Trop Anim Health Prod DOI 10.1007/s11250-014-0540-6
REGULAR ARTICLES
Molecular detection of Rift Valley fever virus in serum samples from selected areas of Tanzania Augustino Alfred Chengula & Christopher Jacob Kasanga & Robinson Hammerthon Mdegela & Raphael Sallu & Mmeta Yongolo
Accepted: 9 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease affecting domestic animals and humans caused by the Rift Valley fever virus (RVFV). The virus belongs to the genus Phlebovirus of the family Bunyaviridae. The main aim of this study was to detect the presence of antibodies to RVFV as well as the virus in the serum samples that were collected from livestock during the 2006/2007 RVF outbreaks in different locations in Tanzania. Analysis of selected samples was done using a RVF-specific inhibition enzyme-linked immunosorbent assay (I-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Genomic viral RNA was extracted directly from serum samples using a QIAamp® Viral RNA Mini Kit (QIAGEN), and a one-step RT-PCR protocol was used to amplify the S segment of RVFV. Positive results were obtained in 39.5 % (n=200) samples using the RVF I-ELISA, and 17.6 % (n=108) of samples were positive by RT-PCR. I-ELISA detected 41 (38.7 %), 32 (39.0 %), and 6 (50.0 %) positive results in cattle, goats, and sheep sera, respectively, whereas the RT-PCR detected 11 (0.2 %), 7 (0.2 %), and 1 (0.1 %) positive results in cattle, goats, and sheep sera, respectively. These findings have demonstrated the presence of RVFV in Tanzania during A. A. Chengula (*) : C. J. Kasanga Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania e-mail: [email protected] R. H. Mdegela Department of Veterinary Medicine and Public Health, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania R. Sallu : M. Yongolo Tanzania Veterinary Laboratory Agency, Ministry of Livestock Development and Fisheries, P. O. Box 9254, Dar es Salaam, Tanzania
the 2006/2007 RVF outbreaks. To our knowledge, this is the first report to detect RVFV in serum samples from domestic animals in Tanzania using PCR technique. Therefore, a detailed molecular study to characterize the virus from different geographical locations in order to establish the profile of strains circulating in the country and develop more effective and efficient control strategies should be done. Keywords Rift Valley fever . RVF outbreaks . Domestic animals . Mosquitoes . ELISA . RT-PCR
Introduction Rift Valley fever (RVF) is an arboviral disease endemic in most sub-Saharan African countries transmitted by mosquitoes especially of the genus Aedes and Culex (Davies and Martin 2006) The disease affects primarily ruminant animals leading to major socioeconomic losses (Rich and Wanyoike 2010; Sindato et al. 2012). In Tanzania, the effects of RVF were high in sheep and goats followed by cattle mainly due to deaths and abortions (Chengula et al. 2013). Other clinical manifestation that was observed during the outbreak in domestic animals included high fever, discharges from the nose and eyes, hemorrhagic diarrhea, abdominal pain, and vomiting. In human, the clinical symptoms included death (case fatality rate was 28.2 %), fever, encephalopathy, hemorrhage, and retinopathy (Mohamed et al. 2010). Rift Valley fever virus (RVFV) of the family Bunyaviridae and genus Phlebovirus is responsible for causing the disease in animals. The virus has a negative-sense single-stranded RNA genome (approximately 11.9 kb in size) and is composed of three segments designated as S (small, 1690 nt), M (medium, 3885 nt), and L (large, 6404 nt), packaged together in the virion (Elliott 1990, 1997; Robert 2004; Bird et al. 2007a). The enveloped virion is composed of a lipid bilayer
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containing the carboxy-terminal glycoprotein (Gc) and the amino-terminal glycoprotein (Gn) (Freiberg et al. 2008; Sherman et al. 2010; Bouloy and Weber 2010). The co-packaging of these RNAs to produce infectious RVFV has been suggested to be driven by specific intermolecular interactions among the three viral RNAs (Terasaki et al. 2010). Each segment is enclosed in a separate nucleocapsid within the virion (Garcia et al. 2001; Dighe et al. 2010). Segment S codes for two proteins (the nucleoprotein N and the nonstructural NSs protein) by using an ambisense strategy and L codes for the 237-kDa viral RNA-dependent RNA polymerase in a single 6.4-kb open reading frame (ORF) (Sall et al. 1998; Bouloy and Weber 2010). Segment M codes at least four viral proteins in a single ORF: the two major envelope surface glycoproteins, the Gc and Gn (named the 55-kDa Gn and 58-kDa Gc synthesized as part of polyprotein precursors), a 78-kDa fusion of the NSm, and the14-kDa protein NSm of unknown function (Accardi et al. 2001; Lagerqvist et al. 2009). Rift Valley fever is a potential threat to African countries where it is endemic in many countries (Thiongane et al. 1991; Robert 2004). Since its reemergence in Egypt in 1977, epidemics have occurred in Mauritania (1987 to 1998), Madagascar (1990/1991 and 2007/2008), Egypt (1993), and eastern Africa in Kenya, Somalia, and Tanzania (1977/1978 and 2006/2007), Yemen and Saudi Arabia (2000), and in South Africa (2007/2008 and 2010/2011) (Sall et al. 1999; CDC 2000; OIE 2010; Archer et al. 2011; FAO 2011). The seroprevalence of RVF in Kenya during the 2006–2007 outbreak was reported by LaBeaud et al. (2008) to be 13 %. A detailed molecular epidemiology during that outbreak (2006/2007) which affected also Tanzania was done in Kenya by only using reverse transcription polymerase chain reaction (RTPCR) (Munyua et al. 2010; Pepin et al. 2010). The disease has reappeared in Madagascar in 2008/2009, and analysis of partial sequences from RVFV strains showed to be similar to the strains circulating in Kenya during the 2006/2007 outbreak (Andriamandimby et al. 2010). An extensive genomic analyses carried out by Bird et al. (2007a, b) using samples from the 2006–2007 RVF outbreak in East Africa demonstrated the concurrent circulation of multiple virus lineages with a common ancestry from the 1997–1998 East African RVF outbreaks. The increase in genomic diversity, 2 to 4 years prior to the 2006/2007 outbreaks and the interepidemic seropositivity from recent studies indicates an ongoing RVFV activity and evolution during the interepidemic period (Sall et al. 1998; Sumaye et al. 2013; Turell et al. 1990). Diagnosis of RVFV is based on clinical signs, virus isolation, enzyme-linked immunosorbent assay (ELISA), and molecular techniques (RT-PCR and nucleic acid sequencing) (Garcia et al. 2001; Sall et al. 2002; FAO 2003). The latter is more sensitive and reproducible and, as such, provides a promising option for diagnosis and detection of viral RNA
of RVFV (Garcia et al. 2001; Le Roux et al. 2009). Since viral RNA and IgM detection by RT-PCR and ELISA, respectively, are more rapid than virus isolation, Sall et al. (2002) recommended using these techniques/assays in parallel as diagnostic methods for RVFV when there is an outbreak. During the outbreak in Tanzania, the diagnosis was based on serological methods in domestic animals and human serum, and tissues samples were transported to the Kenya Medical Research Institute/Centers for Disease Control and Prevention (KMRI/CDC) laboratory in Nairobi (Mohamed et al. 2010). In this laboratory, samples were tested for IgM and IgG antibodies using ELISA, and tissue samples were tested for viral RNA by using real-time RT-PCR. During the 2006/2007 RVF outbreak in Kenya and Tanzania, molecular epidemiology using RT-PCR was performed only on samples from domestic animals from Kenya (Pepin et al. 2010), while in Tanzania, molecular epidemiological studies were performed only in humans (Mohamed et al. 2010; Nderitu et al. 2011). Therefore, there is a lack of information on the circulating RVFV field strains in animals in Tanzania which could assist in vaccination strategies. Thus, the main aim of this study was to detect RVF antibodies and the virus in serum samples collected during the 2006/2007 outbreak in selected areas of Tanzania using RVF inhibition ELISA and RT-PCR. The information obtained in this study will lead to an improved understanding on RVFV and generate baseline data for further studies on the virus in Tanzania.
Materials and methods Samples This study used serum samples from cattle, goats, and sheep obtained from the Tanzania Veterinary Laboratory Agency (TVLA, BSL2) in Dar es Salaam which were collected during the 2006/2007 RVF outbreaks. Serum samples were collected by livestock field officers under the supervision of Veterinary Investigation Centres (VICs) and transported on ice to TVLA where they were stored between −50 and −35 °C. Regions where samples were collected included Mwanza, Arusha, Tabora, Dodoma, Tanga, Mbeya, Iringa, Pwani, Dar es Salaam, Lindi, and Mtwara. RVF inhibition ELISA Screening of serum samples for detection of IgG antibody levels against RVFV was done using a RVF-specific inhibition ELISA (I-ELISA) as described previously (Paweska et al. 2005). The optical density (OD) was measured at 405 nm. The specific activity of each serum (net OD) was calculated by subtracting the nonspecific background OD in the wells with control antigen from the specific OD in wells with virus
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antigen. The mean OD readings for replicate tests were converted to a percent inhibition (PI) value using the following equation: [100−(mean net OD of test sample/mean net OD of negative control)×100]. Cutoff values (expressed as a PI of an internal negative control) used were 41.9, 41.4, and 38.4 for cattle, goat, and sheep, respectively. In this test, 200 sera were analyzed for detection of IgG for RVF.
Results for samples from each test clustered by location where samples were collected are shown in Table 1. All positive samples by RVF I-ELISA were negative by RT-PCR, and 65.5 % (n=29) of negative samples by I-ELISA were positive by RT-PCR. The prevalence of RVF based on IELISA and RT-PCR as clustered by species is shown in Table 2.
Extraction of RVFV RNA Discussion Genomic viral RNA was directly extracted from 108 sera, 79 samples were positive from the RVF I-ELISA, while 29 negative samples were used. Basic RNA extraction was performed using the QIAamp® Viral RNA Mini Kit (QIAGEN, Germany) in accordance with the manufacturer’s instructions. The extracted RNA was then stored at −20 °C until its use. Reverse transcription polymerase chain reaction A one-step RT-PCR primers, first described by Garcia et al. (2001), were used for synthesis and amplification of DNA based on the one-step RT-PCR protocol (with modification) of Salim et al. (2010). A reaction mix was prepared in a single tube by adding 12.5 μl of 2× reaction mix (containing dNTPs and MgSO4), 1.0 μl of 25× reverse transcriptase-Taq polymerase mix, 2.0 μl of 30 nmol of each S segment genespecific oligonucleotide primer, and 1.7 μl of enhancer. A forward primer S432 (5′-ATG ATG ACA TTA GAA GG GA-3′) and a reverse primer NS3m (5′ATG CTG GGA AGT GAT GAG-3′) were used to amplify a 298-bp S RNA segment gene of RVFV. The total volume of the reaction was 20.0 μl by adding RNase-free water. Target RNA (5.0 μl) was added last to make a final volume of 25 μl. The cycling conditions were set at 37 °C for 30 min (a reverse transcription step), an initial denaturation of 10 min at 95 °C followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, extension at 72 °C for 30 s, a final extension of 10 min at 72 °C, and finally holding at 4 °C. Finally, PCR products were visualized and photographed after agarose gel electrophoresis. A live attenuated freezedried Rift Valley fever viral vaccine (RIFTVAX™, Nairobi, Kenya) and RNase-free water instead of template were used as positive and negative controls, respectively. Products were sized against a 100-bp molecular weight DNA marker (GelPilot 100-bp Ladder, QIAGEN).
Results In this study, RVF inhibition ELISA used a total of 106, 82, and 12 serum samples from cattle, goats, and sheep, respectively. On the other hand, RT-PCR used a total of 54, 36, and 18 serum samples from cattle, goats, and sheep, respectively.
Epidemics of RVF in Tanzania normally starts on the northern part (especially in Ngorongoro region) in the villages along the floor of the eastern arm of the Rift Valley (Chengula et al. 2013; Davies and Martin 2006). Documented reports from Tanzania indicate that the disease was reported for the first time in 1930, followed by periodic epidemics at an interval of 10–20 years: 1947, 1957, 1977, 1997, and the latest in 2007 (Sindato et al. 2012). These outbreaks were preceded by a season of heavy rainfall (Anyamba et al. 2010; Davies and Martin 2006; FAO EMPRES WATCH 2007). Despite all these outbreaks that have impacted greatly on domestic animals and humans, only one molecular study in humans, using real-time RT-PCR, has been documented (Mohamed et al. 2010). Also, two human samples (from Dodoma and Tanga in Tanzania) that were sent to Kenya for diagnostic purposes have recently been sequenced together with others from Kenya and Somalia (Nderitu et al. 2011). The latest disease outbreak of 2007 covered a wider area of Tanzania (11 regions of mainland) affecting domestic animals and humans (Sindato et al. 2012). Molecular detection of RVFV using RT-PCR is recommended due to it being rapid and effective especially during the early stages of the disease when antibodies are not detectable (van Vuren et al. 2007; Shoemaker et al. 2002). Recently, a realtime RT-PCR that can detect all the three segments (S, M, and Table 1 Proportion (percentage) of positive serum samples based on ELISA (N=200) and RT-PCR (N=108) tests clustered by location Region
Date of collection
ELISA, n (%)
RT-PCR, n (%)
Dodoma Tabora
29 Mar–4 Apr 2007 26–30 Mar 2007
33 (45.5) 53 (34.0)
20 (10.0) 34 (44.1)
Manyara Arusha Tanga Mwanza Mtwara Iringa Mbeya Lindi Pwani
21 Oct 2007 21 Oct 2007 9 May 2007 12 Apr 2007 12–18 Apr 2007 17 Sep 2007 18 Apr 2007 12 Apr 2007 22 Nov 2007
20 (50.0) 10 (30.0) 5 (20.0) 11 (9.1) 17 (58.8) 11 (36.4) 15 (33.3) 6 (50.0) 19 (47.4)
10 (0.0) 7 (0.00) 2 (50.0) 7 (0.0) 10 (0.0) 4 (0.0) 5 (0.0) 3 (33.3) 6 (0.0)
N total samples of all regions, n total samples of each region
Trop Anim Health Prod Table 2 Proportion of positive serum samples from different animal species tested by ELISA and RT-PCR methods Species
ELISA Total sample
RT-PCR Positive (%)
Total sample
Positive (%)
Cattle
106
38.7
53
20.8
Goat Sheep Overall
82 12 200
39.0 50.0 39.5
38 17 108
18.4 5.9 17.6
L) of the RVFV genome has been developed (Wilson et al. 2013) to facilitate rapid detection of the virus. In this study, RVF I-ELISA test detected 39.5 % (n=200) positive serum samples that were collected during the 2006– 2007. All serum samples (79) which were IgG-positive and 29 IgG-negative were further tested using conventional RT-PCR for which 17.6 % (n=108) of samples tested were found to be positive for RVFV genome. All RT-PCR positive samples that tested negative by RVF I-ELISA test represent acute infections, as IgG antibodies are detectable approximately 1 month or more, post infection (Bird et al. 2008; OIE 2008). Positive samples by RT-PCR were obtained from samples collected between March and May 2007, possibly because they were collected within the RVF outbreak period. Some areas (Manyara, Arusha, Iringa, and Pwani) where samples were collected after the outbreak (after June 2007) indicated the presence of IgG antibodies using I-ELISA, but there were no viral genomes detected. The antibodies may be due to the virus activity in the animals during the outbreak or interepidemic virus activity especially for samples collected 2 months later after the outbreak. Interepidemic virus activity in Tanzania has been reported in Mbeya from human blood samples (Heinrich et al. 2012). In livestock, the interepidemic transmission of RVF has been reported in areas experiencing annual flooding (Kilombero River Valley in Tanzania) by Sumaye et al. (2013). The interepidemic virus activity in livestock has also been reported in other countries (LaBeaud et al. 2008). Recently, an evidence of interepidemic transmission of RVF in African buffalo has been documented in South Africa (LaBeaud et al. 2011). The virus tends to circulate at low levels in domestic animals and humans during the interepidemic period in RVF endemic countries (FAO EMPRES WATCH 2007). Understanding the distribution of the potential vectors of RVFV has a biological and epidemiological significance in relation to disease outbreak hotspots. A model for predicting the distribution of RVFV in East Africa targeting Aedes aegypti and Culex pipiens complex, potential vectors for maintenance and amplification of the virus respectively, has been developed (Mweya et al. 2013). The model can also provide good information on suitable areas for collecting vectors of RVF during
interepidemic periods to determine the virus replication in the vectors. The diversity of RVFV strains isolated within an epidemic and in the endemic setting may not be the same, being low in the endemic setting (Bird et al. 2007b). This emphasizes on molecular monitoring of virus evolution for strategic control of RVF especially on vaccination of animals using the right vaccine candidate. Livestock, for a long time now, have been vaccinated using a modified live virus strain (Smithburn strain) which is said to be cost-effective commercial practice due to it being less expensive to produce (Davies 2010). Although the evolution rate of the virus is considered to be slow, the isolates from Tanzania (TAN/Tan-001/07 and TAN/Dod-002/07) during the 2006–2007 outbreak were found to be different from those circulated in Kenya (Nderitu et al. 2011). In addition, the isolates from the 2006–2007 outbreak were more distant from the older RVFV isolates, suggesting an ongoing slow evolution clock for the RVFV. Thus, molecular information of virus is a very important key for understanding the evolution of the virus. So far, the circulating virus strains in domestic animals in Tanzania are not yet known. There is a need of charactering the virus during epidemics and interepidemics in order to understand what is going on during the two periods for effective control strategies. It is important to use information obtained during molecular and serological surveillance together with global early warning forecasts issued by the United Nations Food and Agriculture Organization (FAO) to control RVF outbreaks. In the latest outbreak of East Africa (2006/2007), the FAO issued an early warning forecast predicting possible RVF outbreak for the Horn of Africa on November 2006 (Anyamba et al. 2010; Martin et al. 2007). However, there was no time frame for the countries to prepare themselves as the warning was issued in short time just before cases for both human and livestock to appear in the same November 2006 (FAO EMPRES WATCH 2012; Jost et al. 2010). Rift Valley fever remains to be threat to livestock keepers and non-livestock keepers and the nation at large due to its socioeconomic effects (Dar and McIntyre 2013; ElBahnasawy et al. 2013; Otte et al. 2004). In 2007, the disease led to disrupted livelihood and markets for livestock products that resulted from a ban of livestock slaughter and exportation. In Tanzania, during RVF outbreak, the average price of bulls for slaughter dropped by 33 %, the internal flow of marketed cattle decreased by 40 %, and at least 75 % of exports were not allowed to leave the country (FAO EMPRES WATCH 2012), since the virus can be found in different species from mosquitoes, animals (domestic and wildlife animals) to human. A collaborative control strategy involving different sectors applying the concept of “one health” will have a fast and great impact especially during epidemics. Lack of coordination and intersectorial collaboration during the control of 2006–2007 RVF outbreak in Tanzania has been reported as one of the challenges for controlling the disease (Chengula et al. 2013).
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Using the I-ELISA and RT-PCR, this study has found that there were IgG antibodies and viral genomes of RVFV in the sera collected from livestock during the 2006–2007 outbreak. Based on our knowledge, this is the first documentation on the detection of RVFV in serum samples using RT-PCR techniques. A serological surveillance to monitor virus activities in animals and humans is important, together with virus activities in mosquitoes transmitting the disease, and the animals (wildlife and domestic animals). The information will also be useful during planning the control strategies of RVF by the responsible organs. Since RVFV strains circulating in Tanzania are not known, the study suggests carrying out detailed molecular studies to characterize the viruses circulating in the country. Acknowledgments The authors highly acknowledge the Regional Universities Forum (RUFORUM) who funded the project, the Director of TVLA, and all members of the virology section at TVLA, Dar es Salaam. Conflict of interest The authors do declare that they have no conflict of interest.
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outbreak in Kenya. American Journal of Tropical Medicine and Hygiene, 83, 52–57. Robert, S. (2004). Bunyaviridae. In: Principles and Practice of Clinical Virology, Fifth Edition. “Edited by” (A. J. Zuckerman, J. E., Banatvala, J. R., Pattison, P. D., Griffiths and Schoub, B.D.) John Wiley & Sons Ltd, West Sussex, England pp 556-588 Salim, R. W., Khairalla, K. M. S., Eljamal, A. A., Karrar, A.E. and Aradaib, A. E., 2010. A Single Tube RT-PCR amplification for detection of Rift valley fever virus. Research Journal of Medical Sciences, 4 (3), 146-151. Sall, A.A., Zanotto, P.M., Vialat, P., Sène, O.K. and Bouloy, M. 1998. Molecular epidemiology and emergence of Rift Valley fever. Memórias do Instituto Oswaldo Cruz, 93, 609–614. Sall, A. A., Zanotto, P. M.A., Sène, O. K., Zeller, H. G., Digoutte, J. P., Thiongane, Y. and Bouloy, M., 1999. Genetic reassortment of Rift Valley fever virus in nature. Journal of Virology, 73 (10), 8196-8200. Sall, A.A., Macondo, E.A., Sène, O.K., Diagne, M., Sylla, R., Mondo, M., Girault, L., Marrama, L., Spiegel, A., Diallo, M., Bouloy, M. and Mathiot, C. 2002. Use of Reverse Transcriptase PCR in Early Diagnosis of Rift Valley Fever. Clinical and Diagnostic Laboratory Immunology, 9, 713–715. Sherman, M.B., Freiberg, A.N., Holbrook, M.R. and Watowich, J., 2010. Single-particle cryo-electron microscopy of Rift Valley fever virus. Journal of Virology, 387, 11–15. Shoemaker, T., Boulianne, C., Vincent, M.J., Pezzanite, L., Al-Qahtani, M.M., Al-Mazrou, Y., Khan, A.S., Rollin, P.E., Swanepoel, R., Ksiazek, T.G. and Nichol, S.T., 2002. Genetic analysis of viruses associated with emergence of Rift Valley fever in Saudi Arabia and Yemen, 2000-01. Emerging Infectious Diseases, 8, 1415–1420. Sindato, C., Karimuribo, E., Mboera, E.G., 2012. The epidemiology and socio-economic impact of Rift Valley fever epidemics in Tanzania: A review. Onderstepoort Journal of Veterinary Research, 79 (2). doi:10.4102/ojvr.v79i2.467. Sumaye, R.D., Geubbels, E., Mbeyela, E. and Berkvens, D., 2013. Interepidemic Transmission of Rift Valley Fever in Livestock in the Kilombero River Valley, Tanzania: A Cross-Sectional Survey. PLoS Neglected Tropical Diseases, 7(8), e2356. doi:10.1371/ journal.pntd.0002356. Terasaki, K., Murakami, S., Lokugamage, K.G., Makino, S., 2010. Mechanism of tripartite RNA genome packaging in Rift Valley fever virus. Proceedings of the National Academy of Sciences, 108 (2), 804-809. Thiongane, Y., Gonzalez, J.P., Fati, A. and Akakpo, J. A., 1991. Changes in Rift Valley fever neutralizing antibody prevalence among small domestic ruminants following the 1987 outbreak in the Senegal River basin. Research in Virology, 142, 67–70. Turell, M.J., Saluzzo, J.F., Tammariello, R.F., Smith, J.F. 1990. Generation and transmission of Rift Valley fever viral reassortants by the mosquito Culex pipiens. Journal of General Virology, 71 (10), 2307–2312. Wilson, W.C., Romito, M., Jasperson, D.C., Weingartl, H., Binepal, Y.S., Maluleke, M.R., Wallace, D.B., Jansen van Vuren, P. and Paweska, J.T., 2013. Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments. Journal of Virological Methods, 193, 426–431.
REGULAR ARTICLES
Molecular detection of Rift Valley fever virus in serum samples from selected areas of Tanzania Augustino Alfred Chengula & Christopher Jacob Kasanga & Robinson Hammerthon Mdegela & Raphael Sallu & Mmeta Yongolo
Accepted: 9 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease affecting domestic animals and humans caused by the Rift Valley fever virus (RVFV). The virus belongs to the genus Phlebovirus of the family Bunyaviridae. The main aim of this study was to detect the presence of antibodies to RVFV as well as the virus in the serum samples that were collected from livestock during the 2006/2007 RVF outbreaks in different locations in Tanzania. Analysis of selected samples was done using a RVF-specific inhibition enzyme-linked immunosorbent assay (I-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Genomic viral RNA was extracted directly from serum samples using a QIAamp® Viral RNA Mini Kit (QIAGEN), and a one-step RT-PCR protocol was used to amplify the S segment of RVFV. Positive results were obtained in 39.5 % (n=200) samples using the RVF I-ELISA, and 17.6 % (n=108) of samples were positive by RT-PCR. I-ELISA detected 41 (38.7 %), 32 (39.0 %), and 6 (50.0 %) positive results in cattle, goats, and sheep sera, respectively, whereas the RT-PCR detected 11 (0.2 %), 7 (0.2 %), and 1 (0.1 %) positive results in cattle, goats, and sheep sera, respectively. These findings have demonstrated the presence of RVFV in Tanzania during A. A. Chengula (*) : C. J. Kasanga Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania e-mail: [email protected] R. H. Mdegela Department of Veterinary Medicine and Public Health, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania R. Sallu : M. Yongolo Tanzania Veterinary Laboratory Agency, Ministry of Livestock Development and Fisheries, P. O. Box 9254, Dar es Salaam, Tanzania
the 2006/2007 RVF outbreaks. To our knowledge, this is the first report to detect RVFV in serum samples from domestic animals in Tanzania using PCR technique. Therefore, a detailed molecular study to characterize the virus from different geographical locations in order to establish the profile of strains circulating in the country and develop more effective and efficient control strategies should be done. Keywords Rift Valley fever . RVF outbreaks . Domestic animals . Mosquitoes . ELISA . RT-PCR
Introduction Rift Valley fever (RVF) is an arboviral disease endemic in most sub-Saharan African countries transmitted by mosquitoes especially of the genus Aedes and Culex (Davies and Martin 2006) The disease affects primarily ruminant animals leading to major socioeconomic losses (Rich and Wanyoike 2010; Sindato et al. 2012). In Tanzania, the effects of RVF were high in sheep and goats followed by cattle mainly due to deaths and abortions (Chengula et al. 2013). Other clinical manifestation that was observed during the outbreak in domestic animals included high fever, discharges from the nose and eyes, hemorrhagic diarrhea, abdominal pain, and vomiting. In human, the clinical symptoms included death (case fatality rate was 28.2 %), fever, encephalopathy, hemorrhage, and retinopathy (Mohamed et al. 2010). Rift Valley fever virus (RVFV) of the family Bunyaviridae and genus Phlebovirus is responsible for causing the disease in animals. The virus has a negative-sense single-stranded RNA genome (approximately 11.9 kb in size) and is composed of three segments designated as S (small, 1690 nt), M (medium, 3885 nt), and L (large, 6404 nt), packaged together in the virion (Elliott 1990, 1997; Robert 2004; Bird et al. 2007a). The enveloped virion is composed of a lipid bilayer
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containing the carboxy-terminal glycoprotein (Gc) and the amino-terminal glycoprotein (Gn) (Freiberg et al. 2008; Sherman et al. 2010; Bouloy and Weber 2010). The co-packaging of these RNAs to produce infectious RVFV has been suggested to be driven by specific intermolecular interactions among the three viral RNAs (Terasaki et al. 2010). Each segment is enclosed in a separate nucleocapsid within the virion (Garcia et al. 2001; Dighe et al. 2010). Segment S codes for two proteins (the nucleoprotein N and the nonstructural NSs protein) by using an ambisense strategy and L codes for the 237-kDa viral RNA-dependent RNA polymerase in a single 6.4-kb open reading frame (ORF) (Sall et al. 1998; Bouloy and Weber 2010). Segment M codes at least four viral proteins in a single ORF: the two major envelope surface glycoproteins, the Gc and Gn (named the 55-kDa Gn and 58-kDa Gc synthesized as part of polyprotein precursors), a 78-kDa fusion of the NSm, and the14-kDa protein NSm of unknown function (Accardi et al. 2001; Lagerqvist et al. 2009). Rift Valley fever is a potential threat to African countries where it is endemic in many countries (Thiongane et al. 1991; Robert 2004). Since its reemergence in Egypt in 1977, epidemics have occurred in Mauritania (1987 to 1998), Madagascar (1990/1991 and 2007/2008), Egypt (1993), and eastern Africa in Kenya, Somalia, and Tanzania (1977/1978 and 2006/2007), Yemen and Saudi Arabia (2000), and in South Africa (2007/2008 and 2010/2011) (Sall et al. 1999; CDC 2000; OIE 2010; Archer et al. 2011; FAO 2011). The seroprevalence of RVF in Kenya during the 2006–2007 outbreak was reported by LaBeaud et al. (2008) to be 13 %. A detailed molecular epidemiology during that outbreak (2006/2007) which affected also Tanzania was done in Kenya by only using reverse transcription polymerase chain reaction (RTPCR) (Munyua et al. 2010; Pepin et al. 2010). The disease has reappeared in Madagascar in 2008/2009, and analysis of partial sequences from RVFV strains showed to be similar to the strains circulating in Kenya during the 2006/2007 outbreak (Andriamandimby et al. 2010). An extensive genomic analyses carried out by Bird et al. (2007a, b) using samples from the 2006–2007 RVF outbreak in East Africa demonstrated the concurrent circulation of multiple virus lineages with a common ancestry from the 1997–1998 East African RVF outbreaks. The increase in genomic diversity, 2 to 4 years prior to the 2006/2007 outbreaks and the interepidemic seropositivity from recent studies indicates an ongoing RVFV activity and evolution during the interepidemic period (Sall et al. 1998; Sumaye et al. 2013; Turell et al. 1990). Diagnosis of RVFV is based on clinical signs, virus isolation, enzyme-linked immunosorbent assay (ELISA), and molecular techniques (RT-PCR and nucleic acid sequencing) (Garcia et al. 2001; Sall et al. 2002; FAO 2003). The latter is more sensitive and reproducible and, as such, provides a promising option for diagnosis and detection of viral RNA
of RVFV (Garcia et al. 2001; Le Roux et al. 2009). Since viral RNA and IgM detection by RT-PCR and ELISA, respectively, are more rapid than virus isolation, Sall et al. (2002) recommended using these techniques/assays in parallel as diagnostic methods for RVFV when there is an outbreak. During the outbreak in Tanzania, the diagnosis was based on serological methods in domestic animals and human serum, and tissues samples were transported to the Kenya Medical Research Institute/Centers for Disease Control and Prevention (KMRI/CDC) laboratory in Nairobi (Mohamed et al. 2010). In this laboratory, samples were tested for IgM and IgG antibodies using ELISA, and tissue samples were tested for viral RNA by using real-time RT-PCR. During the 2006/2007 RVF outbreak in Kenya and Tanzania, molecular epidemiology using RT-PCR was performed only on samples from domestic animals from Kenya (Pepin et al. 2010), while in Tanzania, molecular epidemiological studies were performed only in humans (Mohamed et al. 2010; Nderitu et al. 2011). Therefore, there is a lack of information on the circulating RVFV field strains in animals in Tanzania which could assist in vaccination strategies. Thus, the main aim of this study was to detect RVF antibodies and the virus in serum samples collected during the 2006/2007 outbreak in selected areas of Tanzania using RVF inhibition ELISA and RT-PCR. The information obtained in this study will lead to an improved understanding on RVFV and generate baseline data for further studies on the virus in Tanzania.
Materials and methods Samples This study used serum samples from cattle, goats, and sheep obtained from the Tanzania Veterinary Laboratory Agency (TVLA, BSL2) in Dar es Salaam which were collected during the 2006/2007 RVF outbreaks. Serum samples were collected by livestock field officers under the supervision of Veterinary Investigation Centres (VICs) and transported on ice to TVLA where they were stored between −50 and −35 °C. Regions where samples were collected included Mwanza, Arusha, Tabora, Dodoma, Tanga, Mbeya, Iringa, Pwani, Dar es Salaam, Lindi, and Mtwara. RVF inhibition ELISA Screening of serum samples for detection of IgG antibody levels against RVFV was done using a RVF-specific inhibition ELISA (I-ELISA) as described previously (Paweska et al. 2005). The optical density (OD) was measured at 405 nm. The specific activity of each serum (net OD) was calculated by subtracting the nonspecific background OD in the wells with control antigen from the specific OD in wells with virus
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antigen. The mean OD readings for replicate tests were converted to a percent inhibition (PI) value using the following equation: [100−(mean net OD of test sample/mean net OD of negative control)×100]. Cutoff values (expressed as a PI of an internal negative control) used were 41.9, 41.4, and 38.4 for cattle, goat, and sheep, respectively. In this test, 200 sera were analyzed for detection of IgG for RVF.
Results for samples from each test clustered by location where samples were collected are shown in Table 1. All positive samples by RVF I-ELISA were negative by RT-PCR, and 65.5 % (n=29) of negative samples by I-ELISA were positive by RT-PCR. The prevalence of RVF based on IELISA and RT-PCR as clustered by species is shown in Table 2.
Extraction of RVFV RNA Discussion Genomic viral RNA was directly extracted from 108 sera, 79 samples were positive from the RVF I-ELISA, while 29 negative samples were used. Basic RNA extraction was performed using the QIAamp® Viral RNA Mini Kit (QIAGEN, Germany) in accordance with the manufacturer’s instructions. The extracted RNA was then stored at −20 °C until its use. Reverse transcription polymerase chain reaction A one-step RT-PCR primers, first described by Garcia et al. (2001), were used for synthesis and amplification of DNA based on the one-step RT-PCR protocol (with modification) of Salim et al. (2010). A reaction mix was prepared in a single tube by adding 12.5 μl of 2× reaction mix (containing dNTPs and MgSO4), 1.0 μl of 25× reverse transcriptase-Taq polymerase mix, 2.0 μl of 30 nmol of each S segment genespecific oligonucleotide primer, and 1.7 μl of enhancer. A forward primer S432 (5′-ATG ATG ACA TTA GAA GG GA-3′) and a reverse primer NS3m (5′ATG CTG GGA AGT GAT GAG-3′) were used to amplify a 298-bp S RNA segment gene of RVFV. The total volume of the reaction was 20.0 μl by adding RNase-free water. Target RNA (5.0 μl) was added last to make a final volume of 25 μl. The cycling conditions were set at 37 °C for 30 min (a reverse transcription step), an initial denaturation of 10 min at 95 °C followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, extension at 72 °C for 30 s, a final extension of 10 min at 72 °C, and finally holding at 4 °C. Finally, PCR products were visualized and photographed after agarose gel electrophoresis. A live attenuated freezedried Rift Valley fever viral vaccine (RIFTVAX™, Nairobi, Kenya) and RNase-free water instead of template were used as positive and negative controls, respectively. Products were sized against a 100-bp molecular weight DNA marker (GelPilot 100-bp Ladder, QIAGEN).
Results In this study, RVF inhibition ELISA used a total of 106, 82, and 12 serum samples from cattle, goats, and sheep, respectively. On the other hand, RT-PCR used a total of 54, 36, and 18 serum samples from cattle, goats, and sheep, respectively.
Epidemics of RVF in Tanzania normally starts on the northern part (especially in Ngorongoro region) in the villages along the floor of the eastern arm of the Rift Valley (Chengula et al. 2013; Davies and Martin 2006). Documented reports from Tanzania indicate that the disease was reported for the first time in 1930, followed by periodic epidemics at an interval of 10–20 years: 1947, 1957, 1977, 1997, and the latest in 2007 (Sindato et al. 2012). These outbreaks were preceded by a season of heavy rainfall (Anyamba et al. 2010; Davies and Martin 2006; FAO EMPRES WATCH 2007). Despite all these outbreaks that have impacted greatly on domestic animals and humans, only one molecular study in humans, using real-time RT-PCR, has been documented (Mohamed et al. 2010). Also, two human samples (from Dodoma and Tanga in Tanzania) that were sent to Kenya for diagnostic purposes have recently been sequenced together with others from Kenya and Somalia (Nderitu et al. 2011). The latest disease outbreak of 2007 covered a wider area of Tanzania (11 regions of mainland) affecting domestic animals and humans (Sindato et al. 2012). Molecular detection of RVFV using RT-PCR is recommended due to it being rapid and effective especially during the early stages of the disease when antibodies are not detectable (van Vuren et al. 2007; Shoemaker et al. 2002). Recently, a realtime RT-PCR that can detect all the three segments (S, M, and Table 1 Proportion (percentage) of positive serum samples based on ELISA (N=200) and RT-PCR (N=108) tests clustered by location Region
Date of collection
ELISA, n (%)
RT-PCR, n (%)
Dodoma Tabora
29 Mar–4 Apr 2007 26–30 Mar 2007
33 (45.5) 53 (34.0)
20 (10.0) 34 (44.1)
Manyara Arusha Tanga Mwanza Mtwara Iringa Mbeya Lindi Pwani
21 Oct 2007 21 Oct 2007 9 May 2007 12 Apr 2007 12–18 Apr 2007 17 Sep 2007 18 Apr 2007 12 Apr 2007 22 Nov 2007
20 (50.0) 10 (30.0) 5 (20.0) 11 (9.1) 17 (58.8) 11 (36.4) 15 (33.3) 6 (50.0) 19 (47.4)
10 (0.0) 7 (0.00) 2 (50.0) 7 (0.0) 10 (0.0) 4 (0.0) 5 (0.0) 3 (33.3) 6 (0.0)
N total samples of all regions, n total samples of each region
Trop Anim Health Prod Table 2 Proportion of positive serum samples from different animal species tested by ELISA and RT-PCR methods Species
ELISA Total sample
RT-PCR Positive (%)
Total sample
Positive (%)
Cattle
106
38.7
53
20.8
Goat Sheep Overall
82 12 200
39.0 50.0 39.5
38 17 108
18.4 5.9 17.6
L) of the RVFV genome has been developed (Wilson et al. 2013) to facilitate rapid detection of the virus. In this study, RVF I-ELISA test detected 39.5 % (n=200) positive serum samples that were collected during the 2006– 2007. All serum samples (79) which were IgG-positive and 29 IgG-negative were further tested using conventional RT-PCR for which 17.6 % (n=108) of samples tested were found to be positive for RVFV genome. All RT-PCR positive samples that tested negative by RVF I-ELISA test represent acute infections, as IgG antibodies are detectable approximately 1 month or more, post infection (Bird et al. 2008; OIE 2008). Positive samples by RT-PCR were obtained from samples collected between March and May 2007, possibly because they were collected within the RVF outbreak period. Some areas (Manyara, Arusha, Iringa, and Pwani) where samples were collected after the outbreak (after June 2007) indicated the presence of IgG antibodies using I-ELISA, but there were no viral genomes detected. The antibodies may be due to the virus activity in the animals during the outbreak or interepidemic virus activity especially for samples collected 2 months later after the outbreak. Interepidemic virus activity in Tanzania has been reported in Mbeya from human blood samples (Heinrich et al. 2012). In livestock, the interepidemic transmission of RVF has been reported in areas experiencing annual flooding (Kilombero River Valley in Tanzania) by Sumaye et al. (2013). The interepidemic virus activity in livestock has also been reported in other countries (LaBeaud et al. 2008). Recently, an evidence of interepidemic transmission of RVF in African buffalo has been documented in South Africa (LaBeaud et al. 2011). The virus tends to circulate at low levels in domestic animals and humans during the interepidemic period in RVF endemic countries (FAO EMPRES WATCH 2007). Understanding the distribution of the potential vectors of RVFV has a biological and epidemiological significance in relation to disease outbreak hotspots. A model for predicting the distribution of RVFV in East Africa targeting Aedes aegypti and Culex pipiens complex, potential vectors for maintenance and amplification of the virus respectively, has been developed (Mweya et al. 2013). The model can also provide good information on suitable areas for collecting vectors of RVF during
interepidemic periods to determine the virus replication in the vectors. The diversity of RVFV strains isolated within an epidemic and in the endemic setting may not be the same, being low in the endemic setting (Bird et al. 2007b). This emphasizes on molecular monitoring of virus evolution for strategic control of RVF especially on vaccination of animals using the right vaccine candidate. Livestock, for a long time now, have been vaccinated using a modified live virus strain (Smithburn strain) which is said to be cost-effective commercial practice due to it being less expensive to produce (Davies 2010). Although the evolution rate of the virus is considered to be slow, the isolates from Tanzania (TAN/Tan-001/07 and TAN/Dod-002/07) during the 2006–2007 outbreak were found to be different from those circulated in Kenya (Nderitu et al. 2011). In addition, the isolates from the 2006–2007 outbreak were more distant from the older RVFV isolates, suggesting an ongoing slow evolution clock for the RVFV. Thus, molecular information of virus is a very important key for understanding the evolution of the virus. So far, the circulating virus strains in domestic animals in Tanzania are not yet known. There is a need of charactering the virus during epidemics and interepidemics in order to understand what is going on during the two periods for effective control strategies. It is important to use information obtained during molecular and serological surveillance together with global early warning forecasts issued by the United Nations Food and Agriculture Organization (FAO) to control RVF outbreaks. In the latest outbreak of East Africa (2006/2007), the FAO issued an early warning forecast predicting possible RVF outbreak for the Horn of Africa on November 2006 (Anyamba et al. 2010; Martin et al. 2007). However, there was no time frame for the countries to prepare themselves as the warning was issued in short time just before cases for both human and livestock to appear in the same November 2006 (FAO EMPRES WATCH 2012; Jost et al. 2010). Rift Valley fever remains to be threat to livestock keepers and non-livestock keepers and the nation at large due to its socioeconomic effects (Dar and McIntyre 2013; ElBahnasawy et al. 2013; Otte et al. 2004). In 2007, the disease led to disrupted livelihood and markets for livestock products that resulted from a ban of livestock slaughter and exportation. In Tanzania, during RVF outbreak, the average price of bulls for slaughter dropped by 33 %, the internal flow of marketed cattle decreased by 40 %, and at least 75 % of exports were not allowed to leave the country (FAO EMPRES WATCH 2012), since the virus can be found in different species from mosquitoes, animals (domestic and wildlife animals) to human. A collaborative control strategy involving different sectors applying the concept of “one health” will have a fast and great impact especially during epidemics. Lack of coordination and intersectorial collaboration during the control of 2006–2007 RVF outbreak in Tanzania has been reported as one of the challenges for controlling the disease (Chengula et al. 2013).
Trop Anim Health Prod
Using the I-ELISA and RT-PCR, this study has found that there were IgG antibodies and viral genomes of RVFV in the sera collected from livestock during the 2006–2007 outbreak. Based on our knowledge, this is the first documentation on the detection of RVFV in serum samples using RT-PCR techniques. A serological surveillance to monitor virus activities in animals and humans is important, together with virus activities in mosquitoes transmitting the disease, and the animals (wildlife and domestic animals). The information will also be useful during planning the control strategies of RVF by the responsible organs. Since RVFV strains circulating in Tanzania are not known, the study suggests carrying out detailed molecular studies to characterize the viruses circulating in the country. Acknowledgments The authors highly acknowledge the Regional Universities Forum (RUFORUM) who funded the project, the Director of TVLA, and all members of the virology section at TVLA, Dar es Salaam. Conflict of interest The authors do declare that they have no conflict of interest.
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