Improved tools for the diagnosis of Rift Valley fever and serological evidence of the disease in sheep and goats in the inter-epidemic period in the Zambézia Province, Mozambique

Rift Valley fever (RVF) is primarily a mosquito-borne disease of domestic ruminants and humans caused by a virus of the family Bunyaviridae</i., genus Phlebovirus. The causative agente, RVF virus (RVFV), has been responsible for large epidemics in livestock in Africa and in the Arabic Peninsula with significant economic impact due to high mortality rates particularly in new-born lambs and kids and abortion in sheep, goats and cattle. The disease in humans is characterized by a mild to moderate febrile illness but severe complications such as ocular sequelae, encephalitis or a fatal haemorrhagic state, may occur in a low proportion of individuals. Apart from reports of the disease in 1969 and 1999 when RVF caused abortions and deaths in cattle and water buffalos respectively and the survey that was conducted in humans in 1981-83 that found a seroprevalence of 2% in pregnant woman, there are no other records of the disease in Mozambique. At the beginning of this study the available enzyme-linked immunosorbent assay (ELISA) was based on whole inactivated virus which binds poorly to the immunoplates and requires containment facilities to reduce the risk of exposure of laboratory staff to infection with the virulent virus. The objectives of this study were to: <ul> <li> develop and evaluate new RVF reagents for safe, rapid and accurate diagnosis and surveillance of RVF; and</li> <li> apply the newly developed assays to investigate the circulation of RVFV in sheep and goats in the Zambézia Province, Mozambique.</li> </ul> Chapter 1. This is the general introduction and deals particularly with aspects of the characteristic of RVFV; vectors and maintenance of RVFV; major recent epidemics; RVF in Mozambique; RVF in domestic animals and humans; and diagnostic assays. Chapter 2 describes the cloning and expression of RVFV nucleocapsid (N) protein and evaluation of a N-protein based indirect ELISA for the detection of specific IgG and IgM antibodies in domestic ruminants. The recombinant nucleocapsid protein of RVFV was cloned, expressed in the bacterial expression vector pET43b and evaluated as antigen in an indirect ELISA using sera from experimentally infected sheep (n = 128), vaccinated sheep (n = 240), and field-collected sera from sheep (n = 251), goats (n =362) and cattle (n =100). Using virus neutralization (VN) as gold standard very high diagnostic sensitivity and specificity (both 100%) was found in goats when using the anti-species IgG conjugate. Protein G was also tested in the same ELISA format as a detection system, and the sensitivity and specificity in goats were 99,4% and 99,5%, in sheep field sera both 100%, and in cattle 100% and 98,3%, respectively. The indirect ELISA based on recombinant nucleocapsid protein has the potential to complement the conventional assays for serodiagnosis of RVF. Advantages of the N-protein are its safety, stability and cost-effectiveness in use and production. Chapter 3 compares the performance of the indirect ELISA based on the recombinant nucleocapsid protein with the inactivated whole virus based IgG sandwich ELISA for the detection of RVFV antibodies in field-collected sera sample from sheep (n=605) and goat (n=657). A good agreement between both tests was found when testing for the presence of specific anti-RVFV IgG in the sheep (kappa=0,92) and goat sera (kappa = 0,99). Although the sandwich ELISA was slightly more sensitive than the indirect ELISA for the detection of IgG to RVFV the simpler and shorter procedure as well as the safety and high quality of the recombinant nucleocapsid protein antigen makes the indirect ELISA more suitable for surveillance studies. Chapter 4 describes the generation and characterization of monoclonal antibodies against RVFV nucleoprotein to improve existing diagnostic assays. Monoclonal antibodies specific for the nucleocapsid protein of RVFV were produced, isotyped and characterized. Four antibodies selected that belonged to the class IgG and the subclass IgG2a, showed a strong reaction with denaturated nucleocapsid recombinant protein in Western blot and recognized the antigen in an inactivated preparation of whole RVFV in Western blot and in an ELISA. Cross-reactivity with extracts of genetically related and non-related viruses including Bunyamwera virus and Calovo virus (Bunyaviridae family), West Nile virus and Dengue virus II (Flaviviridae family) and Sindbis and Chikungunya viruses (Togaviridae family), could not be shown. These monoclonal antibodies represent a useful tool for the development of rapid diagnostic assays for early diagnosis of RVF. Chapter 5 deals with the evidence of RVFV circulation in the inter-epidemic period in the Zambézia Province, Mozambique based on serological studies. During the last two decades several outbreaks of RVF have been reported in countries in Southern Africa. No clinical disease has been reported in Mozambique during this period. In a serological study conducted in 2007 in five districts of Zambézia Province, Mozambique of a total of 654 small ruminants sampled (277 sheep and 377 goats), 35,8% of sheep sera and 21,2% of goat sera were positive to RVFV. In 2010, a cross-sectional survey was conducted in 313 sheep and 449 goats in two districts (Mopeia and Nicoadala) of the same province. This study revealed an overall seropositivity rate of 9,2% in sheep and 11,6% in goat and an increased likelihood of being seropositive in older animals (OR = 7,3; p < 0,001) with a IgG ELISA. Twenty nine out of 240 animals assessed by IgM ELISA were positive, suggesting recent exposure to RVFV. However, a study carried out between September 2010 and April 2011 in a cohort of 125 animals (74 sheep and 51 goats) failed to demonstrate sero-conversion. The results of the study indicate that RVFV circulates subclinically in domestic small ruminants in Zambézia. Chapter 6. deals with concluding remarks and recommendations. Copyright