Serological Evidence for the Circulation of Rift Valley Fever Virus in Domestic Small Ruminants in Southern Gabon

Introduction

The rift valley fever virus (RVFV), a member of the Bunyaviridae family, genus Phlebovirus, is the causative agent of Rift Valley fever (RVF), a zoonotic disease mainly affecting sheep, goats, cattle, camels, water buffaloes, and humans. RVF is characterized by a high mortality rate (90–100%) in young domesticated animals (lambs, puppies, kittens). In pregnant sheep, the mortality and abortion rates vary from 5% to almost 100% in different outbreaks and between different flocks. The death rate in cattle is usually <10%. In humans, many epidemics have been reported in North African (Egypt) (Abd el-Rahim et al. 1999), East African (Kenya, Somalia, Sudan, and Tanzania) (Himeidan et al. 2014), West African (Mauritania, Senegal, and Niger) (Faye et al. 2014, Sow et al. 2014, WHO 2016), South African (South Africa) (Pienaar and Thompson 2013) countries, and in Madagascar (Andriamandimby et al. 2010) and the Arabian Peninsula (Al-Afaleq et al. 2011). In Chad, an outbreak occurred in human populations (Ringot et al. 2004), whereas in other countries of Central Africa, no RVF outbreaks have been reported to date. However, seroprevalence studies have shown RVFV circulation at very low levels in humans and animals in the Central African Republic and in humans in Cameroon and Gabon (Gonzalez et al. 1989, Pourrut et al. 2010).

Indeed, in Gabon, RVFV-specific antibodies have been episodically found in humans. In 2010, evidence of the circulation of RVFV in several villages of Nyanga province, located in southwest Gabon, was provided by the detection of RVFV-specific IgG in human rural populations at a level ranging from 0% to 13% (Pourrut et al. 2010). Moreover, for many years, several cases of abortions and stillbirths were recorded in traditional farms rearing small ruminants in the Mongo County in Nyanga province.

The aim of this study was to search for RVFV-specific antibodies and RNA in populations of sheep and goats from the Mongo County to determine their RVF seroprevalence and the circulation of the RVFV in this area.

Materials and Methods

A cross-sectional study was conducted from June to September 2014, during the dry season, in nine localities in the Mongo County (Fig. 1) (latitude: 3°26′S; longitude: 11°44′E) located about 65 km from Tchibanga (the main town of Nyanga province, southwest Gabon), as previously described (Maganga et al. 2016).

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FIG. 1.

Geographical location of the study area. Opened circles represent the nine selected villages of Mongo County. Stars show the sites in which IgG against RVFV-positive serum samples were detected. RVFV, Rift Valley fever virus.

No census of livestock exists for this region; the animals were selected on the willingness of the livestock keepers to cooperate in the study. Thus, the sample size depended on the livestock keepers' availability and their ability to hold their animals. Animal sampling, blood, and data collection were conducted as previously described by Maganga et al. (2016). The age of animals was determined through dentition according to Vatta et al. (2007). Animals were classified as young (under 3 years old) and adult, that is to say, mature sheep and goats aged >3 years.

The serum samples of sheep and goats were tested for the presence of IgG antibodies specific to the RVFV using the inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against RVF in humans, domestic, and wildlife ruminants (Biological Diagnostic Supplies Limited, United Kingdom), as previously described (Sumaye et al. 2013). The results were considered positive if the cutoff values of percent inhibition (PI) were, respectively, ≥41.4 and 38.4 in goats and sheep according to the manufacturer's instructions.

Total RNAs were then extracted from plasma. RNA extraction was performed using the QIAamp viral mini kit (QIAGEN) following the manufacturer's instructions.

Synthesis of complementary DNA (cDNA) was performed using the High-Capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions, in a total volume of 50 μL for 10 min at 25°C, followed by 60 min at 37°C. The search for the RVFV was performed using a real-time PCR targeting the G2 gene in an M RNA segment (Drosten et al. 2002) and a nested one-step RT-PCR using phlebovirus universal primers targeting partial segments of the polymerase gene (Sanchez-Seco et al. 2003).

The risk of seropositivity was assessed by logistic regression as previously described (Maganga et al. 2016).

For this study, permissions were obtained from the local administrative authorities (Prefect and heads of the villages). All the keepers of the small ruminants used for blood sampling gave their oral consent.

Results

A total of 201 small ruminants were sampled across nine sites, including 106 goats (28 males and 78 females, 47 young and 59 adults) and 95 sheep (17 males and 78 females, 28 young and 67 adults). In all, 38% (8/21) of the females in goats and 62% of the females in sheep (13/21) had problems of abortions and/or stillbirths. These sheep and goats for slaughter/meat were bred in free range.

The overall IgG seroprevalence against the RVFV in the small ruminant population was 6.47% (13/201). Sheep had the highest IgG rate (8.42%) compared with goats (4.72%). IgG antibodies were detected only in females. Among them, 13 in 156 (8.33%) showed IgG antibodies specific to the RVFV, with 10.25% (8/78) in sheep and 6.41% (5/78) in goats. The adults had a much higher seroprevalence (9.52%) than the young (1.33%) (p = 0.026). The RVFV IgG rate was 8.5% (5/59) and 10.5% (7/67) in adult goats and sheep, respectively, and 0% (0/47) and 3.5% (1/28), respectively, in young goats and sheep. IgG antibodies against the RVFV were found in 1 of the 21 animals that had problems of abortions and/or stillbirths. No RVFV RNA was detected in any of the 201 animal plasmas.

The overall RVFV IgG rates ranged from 0% to 18.5%. IgG antibodies specific to RVFV were detected in animals from five sites: PC Nyanga (3.2%); Dilemba (3%); Bibora (13.16%); Bayadi (4.17%); and Moulengui-Binza (18.5%) (Table 1 and Fig. 1). There was no significant difference in prevalence between sheep and goats and between the sampling sites (data not shown). Univariate and multivariate logistic regression analyses showed that age, species, sex, and locality were not significant risk factors (data not shown).

Discussion

In this study, we report for the first time the presence of IgG antibodies against RVFV in animals from the Mongo County in Gabon. A serological prevalence of 6.47% was found in the small ruminant population of this rural area. However, the circulation of other phleboviruses in Gabon cannot be excluded, so additional test such as the virus neutralization assay could be performed to confirm the specificity of the C-ELISA used.

Nanyingi et al. (2016) reported higher seroprevalence in sheep and goats in Kenya than that found in this study, respectively, 32.2% and 25.8%, also by using a commercial indirect competitive enzyme-linked immunosorbent assay. Using the same type of assay as well, Sumaye et al. (2013) found seroprevalence of 11.86% and 11.37%, respectively, in goats and sheep during a cross-sectional survey in Tanzania. The variations in seroprevalence reported in the different studies are described to be influenced by the age of the animals, the season and timing of sampling (during a disease outbreak or an interepizootic period), the diagnostic test used, breeding practices, including the mixing of animals, virus maintenance, and the persistence of ecological factors.

The serological prevalence observed in sheep (8.42%) and goats (4.72%) of the Mongo County is related to those observed in some endemic countries, such as Senegal, Mauritania, and Burkina Faso, during interepizootic periods (2% to 10%) (Lernout et al. 2013), and Tanzania (Sumaye et al. 2013).

The significantly higher IgG anti-RVFV seroprevalence in adults could suggest a more ancient circulation of the virus and therefore the likely absence of a recent circulation of the RVFV in this region. Indeed, Jeanmaire et al. (2011) and Nanyingi et al. (2016) showed that prevalence increased with the age of the animal.

The sampling period could have led to an underestimation of the RVFV IgG seroprevalence in the Mongo Country and could also explain the lack of detection of the RVFV. Indeed, RVFV is transmitted by blood-sucking mosquitoes whose development depends on the presence of ponds. Pourrut et al. (2010) showed a high serological prevalence of the RVFV in the region of lakes, suggesting that the season and the type of ecosystem are important factors in the development of vectors of RVF.

The findings of this study highlight the risk of RVF for domestic ruminants bred in this region and for the human rural population living in contact with these animals.

This study emphasizes the need to develop adequate control measures to limit this threat, such as (1) awareness campaigns for livestock keepers on the risk of RVFV infection through the manipulation of aborted fetuses and (2) further virological and serological surveys of the RVFV during rainy season and in wildlife (bats and wild ruminants), hematophagous arthropods, other domestic ruminants such as cattle, and in humans to detect and characterize the RVFV in the region and provide information on the strain(s) circulating in the country.