Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

Lumpy skin disease: attempted propagation in tick cell lines and presence of viral DNA in field ticks collected from naturally-infected cattle.

Lumpy skin disease (LSD) is of substantial economic importance for the cattle industry in Africa and the Near and Middle East. Several insect species are thought to transmit the disease mechanically. Recent transmission studies have demonstrated the first evidence for a role of hard (ixodid) ticks as vectors of lumpy skin disease virus (LSDV). The aim of this study was to attempt in vitro growth of the virus in Rhipicephalus spp. tick cell lines and investigate in vivo the presence of the virus in ticks collected from cattle during LSD outbreaks in Egypt and South Africa. No evidence was obtained for replication of LSDV in tick cell lines although the virus was remarkably stable, remaining viable for 35 days at 28°C in tick cell cultures, in growth medium used for tick cells and in phosphate buffered saline. Viral DNA was detected in two-thirds of the 56 field ticks, making this the first report of the presence of potentially virulent LSDV in ticks collected from naturally infected animals.

Attempted mechanical transmission of lumpy skin disease virus by biting insects.

The mosquitoes Anopheles stephensi Liston and Culex quinquefasciatus Say (Diptera: Culicidae), the stable fly Stomoxys calcitrans Linnaeus (Diptera: Muscidae) and the biting midge Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were allowed to feed on either lumpy skin disease (LSD) infected animals or through a membrane on a bloodmeal containing lumpy skin disease virus (LSDV). These arthropods were then allowed to refeed on susceptible cattle at various intervals after the infective feed. Virus was detected in the insects by polymerase chain reaction immediately after feeding and at sufficiently high titre to enable transmission to occur. However, no transmission of virus from infected to susceptible animals by An. stephensi, S. calcitrans, C. nubeculosus and Cx. quinquefasciatus was observed.

Importance of thymidine kinase activity for normal growth of lumpy skin disease virus (SA-Neethling).

In order to study the importance of an intact thymidine kinase (TK) gene for the vaccine strain of a southern African capripoxvirus, namely, lumpy skin disease virus (LSDV) (type SA-Neethling), a TK disruption recombinant was generated expressing the Escherichia coli beta-galactosidase (lacZ) reporter gene. A comparative growth study of the recombinant and wild-type (wt) LSDV in TK-positive primary and secondary cells and TK-negative secondary cells was performed. It was found that although recombinant and wt virus both grew in TK-positive cells without selection, the recombinant was unable to grow in TK-negative cells (with or without selection), indicating that TK activity is important, if not essential, for normal growth of LSDV.

Alternative cell lines for the propagation of lumpy skin disease virus.

In our Institute lumpy skin disease virus is grown on primary lamb testis cells for isolation, identification and vaccine production. However, the availability of lambs in Kenya has been seriously reduced over the past few years. This has led to an increase in the cost of using primary lamb testis cells. This study was undertaken to investigate other primary cell lines, which are easily available and provide an equivalent or better yield of lumpy skin disease virus. Foetal bovine muscle (FBM) cells were found to be an adequate alternative for lamb testis cells.

Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae).

Aedes aegypti female mosquitoes are capable of the mechanical transmission of lumpy skin disease virus (LSDV) from infected to susceptible cattle. Mosquitoes that had fed upon lesions of LSDV-infected cattle were able to transmit virus to susceptible cattle over a period of 2-6 days post-infective feeding. Virus was isolated from the recipient animals in 5 out of 7 cases. The clinical disease recorded in the animals exposed to infected mosquitoes was generally of a mild nature, with only one case being moderate. LSDV has long been suspected to be insect transmitted, but these findings are the first to demonstrate this unequivocally, and they suggest that mosquito species are competent vectors.