Rift Valley fever (RVF) is a zoonotic disease that is characterized by periodic and severe outbreaks in humans and animals. Published information on the occurrence of RVF in domestic animals is very scarce in the Democratic Republic of the Congo (DRC). To assess possible circulation of Rift Valley fever virus (RVFV) in cattle in the eastern province of DRC, 450 sera collected from cattle in North Kivu, South Kivu, and Ituri provinces were analyzed using the enzyme-linked immunosorbent assay (ELISA), for the detection of viral Immunoglobulin (Ig) G and M, and reverse transcriptase polymerase chain reaction (RT-PCR), for detection of viral RVF RNA. A cumulative anti-RVF IgG prevalence of 6.22% (95% CI 4.25–8.97) was recorded from the three provinces sampled. In North Kivu and Ituri provinces the anti-RVF IgG prevalence was 12.67% [95% CI 7.80–19.07] and 6% [95% CI 2.78–11.08], respectively, while all the sera collected from South Kivu province were negative for anti-RVF IgG antibodies. Anti-RVF IgM prevalence of 1.8% was obtained among sampled animals in the three provinces. None of the positive anti-RVF IgM samples (n=8) was positive for viral RVFV RNA using RT-PCR. Our findings suggest that RVFV is widely distributed among cattle in eastern province of DRC particularly in North Kivu and Ituri provinces although the epidemiological factors supporting this virus circulation remain unknown in these areas.
Rift Valley fever virus (RVFV) is a vector-borne single stranded zoonotic virus that infects humans and a wide range of vertebrate hosts including cattle, small ruminants, camels, and wildlife species. RVFV belongs to the family Bunyaviridae, genus
RVFV is mainly transmitted among livestock through mosquito bites although vertical transmission between animals is also reported [
During enzootic periods when there is normal rainfall in East Africa, it is assumed that RVFV is maintained through vertical transovarial transmission of floodwater
Several sub-Saharan African tropical and subtropical countries have reported outbreaks of RVFV [
This study was conducted in three provinces (North Kivu, South Kivu, and Ituri) in unvaccinated cattle herds (Figure
South Kivu province is located at 3° 01′ south 28°16′ east, close to Rwanda and Burundi, and its eastern border corresponds to the western Rift Valley. The province is subdivided into 8 territories (Fizi, Idjwi, Kabare, Kalehe, Mwenga, Shabinda, Uvira, and Walungu) and samples were collected from five of these territories (Fizi, Kabare, Mwenga, Shabinda, and Uvira). The mountainous eastern part contrasts with the central and western parts of the province which have high and low plateaus, respectively. The eastern part of province (mountainous Kivu) has a mountain climate characterized by mild temperatures with a dry season lasting for 3-4 months, from June to September. The central and western parts of South Kivu province, particularly Shabunda, Mwenga, and Fizi territories, are a dense rainforest zone with an equatorial humid climate. The Ruzizi lowland is associated with a microclimate comprising a tropical climate and dry trend with low rainfall (± 1,000 mm/year). The average annual rainfall is 1,800 mm, with wide yearly fluctuations. South Kivu province has an altitude ranging from 1,800 to 3,300m. The forest is being cleared for agriculture and firewood around villages and cattle grazing on the high plateaus (e.g., Itombwe Mountains). The vegetation of this area is mostly grassland that intergrades with lowland
Ituri province (one of the four districts of Orientale province in the past) is located at 01°50′ north 29°30′ east, northeast of the Ituri River and on the western side of Lake Albert. It borders Uganda and South Sudan. The province is subdivided into 5 districts (Aru, Mahagi, Djungu, Irumi, and Mambasa) and samples were collected from tree of these territories (Aru, Irumi, and Mahagi). Ituri province is on a high plateau ranged between 1,000 and 2,000m. It has a large tropical forest as well as a savanna landscape. The Ituri forest geographic boundaries are difficult to define as the forest blends in with other forests and swamp regions. The north is limited by savanna, the east by the western Rift Valley, and the south west by a lowland rain forest. The dry season lasts from December to February. The heaviest rains occur in October and early November with significant flooding. Abundant rainfall is recorded in the southwest of the province where it can reach a maximum of 2,000mm. The average temperatures range between 19 and 31°C.
The sample size was determined according to the method described by Martin et al. [
Cattle in this region are not regularly vaccinated against RVF. With farmers’ consent, 10% of cattle from each herd were randomly selected during the rainy season from November to December 2013, from which 450 blood samples were collected (150 in North Kivu, 150 in Ituri, and 150 in South Kivu). Sampled cattle were classified according to age: 1–3 years and 3–7 years. Blood samples were collected in dry vacutainer tubes from the jugular vein. Each vacutainer tube was labeled and individual information of each animal was recorded. Animals were bled from jugular vein.
The collected blood samples were kept overnight at room temperature in cool box at 4°C to allow blood clotting. On the next day, they were centrifuged at 3,000g for 10 minutes to sediment the erythrocytes. Sera were transferred into new vials and labeled before being stored at -20°C until tested.
Serological assays of all samples (n=450) were carried out using anti-RVF nucleoprotein (NP) IgG antibodies using ID screen® RVF competition multispecies ELISA (ID-Vet Innovative Diagnostics, Montpellier, France) according to the manufacturer’s instructions. After adding the stop solution, the optical density (OD) was read at room temperature using an ELISA reader (Thermo Electron Corporation, Multiskan EX®, Shanghai, China) at 450nm.
For the validity of each plate, the mean value of the two negative controls (ODNC) was calculated and the plate was considered valid when ODNC > 0.7. For a valid plate, the mean value of the two positive controls divided by ODNC was < 0.3. For each sample the competition percentage (S/N%) was calculated by dividing (OD sample/ODNC) x 100. Sample was considered positive if the value was equal to or less than 40%. A value greater than 50% was a considered as negative and values between 40 and 50% indicated a doubtful result. In this study, all doubtful samples were considered negative.
To detect recent infection (IgM), all samples were tested using ID screen RVF IgM ELISA (ID-Vet Innovative Diagnostics, Grabels, France), as per manufacturer’s instructions. The test was valid when the mean corrected value of the positive control OD (ODNC) was greater than 0.35 and the ratio of the mean corrected values of the positive and negative controls (ODNC and ODPC) was greater than 3. The sample was considered positive when the percentage of positivity was greater than or equal to 50%, doubtful when between 40% and 50%, and negative when less than or equal to 40%. All doubtful samples were considered as negative in this study.
The QIAamp UltraSens Virus Kit provided by QIAGEN® is designed for rapid, highly sensitive, and efficient recovery of viral RNA and DNA from plasma or serum. QIAamp UltraSens was used for the detection of viral nucleic acids in positive IgM serum following the manufacturer’s instructions. The SuperScript™ One-Step reverse transcriptase polymerase chain reaction (RT-PCR) with Platinum®
PCR amplification was completed using an Gen Amp® machine (PCR System 9700, Singapore) as follows: 50°C for 30 minutes: 1 cycle; 95°C for 10 minutes: 1 cycle; 95°C for 15 seconds, 50°C for 30 seconds, 72°C for 45 seconds: 40 cycles; 72°C for 5 minutes: 1 cycle. 10
Data from the competition and Capture ELISA were analyzed using Statistical Package for Social Sciences (SPSS) version 15.0 and Excel (MS Excel 2007) to calculate the percentage of RVF antibodies. Herds, age, and territory were used as explanatory factors. Confidence intervals of the proportions of detected antibodies were calculated at 95% confidence and a 5% level of significance was used. Chi-square (
A total of 450 sera samples were analyzed, among which 28 were positive for RVFV-IgG antibodies. This finding represents an anti-RVF IgG antibodies prevalence of 6.22% (95% CI 4.25-8.97) as shown in Table
Anti-RVF IgG and IgM prevalence in territories of Eastern regions in the DRC.
Provinces | Territories | Total tested | IgG | IgM | |
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Prevalence (n) | [95% CI] | Prevalence (n) | |||
Beni | 35 | 14.28 (5) | (4.81-30.26) | 0.0 (0) | |
Lubero | 41 | 12.28 (5) | (4.08-26.20) | 0.0 (0) | |
North Kivu | Butembo | 21 | 4.71(1) | (0.12-23.82) | 4.8 (1) |
Masisi | 38 | 15.79 (6) | (6.02-31.25) | 0.0 (0) | |
Rutshuru | 15 | 13.3 (2) | (1.66-40.46) | 13.3 (2) | |
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Sub-total | 150 | 12.67 (19) | (7.8-19.07) | 2 (3) | |
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Aru | 50 | 16.0 (8) | (7.17-29.11) | 2.0 (1) | |
Ituri | Irumi | 50 | 0.0 (0) | (0.0-7.11) | 0.0 (0) |
Mahagi | 50 | 2.0 (1) | (0.05-10.65) | 2.0 (1) | |
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Sub-total | 150 | 6 (9) | (2.78-11.08) | 1.3 (2) | |
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Fizi | 13 | 0.0 (0) | (0.0-24.71) | 0.0 (0) | |
Kabare | 37 | 0.0 (0) | (0.0-9.49) | 2.7 (1) | |
South-Kivu | Mwenga | 39 | 0.0 (0) | (0.0-9.03) | 2.6 (1) |
Shabunda | 28 | 0.0 (0) | (0.0-12.34) | 7.1 (1) | |
Uvira | 33 | 0.0 (0) | (0.0-10.58) | 0.0 (0) | |
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Sub-total | 150 | 0 | - | 2 (3) | |
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TOTAL | 450 | 6.2 (28) | (4.25-8.97) | 1.8 (8) |
Anti-RVF IgG seroprevalence according to group ages of cattle in the Eastern region in DRC using IgG ELISA test.
Age | Total tested | Total Positive | Prevalence (%) |
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1-3 years | 226 | 16 | 7.08 | 0.572 | 0.0449 |
>3-7 years | 224 | 12 | 5.36 |
Anti-RVF IgG seroprevalence according to gender of cattle in the Eastern region in DRC using IgG ELISA test.
Age | Total tested | Total Positive | Prevalence (%) |
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Male | 60 | 1 | 1.67 | 1.6438 | 0.089 |
Female | 390 | 27 | 6.92 |
All samples collected were screened for IgM ELISA. The results showed that 1.8% (8/450) of cattle had anti-RVF IgM antibodies. In North and South Kivu provinces 2.0% (3/150) of cattle had anti-IgM antibodies while, respectively, compared to 1.3% (2/150) that was obtained in Ituri province. No statistical difference (
This study was designed to investigate the circulation of RVF in the Occidental border of Rift Valley. In DRC this area comprises three provinces: Ituri, North Kivu, and South Kivu. In total 450 head of cattle were screened for the presence of anti-RVF IgG antibodies and anti-RVF IgM antibodies, and positive anti-RVF IgM samples were analyzed for the presence of RVFV RNA using RT-PCR technique. Anti-RVF IgG ELISA technique was used to assess the level of exposition of cattle to RVFV in the region since antibodies last for a long period of time while anti-RVF IgM and RT-PCR techniques served for the detection of recent infections.
Results of this study revealed that 6.22% of cattle were positive for anti-RVF IgG in the three provinces. In North Kivu, the prevalence was 12.66% while 6.0% of cattle had anti-RVF IgG in Ituri province. None of the cattle sampled in South Kivu were found with anti-RVF IgG. Higher proportions of anti-IgG-positive sera were observed in Masisi (North Kivu) and Aru (Ituri) territories. Apart from villages, no antibody was found; the prevalence in the rest of the villages ranged from 2% to 16%. The presence of IgG-positive cattle in most of these villages is an indication of a previous contact between these animals and RVFV. Since young animals aged less than three years old were also found and in a larger proportion than adults, with IgG antibodies it can be assumed that the contact with the virus occurred in situ. This can be supported by the circulation of anti-RVF IgM antibodies in the area whereby 1.8% of cattle were found with such antibodies in the three provinces.
In these areas no history of RVF outbreak has so far been documented, so the presence of RVF IgM antibodies in the absence of clinical signs may indicate silent circulation of the virus in the region. Indeed silent circulation of RVFV has previously been described elsewhere in sub-Saharan Africa and suggests that, in some locations, the virus may circulate for decades in the absence of reported outbreaks or identification of clinical cases in humans or animals. In previous reports, virus circulation occurring among apparently healthy humans and animals has been noticed in Somalia [
The results obtained in this study showed that North Kivu and South Kivu had anti-RVF IgM antibodies prevalence of 2.0%, respectively, while Ituri province had 1.3% of prevalence. Anti-RVF IgM antibodies were detected in Butembo and Rutshuru in North Kivu, Aru, and Mahagi in Ituri province where some animals were IgG-positive. However, anti-RVF IgM antibodies positive cattle were also found in Shabunda and Kabare in South Kivu but in absence of anti-RVF IgG antibodies. Since IgG antibodies last longer than IgM antibodies [
Whether the distribution of RVF differs according to age and gender was also assessed in the current study. In previous reports, a correlation between seropositivity and age has been noted in livestock [
To the best of our knowledge, no RVF vaccination campaign has so far been conducted in the provinces where these animals were sampled. As a result all IgG-positive animals were originated from unvaccinated herds suggesting the occurrence of natural exposure. The distribution of RVFV in the study area highlighted spatial variations whereby higher prevalence was found in North Kivu compared to the Ituri and South Kivu provinces. Varying prevalence of RVF was previously reported in other African countries such as 9.35% recorded in Cameroon [
In conclusion, this preliminary study illustrates the increase circulation and spread of RVFV in eastern provinces of DRC. For a better understanding of RVF epidemiology in this part of the country, more studies need to be conducted including monitoring and investigation of the vector dynamics as well as livestock movements within eastern DRC and across the borders shared with RVF endemic countries.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest.
The authors wish to express their gratitude to Welcome Trust through Southern African Centre for Infectious Disease Surveillance (SACIDS) for their financial support of this project. This work is a part of a Ph.D. supported by the Wellcome Trust Grant WT087546MA to the Southern African Centre for Infectious Disease Surveillance (SACIDS).