Determination of the minimum protective dose for bluetongue virus serotype 2 and 8 vaccines in sheep

Recent outbreaks of bluetongue virus (BTV) serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV) released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.The research was done as part of a masters dissertation by J.M. (Onderstepoort Biological Products). The practical work was done by J.M. (Onderstepoort Biological Products) and supervised by E.V. (University of Pretoria). E.V. (University of Pretoria) wrote and submitted the manuscript.Onderstepoort Biological Productshttp://www.jsava.co.zahttp://upetd.up.ac.za/thesis/available/etd-07312009-145842/ab201

Seroprevalence of Rift Valley fever and lumpy skin disease in African buffalo (Syncerus caffer) in the Kruger National Park and Hluhluwe-iMfolozi Park, South Africa

Rift Valley fever and lumpy skin disease are transboundary viral diseases endemic in Africa and some parts of the Middle East, but with increasing potential for global emergence. Wild ruminants, such as the African buffalo (Syncerus caffer), are thought to play a role in the epidemiology of these diseases. This study sought to expand the understanding of the role of buffalo in the maintenance of Rift Valley fever virus (RVFV) and lumpy skin disease virus (LSDV) by determining seroprevalence to these viruses during an inter-epidemic period. Buffaloes from the Kruger National Park (n = 138) and Hluhluwe-iMfolozi Park (n = 110) in South Africa were sampled and tested for immunoglobulin G (IgG) and neutralising antibodies against LSDV and RVFV using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the serum neutralisation test (SNT). The I-ELISA for LSDV and RVFV detected IgG antibodies in 70 of 248 (28.2%) and 15 of 248 (6.1%) buffaloes, respectively. Using the SNT, LSDV and RVFV neutralising antibodies were found in 5 of 66 (7.6%) and 12 of 57 (21.1%), respectively, of samples tested. The RVFV I-ELISA and SNT results correlated well with previously reported results. Of the 12 SNT RVFV-positive sera, three (25.0%) had very high SNT titres of 1:640. Neutralising antibody titres of more than 1:80 were found in 80.0% of the positive sera tested. The LSDV SNT results did not correlate with results obtained by the I-ELISA and neutralising antibody titres detected were low, with the highest (1:20) recorded in only two buffaloes, whilst 11 buffaloes (4.4%) had evidence of co-infection with both viruses. Results obtained in this study complement other reports suggesting a role for buffaloes in the epidemiology of these diseases during inter-epidemic periods.http://www.jsava.co.zatm201

Insights into the pathogenesis of viral haemorrhagic fever based on virus tropism and tissue lesions of natural Rift Valley fever

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive proinflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research.http://www.mdpi.com/journal/virusespm2022Paraclinical SciencesVeterinary Tropical Disease

Occurrence of Babesia felis and Babesia leo in various wild felid species and domestic cats in Southern Africa, based on reverse line blot analysis

Reverse line blot (RLB) is a hybridization assay that can be used to detect various blood parasites and differentiate between them. Results, using the RLB, showed that Babesia felis and Babesia leo occurred as single or mixed infections in various felid species, but most frequently in domestic cats and lions, respectively. Prevalence of infection in free-ranging cheetahs in Namibia was low (7, 5%), whereas 50% of free-ranging lions in South Africa and Swaziland were infected. A large number (52, 9%) of samples tested positive only for Babesia, neither B. felis nor B. leo. This could be an indication of at least one further, as yet undescribed, Babesia species in felids.Mrs. Gerty Pretorius (Clinical Pathology Section, Department of Companion Animal Clinical Studies), and Prof. Moritz van Vuuren (Department of Veterinary Tropical Diseases) submitted blood specimens. This report emanates from project 36-5-613 which was approved by the Research Committee of the Faculty of Veterinary Science and the Animal Use and Care Committee of the University of Pretoria

Detection of bluetongue virus and African horsesickness virus in co-infected cell cultures with NS1 gene probes

The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by means of northern blot hybridization. Under high-stringency conditions both probes hybridized to 22 BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to serotypes that originate from Australia and India. Furthermore, NS1 gene probes of BTV and African horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures. The method detects viral RNA directly in glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification. AHSV could be detected in cells infected with AHSV at a multiplicity of infection of 10¯⁴ PFU/cell in the presence of a hundredfold excess of co-infecting BTV. The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Detection of bluetongue virus RNA in cell cultures and in the central nervous system of experimentally infected mice using in situ hybridization

Two radiolabelled complementary DNA (eDNA) probes (1663 bp and 200 bp res pectively) were prepared from the genome segment that encodes the non-structural protein 1 (NS1) of bluetongue virus serotype 4 (BTV4). The probes were used to optimize the in situ hybridization (ISH) method on baby hamster kidney-21 (BHK-21) cells and to investigate the use of the technique as a diagnostic procedure. Cells were infected with BTV4 at a multiplicity of infection of 0,5 PFU/cell. An intense cytoplasmic hybridization signal could be detected from 3 hours post-infection onwards, reaching a peak at 17 hours. The ISH procedure has potential use as a diagnostic technique, but will probably find a wider application in pathogenesis studies. An in situ hybridization method was also developed for the detection of BTV RNA in the central nervous system of newborn mice after intracranial inoculation with BTV10. Viral RNA-positive cells were detected from day 3 onwards, predominantly in areas where the virus caused necrotic encephalitis.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Molecular differentiation and pathogenicity of Aviadenoviruses isolated during an outbreak of inclusion body hepatitis in South Africa

Fowl adenovirus (FAdV) is a member of the genus Aviadenovirus and causes a number of economically important poultry diseases. One of these diseases, inclusion body hepatitis (IBH), has a worldwide distribution and is characterised by acute mortality (5% – 20%) in production chickens. The disease was first described in the United States of America in 1963 and has also been reported in Canada, the United Kingdom, Australia, France and Ireland, but until now, not in South Africa. Adenoviruses isolated from the first outbreak of IBH in South Africa were able to reproduce the disease in chicken embryo livers. The aim of the present study was to characterise the viruses and determine the pathogenicity of the FAdV strains responsible for the first reported case of IBH in South Africa. Polymerase chain reaction (PCR) amplification of the L1 loop region of the fowl adenovirus hexon gene using degenerate primer pair hexon A/B was used to identify the viruses that were isolated. Restriction fragment length polymorphism (RFLP) of the amplification products was used for the differentiation of 14 isolates of fowl adenovirus. Sequencing of the PCR products followed by amino acid comparison and phylogenetic analysis using the L1 loop region of the hexon protein was done to determine the identity of the isolates. Amino acid sequences of the hexon genes of all the South African isolates were compared with those of reference strains representing FAdV species. Amino acid comparison of 12 South Africa field isolates to FAdV reference strains revealed a high sequence identity (> 93.33%) with reference strains T8-A and 764. Two of the isolates had high sequence identity (93.40%) with reference strains P7-A, C2B and SR48. Phylogenetic analysis of the L1 loop region of the hexon protein of all 14 South African isolates was consistent with their RFLP clusters. The mortality rates of embryos challenged with 106 egg infective doses (EID50) FAdV 2 were 80% – 87% and mortality rates for embryos challenged with 105.95 (EID50) FAdV 8b were 65% – 80%.http://www.jsava.co.zaam201

Bluetongue virus reassortment, an overlooked aspect of viral evolution with potentially serious implications

Bluetongue virus (BTV) is the prototype member of the Orbivirus genus in the family Reoviridae and is the aetiological agent of the arthropod transmitted disease, bluetongue (BT), which affects ruminant and camelid species. The disease is of significant global importance due to its economic impact and effects on animal welfare. Bluetongue virus, a segmented dsRNA virus, exists as a genetically and phenotypically heterogeneous entity in nature and has the ability to reassort its genome segments in vertebrate or vector cells which have concurrently been infected with more than one strain or serotype of the virus. Although the kinetics of BTV reassortment has been described in both in vivo and in vitro studies, relatively little is known about the consequences which the reassortment of different genome segments may have on the phenotypic properties of the virus. It has been speculated that the reassortment of genome segments between phenotypically distinct strains may result in the generation of novel reassortant viruses, which may display either enhanced virulence or transmission characteristics. The purpose of this review is to provide an overview of the mechanisms of viral evolution which underlie the generation of genetic and phenotypic differences among BTV field strains, to discuss the kinetics of BTV reassortment and to highlight documented examples of the effects of reassortment on the phenotype of the virus. Methods by which BTV reassortants may be generated in vitro, as well as possible approaches for evaluating the consequences of reassortment on the phenotypic properties of the virus are also discussed.Norwegian School of Veterinary Science (NVH)http://www.sciencedirect.com/science/journal/03781135hb201

Seminal transmission of lumpy skin disease virus in heifers

It is known that lumpy skin disease virus (LSDV) can be shed in bull semen following infection and that artificial insemination (AI) poses a biosecurity risk. It is however not known whether the use of LSDV infected semen in AI poses a biosecurity risk. The aims of the current study were to investigate whether LSDV, transmitted through semen, can infect cows and embryos.. Two controlled trials were performed simultaneously. Eleven (11) young beef heifers, naïve to LSDV, were synchronized using an OvSynch protocol and inseminated with fresh semen spiked with a field strain of LSDV on day 0. Six (6) of the heifers were superovulated on Day 1 using PMSG, and embryos were flushed from these heifers on Day 6. Blood and serum samples were collected from Day 4 until Day 27 to determine the presence of LSDV by PCR and virus isolation, and the presence of antibodies against LSDV by SNT. The first clinical signs of LSD were noticed on Day 10, followed by severe generalized LSD in 3 heifers, and mild LSD in 2 more heifers. Two heifers were humanely euthanized due to severe unresponsive stranguria. LSDV was detected by PCR, virus isolation or electron microscopy in blood, embryos and organs of experimentally infected animals, and 8 heifers had seroconverted by Day 27. Two control animals were not affected. This is the first report of experimental seminal transmission of LSDV in cattle.NRF. Project number FA 200704250000.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1865-16822015-10-31hb201

Potential link of single nucleotide polymorphisms (SNPs) to virulence of vaccine‐associated field strains of lumpy skin disease virus in South Africa

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, wide‐spread outbreaks of vaccine‐like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990’s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine‐associated. The genetic differences between the phenotypically distinct vaccine and vaccine‐associated strains were 67 single nucleotides polymorphisms (SNPs). This study characterised the location and possible importance of each of these SNPs in their role during virulence and host specificity