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.

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.

Current epidemiology of human plague in Madagascar.

From 1996 to 1998, 5,965 patients with suspected plague were identified in 38 districts of Madagascar (40% of the total population are exposed). Using standard bacteriology, 917 of them were confirmed or presumptive (C + P) cases. However, more than 2,000 plague cases could be estimated using F1 antigen assay. Two out of the 711 Yersinia pestis isolates tested were resistant to chloramphenicol and to ampicillin (both isolates found in the harbour of Mahajanga). Urban plague (Mahajanga harbour and Antananarivo city) accounted for 37.4% of the C + P cases. Bubonic plague represented 97.2% of the cases, and the lethality rate was still high (20%). In comparing the exposed population, plague was more prevalent in males (M:F sex ratio 1.3:1) and patients under 20 years (2.7% babies under two years). Buboes were mainly localised in the inguinal/femoral regions (55.8%). The epidemiological risk factors are discussed.

Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus.

Aspergillus fumigatus secreted an inducible alkaline protease (AlPase) when cultivated in the presence of collagen (200 micrograms/ml) as sole nitrogen and carbon source. Proteolytic activity was maximum at pH 9.0 with azocollagen as substrate. The enzyme, which was the major protein found in the supernate of a liquid culture, was purified by ammonium sulphate precipitation and gel filtration. The Mr was determined to be 33 Kda by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The isoelectric point was estimated to be pH 8.2. Divalent cations strongly inhibited enzyme activity, whereas non-ionic detergents and reducing agents had no effect. A. fumigatus AlPase was totally inhibited by phenylmethanesulphonyl fluoride, antipain, chymostatin and alpha-2-macroglobulin. A. fumigatus AlPase is closely related to the A. oryzae AlPase, a serine protease of the subtilisin family, as attested by the antigen pattern seen by immunoblotting. The high collagenic activity and the ability of A. fumigatus AlPase to digest elastin could play a role in the invasion of the tissues by the fungus.

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.

[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.

[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.

[Update on plague in Madagascar]. / Actualités sur la peste à Madagascar.

After a thirty year period of successful control, bubonic plague showed the first signs of return in Madagascar where a fatal outbreak occurred in Antananarivo in 1978. A second outbreak was observed in Mahajanga in 1991 after more than a half century. In 1997, 459 confirmed or presumptive cases were reported, as compared to 150 to 250 cases during the last years. However the actual extent of this recrudescence must be placed in the perspective of a more efficient control program that has led to better reporting of suspected cases and availability of more accurate diagnostic techniques. Recent research has led to the development of highly effective immunological diagnostic tools (detection of antibodies and F1 antigen) allowing not only better surveillance of the disease in man and animals but also renewed study of the epidemiological cycle in the current environment. In this regard the capacity of several endemic fleas as vectors and the role of the rat Rattus norvegicus and the musk shrew Suncus murinus are currently under investigation. Genetic study of strains collected from 1936 to 1996 has demonstrated the appearance of 3 new ribotypes of Yersinia pestis since 1982 in the zones of strongest plague activity in Madagascar. A strain showing multiresistance to standard therapeutic antibiotic agents was isolated in 1995. Bubonic plaque is a priority health problem in Madagascar but remains a major concern for the rest of the world.

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.

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.

Diagnosis of bubonic plague by PCR in Madagascar under field conditions.

The diagnostic value of a PCR assay that amplifies a 501-bp fragment of the Yersinia pestis caf1 gene has been determined in a reference laboratory with 218 bubo aspirates collected from patients with clinically suspected plague managed in a regional hospital in Madagascar. The culture of Y. pestis and the detection of the F1 antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) were used as reference diagnostic methods. The sensitivity of PCR was 89% (57 of 64) for the Y. pestis-positive patients, and 80.7% (63 of 78) for the F1 Ag-positive patients. The specificity of PCR for the culture-, F1 Ag-, and antibody-negative patients (n = 105) was 100%. Because in Madagascar most patients with plague are managed and their clinical samples are collected in remote villages, the usefulness of PCR was evaluated for routine diagnostic use in the operational conditions of the control program. The sensitivity of PCR was 50% (25 of 50) relative to the results of culture and 35.2% (19 of 54) relative to the results of the F1 Ag immunocapture ELISA. The specificity of PCR under these conditions was 96%. In conclusion, the PCR method was found to be very specific but not as sensitive as culture or the F1 Ag detection method. The limitation in sensitivity may have been due to suboptimal field conditions and the small volumes of samples used for DNA extraction. This technique is not recommended as a routine diagnostic test for plague in Madagascar.

Epidemiological and diagnostic aspects of the outbreak of pneumonic plague in Madagascar.

BACKGROUND: Plague is a re-emerging disease and pneumonic plague is the most feared clinical form. We describe a well-documented outbreak of pneumonic plague in Madagascar. METHODS: Field epidemiological data were collected. Biological tests (microscopy, culture of Yersinia pestis, F1 antigen ELISA and dipstick assays, IgG anti-F1 ELISA) were done on sputum, serum, or necropsy samples. The infection rate among 154 contacts was assessed by anti-F1 serological techniques. FINDINGS: The index case was a bubonic patient with a secondary lung infection, who contaminated a traditional healer and his family. Funeral ceremonies and attendance on patients contaminated other villagers. In total 18 cases were recorded, and eight died. F1 antigen could be detected in sputum by ELISA and dipstick tests as early as the second day after the onset of the symptoms and also 48 h after treatment. Among the contact population 13 of 154 (8.4%) have been exposed to the plague bacillus (symptomless or latent infections). INTERPRETATION: The F1 dipstick assay on sputum is an invaluable diagnostic tool for pneumonic plague. Treatment of patients and chemoprophylaxis of contacts were efficient in stopping the epidemic.

Serodiagnosis of human plague by an anti-F1 capsular antigen specific IgG/IgM ELISA and immunoblot.

Plague is a re-emerging disease endemic in at least 24 countries. Non-endemic countries should be able to confirm plague to prevent outbreaks due to imported cases. We established a combination of a IgG/IgM screening ELISA and a confirmation immunoblot employing F1 capsular antigen (CA) for the serodiagnosis of plague in countries where yersiniosis is present. The ELISA and the immunoblot assay showed a specificity of 96.1% and 100% among sera from healthy German blood donors. This group had a seroprevalence of 39% of anti-yersinia outer protein (YOP) antibodies obviously caused by previous Y. enterocolitica infection. The ELISA detected anti-F1 CA antibodies in 22 and the immunoblot in 20 out of 26 sera of plague vaccinees. Five control sera from bacteriologically confirmed plague cases from Madagascar reacted positively. It can be concluded that anti-YOP antibodies do not affect assays based on purified F1 CA.

[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.