions. The intermediate stage aims at a sustainable and economically profitable reduction of disease burden, while higher stages target elimination. The mixed-record of success and failure in past efforts against African animal trypanosomosis (AAT) makes the development of this PCP a high priority.

Vaccinia virus expression of the VP7 protein of South African bluetongue virus serotype 4 and its use as an antigen in a capture ELISA.

SummaryRecombinant vaccinia viruses expressing the VP7 core protein of South African bluetongue virus serotype 4 (SA-BTV4) were identified by polymerase chain reaction amplification. Expression of VP7 was verified by radio-immunoprecipitation and a F(ab′)2-based ELISA. Antibodies to VP7 were detected in sera from sheep that had been infected with 20 different virulent BTV serotypes by using the vaccinia virus (VV) expressed VP7 as antigen in a capture ELISA. F(ab′)2-immobilised VV-expressed SA-BTV4 VP7 cross-reacted with sera directed against all 9 African horsesickness virus serotypes and epizootic haemorrhagic disease virus serotype 2.

Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique (2011–2016)

The complete sequence of the fusion (F) protein gene from 11 Newcastle disease viruses (NDVs) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all 11 isolates was 112RRRKRF117 indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses clustered within genotype VIIh and showed a higher similarity to NDVs from South Africa, China and Southeast Asia than to viruses previously described in Mozambique in 1994, 1995 and 2005. The identification of these new NDVs has important implications for Newcastle disease management and control in Mozambique.

The role of nuclear technologies in the diagnosis and control of livestock diseases—a review

Nuclear and nuclear-related technologies have played an important role in animal health, particularly in relation to disease diagnosis and characterization of pathogenic organisms. This review focuses primarily on how and where nuclear technologies, both non-isotopic and isotopic methods, have made their impact in the past and where it might be expected they could have an impact in the future. The review outlines the extensive use of radiation attenuation in attempts to create vaccines for a multiplicity of pathogenic organisms and how the technology is being re-examined in the light of recent advances in irradiation techniques and cryopreservation/lyophilization that might obviate some of the problems of maintenance of viable, attenuate vaccines and their transport and use in the field. This approach could be used for a number of parasitic diseases where vaccination has been problematic and where investigations into the development of molecular vaccines have still failed to deliver satisfactory candidates for generating protective immune responses. Irradiation of antigens or serum samples also has its uses in diagnosis, especially when the samples need to be transported across international boundaries, or when handling the pathogens in question when carrying out a test presents serious health hazards to laboratory personnel. The present-day extensive use of enzyme immunoassays and molecular methods (e.g., polymerase chain reaction) for diagnosis and characterization of animal pathogens has its origins in the use of isotope-labeled antigens and antibodies. These isotopic techniques that included the use of 75Se, 32P, 125I, and 35S isotopes enabled a level of sensitivity and specificity that was hitherto unrealized, and it is prescient to remind ourselves of just how successful these technologies were, in spite of their infrequent use nowadays. Finally, the review looks at the potential for stable isotope analysis for a variety of applications—in the tracking of animal migrations, where the migrant are potential carriers of transboundary animal diseases, and where it would be useful to determine the origins of the carrier, e.g., Highly Pathogenic Avian Influenza and its dissemination by wild water fowl. Other applications could be in monitoring sequestered microbial culture (e.g., rinderpest virus) where in the case of accidental or deliberate release of infective culture it would be possible to identify the laboratory from which the isolate originated.

Analysis and Troubleshooting

When designing primers, it is of initial importance to define the target area, and secondly the type of application. The BLAST function from the National Center for Biotechnology Information (NCBI) will help to identify the most suitable gene sequence to be used. There are many software programs; some free on websites/pages on the internet, dedicated to primer design and primer optimisation. In this chapter the most important factors that need to be taken into consideration when designing and optimising primers are highlighted.

Identification of Clade E Avipoxvirus, Mozambique, 2016

Analysis of scab samples collected from poultry during outbreaks of fowlpox in Mozambique in 2016 revealed the presence of clade E avipoxviruses. Infected poultry were from flocks that had been vaccinated against fowlpox virus. These findings require urgent reevaluation of the vaccine formula and control strategies in this country.

New Trends in the Diagnosis and Molecular Epidemiology of Viral Diseases

Despite intensive worldwide control programmes against infectious diseases, including vaccination programmes with the use of DIVA vaccines; mass culling (stamping out policies) and regulation of animal movements; various virus diseases still have a very high negative impact on animal health and welfare. The intensification of animal husbandry; centralisation of large groups of animals in industrial production units; globalization of trade in live animals and/or animal products, bedding and feeds; as well as increased tourism, are all considerable factors in the threat of devastating infectious diseases word-wide. The opening of borders between many countries such as in the European continent contributes greatly to the high-risk situation, where infectious agents may easily travel thousands of miles and then suddenly appear in areas where they are unexpected and probably even unknown. The sudden and unexpected appearance of any infectious disease in a new region, be it a country or a continent, may lead to a delayed or innaccurate diagnosis resulting in the uncontrolled spread of the disease agent to other susceptible populations of animals over large geographic areas. Recent major examples are incidences of foot-and-mouth disease (FMD) in the UK, the extension of rinderpest into the Somali plains and Rift Valley fever (RVF) spread into the Arabian Peninsula. The latest major problem is the occurrence, re-occurrence and rapid spread of influenza virus. All these exemplify the serious economic and social impact of the of highly contagious transboundary animal diseases ( TADs).