Surveillance of Fleas and Their Small Mammal Hosts for Plague Risks in Some Main Seaports of the Islands of the Southwestern Indian Ocean.

Since ancient times, seaports have been the hot spots for plague introduction into free countries. Infected ship rats reached new areas, and epizootics occurred prior to human infection via flea bites. Beginning in the 1920s/1930s, rodent and flea surveillance was carried out as part of plague hazard management in seaports of the world. Nowadays, such activity is not done regularly. In the southwestern Indian Ocean (SWIO) region, plague surveillance is of great importance given plague endemicity in Madagascar and thus the incurred risk for neighboring islands. This study reports animal-based surveillance aimed at identifying fleas and their small mammal hosts in SWIO seaports as well as Yersinia pestis detection. Small mammal trappings were performed in five main seaports of Madagascar (Toamasina and Mahajanga), Mauritius (Port Louis), and the Union of Comoros (Moroni and Mutsamudu). Mammals were euthanized and their fleas collected and morphologically identified before Y. pestis detection. In total, 145 mammals were trapped: the brown rat Rattus norvegicus (76.5%), the black rat Rattus rattus (8.3%), and the Asian house shrew Suncus murinus (15.2%). Fur brushing allowed collection of 1,596 fleas exclusively identified as Xenopsylla cheopis. All tested fleas were negative for Y. pestis DNA. This study shows that both well-known plague mammal hosts and flea vectors occur in SWIO seaports. It also highlights the necessity of carrying out regular animal-based surveillance for plague hazard management in this region.

Population dynamics of plague vector fleas in an endemic focus: implications for plague surveillance.

Plague is a zoonotic vector-borne disease caused by the bacterium Yersinia pestis. In Madagascar, it persists in identified foci, where it is a threat to public health generally from September to April. A more complete understanding of how the disease persists could guide control strategies. Fleas are the main vector for transmission between small mammal hosts and humans, and fleas likely play a role in the maintenance of plague. This study characterized the dynamics of flea populations in plague foci alongside the occurrence of human cases. From 2018 to 2020, small mammals were trapped at sites in the central Highlands of Madagascar. A total of 2,762 small mammals were captured and 5,295 fleas were collected. The analysis examines 2 plague vector species in Madagascar (Synopsyllus fonquerniei and Xenopsylla cheopis). Generalized linear models were used to relate flea abundance to abiotic factors, with adjustments for trap location and flea species. We observed significant effects of abiotic factors on the abundance, intensity, and infestation rate by the outdoor-associated flea species, S. fonquerniei, but weak seasonality for the indoor-associated flea species, X. cheopis. A difference in the timing of peak abundance was observed between the 2 flea species during and outside the plague season. While the present study did not identify a clear link between flea population dynamics and plague maintenance, as only one collected X. cheopis was infected, the results presented herein can be used by local health authorities to improve monitoring and control strategies of plague vector fleas in Madagascar.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. METHODOLOGY/PRINCIPAL FINDINGS: We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. CONCLUSIONS/SIGNIFICANCE: Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Tracking of Mammals and Their Fleas for Plague Surveillance in Madagascar, 2018-2019.

Plague, a zoonotic disease caused by the bacterium Yersinia pestis, remains a major public health threat in Madagascar. To better understand the risk of transmission to humans and to guide targeted plague prevention and control measures, a survey of Y. pestis infection and exposure in mammals and their fleas was implemented. Small mammals were captured in five districts of Madagascar ranging in levels of plague endemicity, as measured by notified cases, from none to active foci. Blood and spleen samples and fleas were collected from small mammals for the detection of anti-Y. pestis F1 antibodies by ELISA, F1 antigens by rapid diagnostic tests, and pla, caf1, and inv genes by polymerase chain reaction. Some rodent fleas were kept alive and reared in the insectary to assess susceptibility to insecticides. Blood was also collected from 15 dogs and tested for anti-F1 antibodies. A total of 557 spleens, 484 sera, and 1,539 fleas were collected from 557 rodents and shrews. Nineteen (3.4%) spleens were positive for F1 antigen, most from Toamasina (N = 13), a historical plague focus. One dog was also found seropositive in Toamasina. Twenty-two (4.5%) serologic specimens from small mammals were positive for anti-F1 antibodies. The flea index was highest in the city of Antananarivo (8.8). No flea was positive for Y. pestis DNA. Flea populations exhibited resistance to various insecticides weakening the efficacy of vector control. This study highlights the potential use of animal-based surveillance to identify the risk of plague transmission in endemic and nonendemic foci for targeted prevention and control.

Illustrated Morphological Keys for Fleas (Siphonaptera) in Madagascar.

Madagascar has an important diversity of fleas (Siphonaptera), which almost species do not exist elsewhere. Scientists have provided huge efforts to improve knowledge on Malagasy fleas since the middle of 1900s particularly by investigating topics such as taxonomy, systematics, biogeography, and flea vector role. Since then, new species discovery has increased and currently, 48 species are recorded which the majority is endemic. Therefore, it becomes necessary to have updated keys to identify species membership. This paper presents morphological-based keys to identify at genus and species levels adult fleas reported as occurring in Madagascar. Illustrations are proposed to make easier the observation of morphological criteria, which may be tricky for sibling species.

Presence of the Oriental Rat Flea (Siphonaptera: Pulicidae) Infesting an Endemic Mammal and Confirmed Plague Circulation in a Forest Area of Madagascar.

The Oriental rat flea, Xenopsylla cheopis (Rothschild 1903), is a cosmopolitan flea usually found infesting domestic rats. This flea is a well-known major human plague vector in Madagascar. As part of field sampling, fleas and small mammals were collected in the village of South Andranofeno and the natural reserve of Sohisika, two sites of the district of Ankazobe, located in the Central Highlands of Madagascar. Rats inside houses and forest small mammals were trapped using Besancon Technical Services and pitfall traps, respectively. Their fleas were collected and preserved for laboratory works. Collected fleas from the village and forest belonged to five species, which were X. cheopis, Synopsyllus fonquerniei (Wagner and Roubaud 1932) (Siphonaptera: Pulicidae), Echidnophaga gallinacea (Westwood 1875) (Siphonaptera: Pulicidae), Ctenocephalides felisstrongylus (Jordan 1925) (Siphonaptera: Pulicidae), Pulex irritans (Linnaeus 1758) (Siphonaptera: Pulicidae). After sampling in the forest zone, one specimen of X. cheopis was unexpectedly collected while infesting an endemic tenrec Setifer setosus (Schreber 1777) (Afrosoricida: Tenrecidae). Polymerase chain reaction (PCR) diagnosis on all collected fleas allowed detecting plague bacterium Yersinia pestis (Lehmann and Neumann 1896) (Enterobacterales: Yersiniaceae) on nine specimens of the endemic flea S. fonquerniei collected inside forest. The presence of the oriental rat flea in forest highlights the connection between human and wild environments due to animal movements and the fact that the rat flea can infest various hosts. As only one specimen of X. cheopis was collected on S. setosus, we hypothesize that flea was carried from the village to forest. Yersinia pestis infection of forest fleas outlines plague circulation in this sylvatic area.

Risk of maritime introduction of plague from Madagascar to Mayotte.

Plague is a rodent-borne disease caused by Yersinia pestis. Most human infections are bubonic plague, as a result of being bitten by infected rodent fleas. Madagascar, Democratic Republic of Congo and Peru are the three most affected countries. Plague was introduced into eastern Madagascar in 1898 by boat from India. It is estimated that the risk of introduction of plague from Madagascar to neighboring islands is very high due to the maritime links. We conducted a study of plague reservoirs and vectors in Longoni Port in Mayotte and Mahajanga Port in Madagascar during two seasons to highlight a non-negligible risk of introduction of Y. pestis to Mayotte. The results showed that two main reservoirs of plague in Madagascar Suncus murinus and Rattus rattus and the main flea vector Xenopsylla cheopis exists in and surrounding the port of Longoni. Y. pestis was isolated from Rattus norvegicus captured close to the port of Mahajanga during this study. Plague bacteria circulate within populations of rodent without causing rodent die-off in Mahajanga. The risk of introduction of plague from Madagascar to Mayotte exists due to the regular exchanges. Continuous surveillance of rat, shrew and flea populations is therefore necessary in all the surrounding countries that have regular exchanges with Madagascar to prevent the spread of the plague.

Species diversity and phylogeny of fleas of small terrestrial mammals in the forests of the Central Highlands of Madagascar.

Fleas are holometabolous insects forming the order of Siphonaptera. Some studies have been carried out on biology and systematic of Malagasy fleas, but little is known about their phylogenetic relationships. In this study, we focused on flea species occurring in the forests of the Central Highlands and also, on the determination of their phylogenetic relationships. Three families, five genera and thirteen species were identified. The family Pulicidae includes four species (Centetipsylla madagascariensis Rothschild, Synopsyllus fonquerniei Wagner Roubaud, S. estradei Klein and S. robici Klein); Leptopsyllidae has eight species (Paractenopsyllus vauceli Klein, P. petiti Klein, P. viettei Klein, P. grandidieri Klein, P. goodmani Duchemin, P. rouxi Duchemin, P. raxworthyi Duchemin Ratovonjato and Tsaractenus rodhaini Duchemin), and Ctenophtalmidae one species (Dinopsyllus brachypecten Smit). All are endemic to Madagascar and each differs geographically. Flea phylogenetic relationships were inferred using four molecular markers (ITS2, mtCOII, 16SrRNA and 12S rRNA) and using Neighbor-Joining, Maximum Parsimony and Bayesian methods with addition of Genbank sequences of exotic species. The Family Pulicidae was monophyletic while the families Leptopsyllidae and Ctenophtalmidae were paraphyletic. Malagasy fleas are homogeneous and all species adhere to current classification schemes.

Genetic structure and gene flow of the flea Xenopsylla cheopis in Madagascar and Mayotte.

BACKGROUND: The flea Xenopsylla cheopis (Siphonaptera: Pulicidae) is a vector of plague. Despite this insect's medical importance, especially in Madagascar where plague is endemic, little is known about the organization of its natural populations. We undertook population genetic analyses (i) to determine the spatial genetic structure of X. cheopis in Madagascar and (ii) to determine the potential risk of plague introduction in the neighboring island of Mayotte. RESULTS: We genotyped 205 fleas from 12 sites using nine microsatellite markers. Madagascan populations of X. cheopis differed, with the mean number of alleles per locus per population ranging from 1.78 to 4.44 and with moderate to high levels of genetic differentiation between populations. Three distinct genetic clusters were identified, with different geographical distributions but with some apparent gene flow between both islands and within Malagasy regions. The approximate Bayesian computation (ABC) used to test the predominant direction of flea dispersal implied a recent population introduction from Mayotte to Madagascar, which was estimated to have occurred between 1993 and 2012. The impact of this flea introduction in terms of plague transmission in Madagascar is unclear, but the low level of flea exchange between the two islands seems to keep Mayotte free of plague for now. CONCLUSION: This study highlights the occurrence of genetic structure among populations of the flea vector of plague, X. cheopis, in Madagascar and suggests that a flea population from Mayotte has been introduced to Madagascar recently. As plague has not been reported in Mayotte, this introduction is unlikely to present a major concern for plague transmission. Nonetheless, evidence of connectivity among flea populations in the two islands indicates a possibility for dispersal by fleas in the opposite direction and thus a risk of plague introduction to Mayotte.

Divergent evolutionary and epidemiological dynamics of cassava mosaic geminiviruses in Madagascar.

BACKGROUND: Cassava mosaic disease (CMD) in Madagascar is caused by a complex of at least six African cassava mosaic geminivirus (CMG) species. This provides a rare opportunity for a comparative study of the evolutionary and epidemiological dynamics of distinct pathogenic crop-infecting viral species that coexist within the same environment. The genetic and spatial structure of CMG populations in Madagascar was studied and Bayesian phylogeographic modelling was applied to infer the origins of Madagascan CMG populations within the epidemiological context of related populations situated on mainland Africa and other south western Indian Ocean (SWIO) islands. RESULTS: The isolation and analysis of 279 DNA-A and 117 DNA-B sequences revealed the presence in Madagascar of four prevalent CMG species (South African cassava mosaic virus, SACMV; African cassava mosaic virus, ACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV), and of numerous CMG recombinants that have, to date, only ever been detected on this island. SACMV and ACMV, the two most prevalent viruses, displayed low degrees of genetic diversity and have most likely been introduced to the island only once. By contrast, EACMV-like CMG populations (consisting of East African cassava mosaic virus, EAMCKV, EACMCV and complex recombinants of these) were more diverse, more spatially structured, and displayed evidence of at least three independent introductions from mainland Africa. Although there were no statistically supported virus movement events between Madagascar and the other SWIO islands, at least one mainland African ACMV variant likely originated in Madagascar. CONCLUSIONS: Our study highlights both the complexity of CMD in Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. Given that more distinct CMG species and recombinants have been found in Madagascar than any other similarly sized region of the world, the risks of recombinant CMG variants emerging on this island are likely to be higher than elsewhere. Evidence of an epidemiological link between Madagascan and mainland African CMGs suggests that the consequences of such emergence events could reach far beyond the shores of this island.

Asystasia mosaic Madagascar virus: a novel bipartite begomovirus infecting the weed Asystasia gangetica in Madagascar.

Here, we describe for the first time the complete genome sequence of a new bipartite begomovirus in Madagascar isolated from the weed Asystasia gangetica (Acanthaceae), for which we propose the tentative name asystasia mosaic Madagascar virus (AMMGV). DNA-A and -B nucleotide sequences of AMMGV were only distantly related to known begomovirus sequence and shared highest nucleotide sequence identity of 72.9 % (DNA-A) and 66.9 % (DNA-B) with a recently described bipartite begomovirus infecting Asystasia sp. in West Africa. Phylogenetic analysis demonstrated that this novel virus from Madagascar belongs to a new lineage of Old World bipartite begomoviruses.

Molecular characterization of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar.

Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name "Cassava mosaic alphasatellite".

East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus.

BACKGROUND: Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands. RESULTS: The isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG's presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components. CONCLUSIONS: Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.

A novel cassava-infecting begomovirus from Madagascar: cassava mosaic Madagascar virus.

Cassava mosaic geminiviruses (CMGs) are implicated in cassava mosaic disease (CMD), the main constraint to cassava production in Africa. Here, we report the complete nucleotide sequences of the DNA-A and DNA-B of a newly characterized CMG found infecting cassava in Madagascar, for which we propose the tentative name cassava mosaic Madagascar virus. With the exception of two recombinant regions that resembled a CMG, we determined that the non-recombinant part of the DNA-A component is distantly related to the other CMGs. Whereas the DNA-B component possesses one recombinant region originating from an unidentified virus, the rest of the genome was seen to be closely related to members of the species East African cassava mosaic Zanzibar virus (EACMZV). Phylogenetic analysis based on complete genome sequences demonstrated that DNA-A and DNA-B components are outliers related to the clade of EACMV-like viruses and that DNA-A is related to the monopartite tomato leaf curl begomoviruses described in islands in the south-west Indian Ocean.

Evolution of African cassava mosaic virus by recombination between bipartite and monopartite begomoviruses.

BACKGROUND: Cassava mosaic disease (CMD) is a major constraint on cassava cultivation in Africa. The disease is endemic and is caused by seven distinct cassava mosaic geminiviruses (CMGs), some of them including several variants. FINDINGS: From cassava leaf samples presenting CMD symptoms collected in Burkina Faso, four DNA-A begomovirus components were cloned and sequenced, showing 99.9% nucleotide identity among them. These isolates are most closely related to African cassava mosaic virus (ACMV) but share less than 89% nucleotide identity (taxonomic threshold) with any previously described begomovirus. A DNA-B genomic component, sharing 93% nucleotide identity with DNA-B of ACMV, was also characterized. Since all genomic components have a typical genome organization of Old World bipartite begomoviruses, this new species was provisionally named African cassava mosaic Burkina Faso virus (ACMBFV). Recombination analysis of the new virus demonstrated an interspecies recombinant origin, with major parents related to West African isolates of ACMV, and minor parents related to Tomato leaf curl Cameroon virus and Cotton leaf curl Gezira virus. CONCLUSION: This is the first report of an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses.