The Re-Emergence of Rift Valley Fever in Mananjary District, Madagascar in 2021: A Call for Action.

An epizootic of rift valley fever (RVF) was suspected on 21 February 2021 in various districts of Madagascar, with a lab confirmation on 1 April 2021. A cross-sectional survey aiming to detect cases of RVF in humans and to study the circulation of rift valley fever virus (RVFV) in animals was conducted from 22 April to 4 May 2021 in the district of Mananjary. Blood samples from cattle and humans were tested using serological and molecular techniques. In cattle, the circulation of RVFV was confirmed between 5 February and 4 May 2021. The positivity rates of anti-RVFV IgG and IgM were 60% and 40%, respectively. In humans, the circulation of RVFV was observed from 1 April to 5 May 2021. The positivity rate of RVFV was estimated to be 11.7% by combining the results of the molecular and serological approaches. Of the 103 individuals who agreed to participate in the survey, 3 were determined to be positive by RT-PCR, and 10 had anti-RVFV IgM. Among them, one was positive for both. Given that previous studies have reported the circulation of RVFV during inter-epidemic periods and the occurrence of outbreaks due to imported RVFV in Madagascar, our findings suggest the importance of strengthening RVF surveillance from a "One Health" perspective by conducting syndromic and risk-based surveillance at the national and regional levels.

Comprehensive Characterization of Viral Diversity of Female Mosquitoes in Madagascar.

The diversity and circulation of arboviruses are not much studied in Madagascar. The fact is that arboviral emergences are rarely detected. The existing surveillance system primarily relies on serological detection and records only a few human infections annually. The city of Mahajanga, however, experienced a confirmed dengue fever epidemic in 2020 and 2021. This study aimed to characterize and analyze the virome of mosquitoes collected in Mahajanga, near patients with dengue-like syndromes to detect known and unknown viruses as well as investigate the factors contributing to the relative low circulation of arboviruses in the area. A total of 4280 mosquitoes representing at least 12 species from the Aedes, Anopheles, and Culex genera were collected during the dry and the rainy seasons from three sites, following an urbanization gradient. The virome analysis of 2192 female mosquitoes identified a diverse range of viral families and genera and revealed different patterns that are signatures of the influence of the mosquito genus or the season of collection on the composition and abundance of the virome. Despite the absence of known human or veterinary arboviruses, the identification and characterization of viral families, genera, and species in the mosquito virome contribute to our understanding of viral ecology and diversity within mosquito populations in Madagascar. This study serves as a foundation for ongoing surveillance efforts and provides a basis for the development of preventive strategies against various mosquito-borne viral diseases, including known arboviruses.

Seroprevalence of IgG Antibodies Directed against Dengue, Chikungunya and West Nile Viruses and Associated Risk Factors in Madagascar, 2011 to 2013.

Arboviruses have been shown to circulate in Madagascar, including West Nile, dengue, and chikungunya viruses, though the extent of their circulation remains poorly documented. We estimated the seroprevalence of these three arboviruses in Madagascar and determined risk factors associated with seropositivity. Serum samples obtained from 1680 individuals surrounding the Sentinel Health Centers network in all regions of the country were analyzed using ELISA and hemagglutination inhibition assays for dengue, chikungunya, and West Nile viruses IgG antibodies, and multivariate logistic regression models were run. Overall, 6.5% [IC 95% 3.2-9.9] were seropositive for dengue virus, predominantly of Dengue serotype 1, 13.7% [IC 95% 6.5-20.9] for chikungunya virus, and 12.7% [IC 95% 9.0-16.5] for West Nile virus. There was no association with age, showing that dengue and chikungunya viruses were likely recently introduced. Eastern and Northern parts were more affected by dengue and chikungunya viruses, while West Nile virus seemed to circulate in all parts of the country. Dengue and chikungunya seropositivity were notably associated with high levels of vegetation, as well as frequent work in the forest, and West Nile seropositivity with the presence of cultivated areas, as well as standard of living. This analysis gives a new insight into arboviruses circulation and transmission patterns in Madagascar.

Discrimination of Uranotaenia species (Diptera: Culicidae) from Madagascar based on morphology and wing morphometric traits.

The genus Uranotaenia (Diptera: Culicidae) has been well documented in Madagascar where it includes 73 species, 89.4% being endemic. However, one problem is that most species are morphologically similar in the adult stage. Here, 713 Uranotaenia specimens collected in the tropical forests of Anorana and Maromizaha between 2008 and 2014 were examined. Using the dichotomous keys for the Uranotaenia fauna of Madagascar published in 2004, three species were identified: Uranotaenia neireti (220), Ur. alboabdominalis (110) and Ur. mayottensis (28). The other specimens (355) were not identifiable and were classified as Uranotaenia sp1. Using wing morphometry, the four taxa were classified into four morphogroups. Within the Uranotaenia sp1 group, specimens from the Anorana forest and those from the Maromizaha forest overlapped. This result suggests that wing morphometric traits could be a good marker to distinguish Uranotaenia species in Madagascar.

Avian malaria on Madagascar: bird hosts and putative vector mosquitoes of different Plasmodium lineages.

BACKGROUND: Avian malaria occurs almost worldwide and is caused by Haemosporida parasites (Plasmodium, Haemoproteus and Leucocytozoon). Vectors such as mosquitoes, hippoboscid flies or biting midges are required for the transmission of these parasites. There are few studies about avian malaria parasites on Madagascar but none about suitable vectors. METHODS: To identify vectors of avian Plasmodium parasites on Madagascar, we examined head, thorax and abdomen of 418 mosquitoes from at least 18 species using a nested PCR method to amplify a 524 bp fragment of the haemosporidian mitochondrial cytochrome b gene. Sequences obtained were then compared with a large dataset of haemosporidian sequences detected in 45 different bird species (n = 686) from the same area in the Maromizaha rainforest. RESULTS: Twenty-one mosquitoes tested positive for avian malaria parasites. Haemoproteus DNA was found in nine mosquitoes (2.15%) while Plasmodium DNA was found in 12 mosquitoes (2.87%). Seven distinct lineages were identified among the Plasmodium DNA samples. Some lineages were also found in the examined bird samples: Plasmodium sp. WA46 (EU810628.1) in the Madagascar bulbul, Plasmodium sp. mosquito 132 (AB308050.1) in 15 bird species belonging to eight families, Plasmodium sp. PV12 (GQ150194.1) in eleven bird species belonging to eight families and Plasmodium sp. P31 (DQ839060.1) was found in three weaver bird species. CONCLUSION: This study provides the first insight into avian malaria transmission in the Maromizaha rainforest in eastern Madagascar. Five Haemoproteus lineages and seven Plasmodium lineages were detected in the examined mosquitoes. Complete life-cycles for the specialist lineages WA46 and P31 and for the generalist lineages mosquito132 and PV12 of Plasmodium are proposed. In addition, we have identified for the first time Anopheles mascarensis and Uranotaenia spp. as vectors for avian malaria and offer the first description of vector mosquitoes for avian malaria in Madagascar.

Review of West Nile virus circulation and outbreak risk in Madagascar: Entomological and ornithological perspectives.

West Nile fever (WNF) is a zoonotic disease, occurring nearly globally. In Madagascar, West Nile virus (WNV) was first detected in 1978 from wild birds and the virus is currently distributed across the island, but no epidemic or epizootic period has been recorded. One fatal human case of WNV infection was reported in 2011, suggesting a "tip of the iceberg" phenomenon of a possible WNF epidemic/epizootic on the island. The main objective of this literature-based survey is to review patterns of WNV circulation in Madagascar from the entomological and ornithological points of view. Among the 235 mosquito species described from Madagascar, 29 species are widely associated with WNV infection; 16 of them are found naturally infected with WNV on the island and categorized into major, candidate, and potential vectors of WNV according to their vector capacity. This study upholds the hypothesis that WNV enzooticity is independent of annual movements of migratory birds passing through Madagascar. Moreover, the lack of regular migratory bird flux between Africa and Madagascar would reduce the probability of transmission and the subsequent reintroduction of the virus into locally occurring mosquito species. Given that Palearctic migratory birds are strongly implicated in the transmission of WNV, we highlight notable differences in the movements and species diversity of these birds in Madagascar as compared to eastern and northern Africa. Risk factors from this two-pronged approach are presented for the emergence of WNF outbreak.

An updated checklist of mosquito species (Diptera: Culicidae) from Madagascar.

An updated checklist of 235 mosquito species from Madagascar is presented. The number of species has increased considerably compared to previous checklists, particularly the last published in 2003 (178 species). This annotated checklist provides concise information on endemism, taxonomic position, developmental stages, larval habitats, distribution, behavior, and vector-borne diseases potentially transmitted. The 235 species belong to 14 genera: Aedeomyia (3 species), Aedes (35 species), Anopheles (26 species), Coquillettidia (3 species), Culex (at least 50 species), Eretmapodites (4 species), Ficalbia (2 species), Hodgesia (at least one species), Lutzia (one species), Mansonia (2 species), Mimomyia (22 species), Orthopodomyia (8 species), Toxorhynchites (6 species), and Uranotaenia (73 species). Due to non-deciphered species complexes, several species remain undescribed. The main remarkable characteristic of Malagasy mosquito fauna is the high biodiversity with 138 endemic species (59%). Presence and abundance of species, and their association, in a given location could be a bio-indicator of environmental particularities such as urban, rural, forested, deforested, and mountainous habitats. Finally, taking into account that Malagasy culicidian fauna includes 64 species (27%) with a known medical or veterinary interest in the world, knowledge of their biology and host preference summarized in this paper improves understanding of their involvement in pathogen transmission in Madagascar.

High Prevalence of West Nile Virus in Domestic Birds and Detection in 2 New Mosquito Species in Madagascar.

West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.

Detection in and circulation of Bluetongue virus among domestic ruminants in Madagascar.

So far, no published data was available concerning the circulation of Bluetongue virus (BTV) in Madagascar. During a survey on Rift Valley Fever, we were able to detect a virus belonging to BTV. Therefore, we conducted a study aiming at characterizing molecularly the BTV isolated and assess the importance of circulation of BTV in Madagascar. A total of 4393 sera from ruminants selected randomly by stratification and sampled in 30 districts of Madagascar were tested for BTV. Moreover, 175 cattle were followed during 11 months. Phylogenetic analyses were performed from virus isolated from unfed pools of mosquitoes. Overall, the estimated mean seroprevalence of infection at the national level was 95.9% (95% CI: [95.2-96.5]) in cattle and 83.7% (95% CI: [81.4-85.9]) in small ruminants. Estimation of incidence rate was 54 per 100 cattle-years assuming that the incidence rate is constant all year along. Phylogenetic analyses revealed that BTV detected belong to serotype 2. In conclusion, our results showed that BTV is endemic in Madagascar and highly prevalent among cattle. In our study we did not work on the vector involved in transmission of BTV in cattle. Thus, research should be conducted to better describe epidemiology of BTV in Madagascar including vectors and assess economic impact of the disease associated to BTV infections.

Biology of mosquitoes that are potential vectors of Rift Valley Fever virus in different biotopes of the central highlands of Madagascar.

There were epidemic-epizootics of Rift Valley Fever (RVF) affecting humans and cattle in Madagascar in the district of Anjozorobe in 2008. Little is known about the role of Malagasy mosquitoes in the circulation of RVF virus. Therefore, we investigated the species diversity, dynamics and biology of potential RVF virus vectors in the rainforest, rainforest edge (village of Anorana), and savanna biotope (village of Antanifotsy) of this district between November 2008 and July 2010. We captured 56,605 adults of 35 different species. Anopheles squamosus (Theobald), Anopheles coustani (Laveran), Culex antennatus (Becker), Culex pipiens (L.), and Culex univittatus (Theobald) were the most abundant during the rainy season with Cx. pipiens the most abundant species in the rainforest (47%), and An. squamosus the most abundant species in the rainforest edge and in the savanna biotope (56%, 60%, respectively). Only Cx. univittatus was abundant in the dry season. The parous rate was > 60% throughout the rainy season for An. squamosus and it was > 50% from the middle to the end of the rainy season for Cx. pipiens. Two additional species have been found only at larval stage. Cattle were the most attractive bait for all species, followed by sheep and poultry. Human was the least attractive for all species. Most of the 163 bloodmeals tested were taken from cattle. Three were from poultry, one was from dog and one was a mixed bloodmeal taken from sheep and cattle. These results on vectorial capacity parameters may allow considering the involvement of mosquito transmission of the virus in the district of Anjozorobe during the recent epidemic-epizootic.

Modification of Anopheles gambiae distribution at high altitudes in Madagascar.

In Madagascar, Anopheles gambiae has been found below altitudes of 1,000 m. We sampled An. gambiae sensu lato (sl) between 2008 and 2010 in the Central Highlands of Madagascar at altitudes over 1,200 m. The study site consists of rainforest, rainforest edge, and an open savanna biotope. Anopheles gambiae and An. arabiensis, as well as molecular forms of An. gambiae, were identified molecularly. An. gambiae accounted for 26.7% at the edge of the rainforest and 2.3% in the open savanna biotope. One specimen of this species was caught in the forest. An. arabiensis accounted for 66.3% at the edge of the rainforest and 97.7 % in the open savanna biotope. All An. gambiae adults tested belonged to the S molecular form. An. gambiae is present at high altitudes in Madagascar, with a high prevalence at the rainforest edge. Several factors, including the appearance of new favorable biotopes, recolonization after a reduction of indoor vector control, and climate change, may contribute to its distribution. The changing distribution of An. gambiae may have consequences for the distribution and incidence of malaria in the Malagasy Highlands.

Cytoplasmic incompatibility as a means of controlling Culex pipiens quinquefasciatus mosquito in the islands of the south-western Indian Ocean.

The use of the bacterium Wolbachia is an attractive alternative method to control vector populations. In mosquitoes, as in members of the Culex pipiens complex, Wolbachia induces a form of embryonic lethality called cytoplasmic incompatibility, a sperm-egg incompatibility occurring when infected males mate either with uninfected females or with females infected with incompatible Wolbachia strain(s). Here we explore the feasibility of the Incompatible Insect Technique (IIT), a species-specific control approach in which field females are sterilized by inundative releases of incompatible males. We show that the Wolbachia wPip(Is) strain, naturally infecting Cx. p. pipiens mosquitoes from Turkey, is a good candidate to control Cx. p. quinquefasciatus populations on four islands of the south-western Indian Ocean (La Réunion, Mauritius, Grande Glorieuse and Mayotte). The wPip(Is) strain was introduced into the nuclear background of Cx. p. quinquefasciatus mosquitoes from La Réunion, leading to the LR[wPip(Is)] line. Total embryonic lethality was observed in crosses between LR[wPip(Is)] males and all tested field females from the four islands. Interestingly, most crosses involving LR[wPip(Is)] females and field males were also incompatible, which is expected to reduce the impact of any accidental release of LR[wPip(Is)] females. Cage experiments demonstrate that LR[wPip(Is)] males are equally competitive with La Réunion males resulting in demographic crash when LR[wPip(Is)] males were introduced into La Réunion laboratory cages. These results, together with the geographic isolation of the four south-western Indian Ocean islands and their limited land area, support the feasibility of an IIT program using LR[wPip(Is)] males and stimulate the implementation of field tests for a Cx. p. quinquefasciatus control strategy on these islands.

Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island.

Resistance to insecticides was monitored on Culex pipiens quinquefasciatus mosquitoes collected in twelve localities of La Réunion, a geographically isolated island of the Indian Ocean. This mosquito is of medical concern in the region as a known vector for filariasis and a potential vector for West Nile and Rift Valley Fever viruses. Our bioassays indicated the presence of resistance to all tested insecticides, i.e. organochlorides, organophosphates and pyrethroids. A molecular investigation revealed a higher frequency of resistance genes in the coastal areas compared to elevated rural sites, probably reflecting the different nature of insecticide pressures together with the genetic cost of resistance alleles. A simple molecular test was developed to detect Rdl allele, encoding a gamma-aminobutyric acid (GABA) receptor resistant to dieldrin. Unexpectedly high Rdl frequencies were recorded over the whole island, despite this insecticide having been banned for over 15 years. This resistant allele was also detected for the first time in two samples of Aedes albopictus, a species recently involved in severe Chikungunya epidemics on the island. Rdl selection in these two mosquito species discloses current insecticide pressures in urban areas, from unknown origins, that should be taken into account to develop vector control strategies.