Invasion genetics of a human commensal rodent: the black rat Rattus rattus in Madagascar.

Studies focusing on geographical genetic patterns of commensal species and on human history complement each other and provide proxies to trace common colonization events. On Madagascar, the unintentional introduction and spread of the commensal species Rattus rattus by people may have left a living clue of human colonization patterns and history. In this study, we addressed this question by characterizing the genetic structure of natural populations of R. rattus using both microsatellites and mitochondrial sequences, on an extensive sampling across the island. Such data sets were analysed by a combination of methods using population genetics, phylogeography and approximate Bayesian computation. Our results indicated two introduction events to Madagascar from the same ancestral source of R. rattus, one in the extreme north of the island and the other further south. The latter was the source of a large spatial expansion, which may have initially started from an original point located on the southern coast. The inferred timing of introduction events-several centuries ago-is temporally congruent with the Arabian trade network in the Indian Ocean, which was flourishing from the middle of the first millennium.

AFLP genome scan in the black rat (Rattus rattus) from Madagascar: detecting genetic markers undergoing plague-mediated selection.

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.

Susceptibility to Yersinia pestis experimental infection in wild Rattus rattus, reservoir of plague in Madagascar.

In Madagascar, the black rat, Rattus rattus, is the main reservoir of plague (Yersinia pestis infection), a disease still responsible for hundreds of cases each year in this country. This study used experimental plague challenge to assess susceptibility in wild-caught rats to better understand how R. rattus can act as a plague reservoir. An important difference in plague resistance between rat populations from the plague focus (central highlands) and those from the plague-free zone (low altitude area) was confirmed to be a widespread phenomenon. In rats from the plague focus, we observed that sex influenced plague susceptibility, with males slightly more resistant than females. Other individual factors investigated (weight and habitat of sampling) did not affect plague resistance. When infected at high bacterial dose (more than 105 bacteria injected), rats from the plague focus died mainly within 3-5 days and produced specific antibodies, whereas after low-dose infection (< 5,000 bacteria), delayed mortality was observed and surviving seronegative rats were not uncommon. These results concerning plague resistance level and the course of infection in the black rat would contribute to a better understanding of plague circulation in Madagascar.

Plant-derived recombinant F1, V, and F1-V fusion antigens of Yersinia pestis activate human cells of the innate and adaptive immune system.

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.

CCR5 polymorphism and plague resistance in natural populations of the black rat in Madagascar.

Madagascar remains one of the world's largest plague foci. The black rat, Rattus rattus, is the main reservoir of plague in rural areas. This species is highly susceptible to plague in plague-free areas (low-altitude regions), whereas rats from the plague focus areas (central highlands) have evolved a disease-resistance polymorphism. We used the candidate gene CCR5 to investigate the genetic basis of plague resistance in R. rattus. We found a unique non-synonymous substitution (H184R) in a functionally important region of the gene. We then compared (i) CCR5 genotypes of dying and surviving plague-challenged rats and (ii) CCR5 allelic frequencies in plague focus and plague-free populations. Our results suggested a higher prevalence of the substitution in resistant animals compared to susceptible individuals, and a tendency for higher frequencies in plague focus areas compared to plague-free areas. Therefore, the CCR5 polymorphism may be involved in Malagasy black rat plague resistance. CCR5 and other undetermined plague resistance markers may provide useful biological information about host evolution and disease dynamics.

Isolation and characterization of microsatellites in Rattus rattus.

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.

Comparison of hand-held test kits, immunofluorescence microscopy, enzyme-linked immunosorbent assay, and flow cytometric analysis for rapid presumptive identification of Yersinia pestis.

An in-house immunochromatographic test, Plague BioThreat Alert test strips, ABICAP columns, enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence microscopy were compared for the detection of the fraction 1 capsular antigen of Yersinia pestis, using spiked buffer and clinical specimens. Hand-held test kits proved to be excellent benchtop tools.

Serodiagnosis of human plague by a combination of immunomagnetic separation and flow cytometry.

BACKGROUND: Plague is a severe, highly communicable bacterial disease caused by Yersinia pestis. It is still endemic in more than 20 countries worldwide. Although known as a devastating disease for centuries, laboratory confirmation of clinical suspected cases is still problematic. No standardized and internationally approved test system is commercially available. The aim of this study was the introduction and evaluation of a combination of immunomagnetic separation and flow cytometry for the serodiagnosis of human plague. METHODS: Paramagnetic polystyrene beads were coated with purified F1 capsular antigen (F1 CA) and reacted with sera from plague patients, from 26 laboratory personnel vaccinated against plague and from 102 healthy blood donors (HBD). After incubation with fluorescein isothiocyanate-conjugated anti-human rabbit IgG, particle-associated fluorescence was detected by flow cytometry. RESULTS: Anti-F1 CA antibodies could be demonstrated in all patients with bacteriologically confirmed plague and in 22 sera (84.6%) from vaccinees. Only one serum in the HBD group showed a weakly positive reaction. The total assay time was less than 2 h. CONCLUSIONS: Compared with a recently published combination of an anti-F1 CA enzyme-linked immunosorbent assay (ELISA) and immunoblot, the new assay showed the same sensitivity as the ELISA and almost the same specificity (99.0 versus 100%) as the immunoblot. Allowing a rapid, reliable, and quantitative analysis, immunomagnetic separation combined with flow cytometry might replace both conventional immunoassays.

[Plague control in Madagascar: evaluation of the efficacy of Kartman baitboxes in urban areas]. / Lutte contre ia peste à Madagascar: évaluation de l'efficacité des boîtes de Kartman en milieu urbain.

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.

Lutte contre la peste à Madagascar : évaluation de l'éfficacité des boîtes de Kartman en milieu urbain

La boîte de Kartman associe, dans un même réceptacle, un rodenticide à action lente et un insecticide à action rapide. Elle offre l'opportunité de lutter contre les réservoirs et contre les vecteurs de la peste. Cette méthode a été évaluée dans deux quartiers de la ville d'Antananarivo en associant la communauté à cette lutte. Le rodenticide utilisé a été le diféthialone 25 ppm (Baraki®) et l'insecticide, un carbamate en poudre à une concentration de 3% (Propoxur ®). Le schéma de l'étude réalisée d'octobre 2002à mai 2003 repose sur une comparaison entre un "quartier traité" et un "quartier témoin".L'analyse a porté sur 4 variables : (1) le nombre quotidien de rats trouvés morts, (2) le nombrequotidien d'appâts non consommés restant dans les boîtes, (3) la prévalence des rats porteurs de puces, et (4) l'index pulicidien des rats. Les variables 3 et 4 ont été obtenues à partir de rats piégés vivants à une fréquence mensuelle. Le nombre de rats morts dans le quartier traité a été de 968 versus 3 dans le quartier témoin. Les autres variables étudiées ont atteint un niveau d'équilibre à partir du 4 ème mois. Ainsi, entre J120 et J180, la moyenne quotidienne du nombre d'appâts non consommés a été de 2,79 dans le quartier traité versus 0,14 dans le quartier témoin, la prévalence des rats porteurs de puces a été de 0% dans le quartier traité (n=2 rats) versus 61% dans le quartier témoin(n=42 rats), et l'index pulicidien de ces rats dans le quartier traité a été 0 versus 5,0 dans le quartier témoin. Cette étude démontre l'efficacité de cette méthode pour atteindre les réservoirs et les vecteurs de la peste urbaine. Sous réserve d'utilisation correcte, la boîte de Kartman a sa place parmi les moyens de lutte contre la peste dans les contextes inter-épidémiques ou épidémiques

[Resurgence of the plague in the Ikongo district of Madagascar in 1998. 1. Epidemiological aspects in the human population]. / Résurgence de la peste dans le district d'Ikongo à Madagascar en 1998. 1. Aspects épidémiologiques dans la population humaine.

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 aged < 15 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.

[Resurgence of the plague in the Ikongo district of Madagascar in 1998. 2. Reservoirs and vectors implicated]. / Résurgence de la peste dans le district d'Ikongo à Madagascar en 1998. 2. Réservoirs et vecteurs impliqués.

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.

Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis.

Plasmid-mediated high-level resistance to multiple antibiotics was reported in a clinical isolate of Yersinia pestis in Madagascar in 1997. We describe a second Y. pestis strain with high-level resistance to streptomycin, isolated from a human case of bubonic plague in Madagascar. The resistance determinants were carried by a self-transferable plasmid that could conjugate at high frequencies to other Y. pestis isolates. The plasmid and the host bacterium were different from those previously associated with multiple-drug resistance, indicating that acquisition of resistance plasmids is occurring in this bacterial species. Emergence of resistance to streptomycin in Y. pestis represents a critical public health problem since this antibiotic is used as the first-line treatment against plague in many countries.

[Recrudescence and geographic extension of the plague in Madagascar from 1980 to 1999]. / Recrudescence et extension géographique de la peste à Madagascar de 1980 à 1999.

Plague was introduced to Madagascar in 1898, and it has been characterized by a predominant distribution to the central highlands in the following decades. An increase of plague cases has been observed in the past 20 years, in particular in the capital, Antananarivo, and in the coastal town, Mahajanga, after long periods of silence in 28 and 63 years, respectively. A total of 2,982 confirmed or presumptive cases were reviewed in order to describe the changes in the epidemiological pattern of the disease from 1980 through 1999. The mean annual number of plague cases has increased from 33 during the 1980-1984 period to 298 during the 1995-1999 period. A similar trend of distribution has been observed from the first period to the second by an increase of endemic districts above 800 m altitude from 17 to 37. However, the lethality rate has in the same 20 years observation period decreased from 41.6% to 20.7%, probably due to re-enforcing measures as part of the national control program.

[The plague at the Tsenabe Isotry market in Antananarivo: a complex epidemiologic situation]. / La peste dans le marché Tsenabe Isotry à Antananarivo: une situation épidémiologique complexe.

The transmission of Yersinia pestis is intense among rats in the wholesale market Tsenabe Isotry in the capital Antananarivo (anti-F1 sero-prevalence 80%, flea index 8.4 for a cut-off risk index of > 1). However, the number of plague-suspected (not laboratory confirmed) human cases has only been 3 in this district during a four years period from 1995 to 1999. A seroepidemiological survey among the market vendors was undertaken in June 1999 to test the hypothesis that the low incidence of human plague is due to acquired immunity. In addition, surveillance of the rat and the flea populations in the market was carried out. Only 3 (3.2%) of 95 screened vendors were anti-F1 IgG positive, whereas the markers of plague transmission among rodents and fleas were still high. This result suggests that the low incidence of human plague was not due to acquired immunity but to other factors such as the limited contact between humans and the rat fleas because of the abundance of rats, the absence of epizootic due to the resistance of rats in the capital and a particular behaviour of the predominant rat Rattus norvegicus.

Recrudescence et extension géographique de la peste à Madagascar de 1980 à 1999

Après avoir touché Madagascar en 1898, la peste a atteint Antananarivo en 1921 et s'est étendue sur les Hautes Terres Centrales où elle persiste jusqu'à nos jours. Une recrudescence des cas a été constatée depuis une vingtaine d'années pendant lesquelles, la peste a réemergé dans la capitale Antananarivo et, dans le port de Mahajanga après respectivement 28 et 63 ans de silence apparent. Le programme national de lutte et de surveillance a, de ce fait, été renforcé. L'évolution de l'endémie dans le temps et dans l'espace au cours de cette période a été analysée à partir de 2 982 cas bactériologiquement confirmés ou probables(sex-ratio H/F : 1,3/1). L'incidence annuelle moyenne des cas de peste confirmés ou probables est passée de 33 pendant la période 1980-1984 à 298 pendant la période 1995-1999. Cette augmentation s'est accompagnée d'une large extension géographique de la zone d'endémie, passant de 17 à 37 districts pour la plupart situés à plus de 800 m d'altitude, sauf le port de Mahajanga. Par contre, le taux de létalité a baissé pendant la même période, passant de 41,6% à 20,7%. Un des objectifs des recherches actuelles est une meilleure compréhension des différents cycles épidémiologiques de la peste à Madagascar, afin d'améliorer les stratégies de lutte

La peste dans le marché Tsenabe Isotry à Antananarivo : une situation épidémiologique complexe

La surveillance de la peste murine en 1995 dans le marché de gros Tsenabe Isotry à Antananrivo a montré une circulation intense du bacille pesteux (80% de rats séropositifs) et un index cheopis (8,4) supérieur au seuil classique de risque épidémique (>1). Pourtant, le nombre de malades suspects déclarés dans ce quartier est très faible (3 cas suspects sans confirmation de 1995 à 1999). Dans le but de vérifier si la faible incidence de la peste humaine pouvait être due à l'acquisition d'une immunité, une enquête séroépidémiologique a été menée auprès des marchands en juin 1999 associée à l'analyse des données de la surveillance des rongeurs entre 1998 ­ 1999. La séroprévalence des porteurs d'anticorps anti-F1 est de 3,2% (3/95 marchands), alors que les marqueurs de la transmission de la peste chez les rongeurs et les puces continuent d'être très élevés. Ces résultats suggèrent que l'incidence de la peste parmi les marchands n'est pas liée à une immunité acquise, mais probablement à d'autres facteurs : le faible contact entre l'homme et les puces de rat en raison de l'abondance des rats, l'absence d'épizootie murine due à la résistance des rats de la capitale et le comportement du rongeur prédominant Rattus norvegicus moins commensal que R. rattus

A miniaturised semiautomated system for the identification of Yersinia species within the genus Yersinia.

Commercially available identification systems based on biochemical reactions of bacteria are not suited for typing the species of the genus Yersinia (Y.) or the biovars (BV) of the species Y. enterocolitica. This failure is caused by the limited number of biochemical reactions applied, resulting in the absence of important discriminatory key reactions. The MICRONAUT identification system (Merlin, Bornheim-Hersel) makes use of dried substrates/enzymes reactions in the wells of a 96-well microtitration plate, reading of the results by a scanner device and typing of the isolate by the calculation of probabilities according to a data base. For this study a special identification panel was designed on which 38 substrates and enzyme reactions were configurated including 20 reactions for the identification of the species of the genus and the Y. enterocolitica biovars. The database was calculated using the results obtained from a total of 250 Yersinia strains of the eleven species of the genus. Reevaluation of the results of these strains revealed an overall sensitivity of 98%, as only four strains were not identified satisfactorily. Considering also questionable results the sensitivity was still 85%. The system was also used to identify Y. pestis isolates, but in this case reading was done visually. The printouts usually cite species designation, identification quality and probabilities. The sealing of the plates in an aluminium bag guarantees long life and long lasting quality. However, an evaluation of the system with a considerable number of strains has to be done in a next step. The 'Yersinia identification set' can replace time-consuming tube testing in the future and is a big step forward towards a sensitive identification of Yersinia isolates in the routine laboratory.

Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and rapid immunogold dipstick.

Plague is still prevalent in more than 20 countries. Two F1 antigen diagnostic assays (an immunocapture ELISA and an immunogold chromatography dipstick) were evaluated using bubo aspirates, serum and urine specimens from patients suspected with plague. The specificity of the two F1 assays was found 100%. Using bacteriology as a gold reference diagnostic assay, 52 patients were Yersinia pestis culture positive and 141 negative. The sensitivity of the F1 ELISA test was 100% in bubo, 52% in serum and 58% in urine specimens. In culture negative patients, the F1 antigen could be found in 10% bubo aspirates, 5% serum and 7% urine specimens of culture negative patients for whom a seroconversion for anti-F1 antibodies was also observed. The sensitivity of the dipstick assay was 98% on bubo aspirates specimens. Compared to the ELISA test, the agreement rate was 97.5% and the correlation coefficient tau = 0.90 (p < 10(-3)). In conclusion, the diagnosis of bubonic plague has to be performed on bubo fluid rather than on serum or urine specimens. Both the F1 ELISA and the dipstick assays are valuable tools for an early diagnosis and for the surveillance of plague.