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Correspondance: Carine Brouat, Fax: +33 (0) 4 9962 3345; E-mail: brouat@mpl.ird.fr; International audience; The black rat (Rattus rattus) is the main reservoir of plague (Yersinia pestis infection) in Madagascar's rural zones. Black rats are highly resistant to plague within the plague focus (central highland), whereas they are susceptible where the disease is absent (low altitude zone). To better understand plague wildlife circulation and host evolution in response to a highly virulent pathogen, we attempted to determine genetic markers associated with plague resistance in this species. To this purpose, we combined a population genomics approach and an association study, both performed on 249 AFLP markers, in Malagasy R. rattus. Simulated distributions of genetic differentiation were compared to observed data in four independent pairs, each consisting of one population from the plague focus and one from the plague-free zone. We found 22 loci (9% of 249) with higher differentiation in at least two independent population pairs or with combining P-values over the four pairs significant. Among the 22 outlier loci, 16 presented significant association with plague zone (plague focus vs. plague-free zone). Population genetic structure inferred from outlier loci was structured by plague zone, whereas the neutral loci dataset revealed structure by geography (eastern vs. western populations). A phenotype association study revealed that two of the 22 loci were significantly associated with differentiation between dying and surviving rats following experimental plague challenge. The 22 outlier loci identified in this study may undergo plague selective pressure either directly or more probably indirectly due to hitchhiking with selected loci.

Contact: Anne Loiseau, Fax: +33 (0)4 9962 3345; E-mail: loiseau@supagro.inra.fr; International audience; We isolated and characterized 10 microsatellite loci in the black rat Rattus rattus (Muridae, Rodentia), a widespread invasive species largely known to cause serious problems in agriculture and human health. Polymorphism was studied in two populations, one from Madagascar and one from Senegal. It ranged from three to 12 alleles in Madagascar, and from two to five alleles in Senegal. Together with the loci previously adapted from Rattus norvegicus, this set of markers should allow the conduct of thorough studies on the genetic structure of natural populations of R. rattus.

Between the 20th October and the 18th November 1998, an outbreak of bubonic plague was declared in a hamlet in the Ikongo district of Madagascar. We conducted an epidemiological survey because of the re-emergence of the disease in this area (the last cases had been notified in 1965) and because of the low altitude compared to the classical Malagasy foci. The outbreak had been preceded by an important rat epizootics during September. A total of 21 cases were registered with an attack rate of 16.7% (21/126) and a lethality rate of 33% (7/21). The disease was more prevalent in males (66% of cases) and children aged15 years, as observed in general throughout the country. The anti-F1 seroprevalence among the contact population was 13.5% (13/96), probably attributable to subclinical infection by Yersinia pestis. No rodent was trapped during the survey, but an endemic hedgehog (Tenrec ecaudatus) was highly seropositive, suggesting a recent transmission of the plague bacillus among this species. The small mammals and vectors possibly involved in these new foci were investigated in May 1999.

Une enquête mammalo-entomologique a été réalisée à la suite de la survenue, à une altitude inhabituellement basse pour Madagascar, d'une épidémie de peste bubonique dans le district d'Ikongo et à la présence d'anticorps anti-F1 chez un hérisson endémique lors d'une précédente étude. Les micromammifères ont été échantillonnés dans deux villages, Antanambao-Vohidrotra (540 m. alt.) et Ambalagoavy (265 m. alt.) à l'aide de pièges grillagés et de pièges Sherman, et dans une forêt avoisinante (750 m. alt.) en y ajoutant des trous-pièges. Les puces ont été récoltées par brossage et pièges à bougies. Les rendements de piégeage étaient faibles. Le rat noir (#Rattus rattus$) a été retrouvé dans les trois sites. Un rat était porteur d'anticorps anti-F1 à Antanambao-Vohidrotra, aucun à Ambalagoavy où coexiste la musaraigne introduite #Suncus murinus$. La forêt voisine du premier village a permis de capturer des #Renrecidae$ ("musaraignes" et "hérissons" endémiques) dont quatre exemplaires sont porteurs d'anticorps anti-F1. Une de ces espèces, #Microgale talazaci$, a été trouvée porteuse de #Paractenopsyllus pauliani$, puce endémique non encore impliquée dans le cycle pesteux. #Synopsyllus fonquerniei$ est la seule espèce de puce pestigène retrouvée dans le premier village tandis que #Xenopsylla cheopis$ n'existe qu'à Ambalagoavy. Bienque les essais d'isolement de #Yersinia pestis$ et la recherche d'antigène F1 aient tous été négatifs, il semble bien que la peste ait circulé à Antanambao-Vohidrotra et dans la forêt mais pas à Ambalagoavy. Ces résultats confirment la circulation de la peste en milieu forestier et son implication dans la survenue de cette épidémie. (Résumé d'auteur)

Une épidémie de peste bubonique est survenue entre le 20 octobre et le 18 novembre 1998 dans un hameau du district d'Ikongo (ex Fort-Carnot) à Madagascar. Nous nous sommes intéressés à cette épidémie en raison de son caractère résurgent (derniers cas notifiés en 1965) et de la situation en faible altitude de ce hameau par rapport aux foyers classiques malgaches. Cette épidémie avait été précédée au cours du mois de septembre d'une épizootie murine importante. Au total, 21 cas ont été recensés, soit un taux d'attaque de 16,7% (21/126), avec létalité de 33% (7/21). La maladie, comme on l'observe au niveau national, a atteint plus fréquemment les sujets masculins (66% des cas) et les enfants de moins de 15 ans (76% des cas). L'enquête sérologique réalisée chez les sujets contacts a montré que 13,5% (13/96) étaient séropositifs en anticorps anti-F1, sans doute en relation avec une infection asymptomatique par #Yersinia pestis$. Aucun rongeur n'a été capturé pendant l'enquête, mais le sérum d'un hérisson endémique (#Tenrec ecaudatus$) présentait un taux d'anticorps anti-F1 très élevé, suggérant une circulation récente du bacille pesteux dans cette espèce animale. Des investigations rodento-entomologiques ont été réalisées en mai 1999 pour préciser les réservoirs et vecteurs impliqués dans ce nouveau foyer. (Résumé d'auteur)

Our survey of mammals and fleas arose as a result of an outbreak of bubonic plague at an usually low altitude in the Ikongo district (Madagascar), while a previous study had found anti-F1 antibodies in an endemic hedgehog. Animals were sampled with live traps in two hamlets (Antanambao-Vohidrotra, 540 m alt. and Ambalagoavy, 265 m alt.) and with pitfall traps in a neighbouring forest (750 m alt.). Rat fleas were collected by brushing the fur and free-living fleas by use of light traps. The introduced shrew Suncus murinus was found only in the village of Ambalagoavy while the black rat (Rattus rattus) was found in all three sites and the only seropositive rat was caught at Antanambao-Vohidrotra. In contrast, among the Tenrecidae (endemic shrews and hedgehogs) found in the forest near the first village, four animals were found seropositive for anti-F1 antibodies. One of them was carrying the endemic flea Paractenopsyllus pauliani, not yet reported as a vector of plague. The endemic vector of plague, Synopsyllus fonquerniei, was found only in the first village of Antanambao-Vohidrotra, and the cosmopolite flea Xenopsylla cheopis only in Ambalagoavy. Although no Yersinia pestis could be isolated and no F1-antigen could be detected in these animals, we found evidence of the recent transmission of plague in Antanambao-Vohidrotra and the nearby forest, but not in Ambalagoavy. These data corroborate with the sylvatic plague cycle hypothesis in Madagascar and its involvement in the outcome of the bubonic plague outbreak in this district.

A method associating an anticoagulant rodenticide and an insecticide called Kartman bait-box aimed both at fighting reservoir and vectors of plague. It was evaluated in two neighbourhoods of Antananarivo (Madagascar) from October 2002 to May 2003. It involved the local community in the control. The study was carried out in Ambodirano-Ampefiloha refered as treated neighbourhood in which the Kartman bait box were laid out with an anticoagulant rodenticide and an insecticide with a rapid action versus a "pilot neighbourhood", Ankorondrano-Andranomahery in which the boxes were provided with non poisoning bait and non insecticidal white powder. The rodenticide used was Baraki (difethialone 25 ppm) and the insecticide was a powder of Propoxur 3%. The evaluation of effectiveness of this method was based on the four following parameters: (1) the number of dead rats collected daily inside and in the vicinity of the houses, (2) the daily number of baits non consumed in the Kartman bait box, (3) the cheopis index of the rats trapped using the BTS trap, and (4) the flea carrier index of the rats captured monthly with BTS trap. The cheopis index and the flea carrier index of the rats were calculated monthly. The number of rats that died in the treated neighbourhood was of 968 versus 3 in the pilot neighbourhood. The other parameters reached a stable level after 3 months. Between days 120 and 180, the mean number of unconsumed baits was 2.79 in the treated neighbourhood versus 0.14 in the pilot neighbourhood, the flea carriage (percentage of parasitized hosts) was 0% versus 61% in the pilot neighbourhood, and the cheopis index was 0.0 versus 5.0 in the pilot neighbourhood. This study demonstrates that Kartman bait-boxes reached the rat borne and the vectors of plague found in urban area. We propose to use this method extensively both during epidemic and inter-epidemic contexts.

The authors report the results of a randomized epidemiological survey aiming to assess the sero-prevalence of plague in the general populationor = 2-year-old in Mahajanga. In 656 sera tested (by ELISA), the prevalence of anti-F1 antibodies was found to be 6.1%@1000 inhabitants, close to the expected prevalence in the area, where plague reappeared in 1991 after 62 years of absence. The study also demonstrated that the shrew, Suncus murinus, is an important reservoir in the plague transmission in Mahajanga.

Plague is still endemic in different regions of the world. Current vaccines raise concern for their side effects and limited protection, highlighting the need for an efficacious and rapidly producible vaccine. F1 and V antigens of Yersinia pestis, and F1-V fusion protein produced in Nicotiana benthamiana administered to guinea pigs resulted in immunity and protection against an aerosol challenge of virulent Y. pestis. We examined the effects of plant-derived F1, V, and F1-V on human cells of the innate immunity. F1, V, and F1-V proteins engaged TLR2 signalling and activated IL-6 and CXCL-8 production by monocytes, without affecting the expression of TNF-alpha, IL-12, IL-10, IL-1beta, and CXCL10. Native F1 antigen and recombinant plant-derived F1 (rF1) and rF1-V all induced similar specific T-cell responses, as shown by their recognition by T-cells from subjects who recovered from Y. pestis infection. Native F1 and rF1 were equally well recognized by serum antibodies of Y. pestis-primed donors, whereas serological reactivity to rF1-V hybrid was lower, and that to rV was virtually absent. In conclusion, plant-derived F1, V, and F1-V antigens are weakly reactogenic for human monocytes and elicit cell-mediated and humoral responses similar to those raised by Y. pestis infection.

Background Plague, a zoonosis caused by Yersinia pestis, is found in Asia and the Americas, but predominantly in Africa, with the island of Madagascar reporting almost one third of human cases worldwide. Plague's occurrence is affected by local climate factors which in turn are influenced by large-scale climate phenomena such as the El Niño Southern Oscillation (ENSO). The effects of ENSO on regional climate are often enhanced or reduced by a second large-scale climate phenomenon, the Indian Ocean Dipole (IOD). It is known that ENSO and the IOD interact as drivers of disease. Yet the impacts of these phenomena in driving plague dynamics via their effect on regional climate, and specifically contributing to the foci of transmission on Madagascar, are unknown. Here we present the first analysis of the effects of ENSO and IOD on plague in Madagascar. Methodology/principal findings We use a forty-eight year monthly time-series of reported human plague cases from 1960 to 2008. Using wavelet analysis, we show that over the last fifty years there have been complex non-stationary associations between ENSO/IOD and the dynamics of plague in Madagascar. We demonstrate that ENSO and IOD influence temperature in Madagascar and that temperature and plague cycles are associated. The effects on plague appear to be mediated more by temperature, but precipitation also undoubtedly influences plague in Madagascar. Our results confirm a relationship between plague anomalies and an increase in the intensity of ENSO events and precipitation. Conclusions/significance This work widens the understanding of how climate factors acting over different temporal scales can combine to drive local disease dynamics. Given the association of increasing ENSO strength and plague anomalies in Madagascar it may in future be possible to forecast plague outbreaks in Madagascar. The study gives insight into the complex and changing relationship between climate factors and plague in Madagascar., Author Summary Plague is a vector-borne bacterial infection with rodents and their fleas as its principal hosts. Transmission to humans occurs via the bite of an infected flea. In the highlands of Madagascar, plague is endemic and more than one hundred human cases are reported every year. Global climate is known to affect many infectious diseases and has been shown to affect plague incidence in other areas of the world. The ENSO and the IOD are global climate drivers affecting rainfall and temperature in Madagascar. Our study investigates the effect of global climate drivers on human plague incidence on the island. We found a link between ENSO, IOD, temperature and precipitation and plague incidence throughout the 48-year time-series although it was not constant over time. The correlation between ENSO and plague turned from weakly positive to strongly negative and then to positive, and the association with the IOD became stronger with time. We demonstrate that during periods of high ENSO intensity, plague incidence is likely to increase via ENSO's impact on temperature and precipitation. This shows that climate indices can be a tool to help predict human plague incidence.