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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.

Plague was introduced to Madagascar in 1898 and continues to be a significant human health problem. It exists mainly in the central highlands, but in the 1990s was reintroduced to the port city of Mahajanga, where it caused extensive human outbreaks. Despite its prevalence, the phylogeography and molecular epidemiology of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We examine island-wide geographic-genetic patterns based upon whole-genome discovery of SNPs, SNP genotyping and hypervariable variable-number tandem repeat (VNTR) loci to gain insight into the maintenance and spread of Y. pestis in Madagascar. We analyzed a set of 262 Malagasy isolates using a set of 56 SNPs and a 43-locus multi-locus VNTR analysis (MLVA) system. We then analyzed the geographic distribution of the subclades and identified patterns related to the maintenance and spread of plague in Madagascar. We find relatively high levels of VNTR diversity in addition to several SNP differences. We identify two major groups, Groups I and II, which are subsequently divided into 11 and 4 subclades, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga, where there is evidence for multiple transfers both from and to the central highlands. The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity.

Mark Achtman and colleagues report the whole-genome sequencing of 11 Yersinia pestis isolates, the causative agent of the plague. Their phylogeographic analysis on a larger dataset of Y. pestis global isolates suggests historical routes of transmission. Plague is a pandemic human invasive disease caused by the bacterial agent Yersinia pestis . We here report a comparison of 17 whole genomes of Y. pestis isolates from global sources. We also screened a global collection of 286 Y. pestis isolates for 933 SNPs using Sequenom MassArray SNP typing. We conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmission routes. Our phylogenetic analysis suggests that Y. pestis evolved in or near China and spread through multiple radiations to Europe, South America, Africa and Southeast Asia, leading to country-specific lineages that can be traced by lineage-specific SNPs. All 626 current isolates from the United States reflect one radiation, and 82 isolates from Madagascar represent a second radiation. Subsequent local microevolution of Y. pestis is marked by sequential, geographically specific SNPs.

Plague is often fatal without prompt and appropriate treatment. It affects mainly poor and remote populations. Late diagnosis is one of the major causes of human death and spread of the disease, since it limits the effectiveness of control measures. We aimed to develop and assess a rapid diagnostic test (RDT) for plague. We developed a test that used monoclonal antibodies to the F1 antigen of Yersinia pestis. Sensitivity and specificity were assessed with a range of bacterial cultures and clinical samples, and compared with findings from available ELISA and bacteriological tests for plague. Samples from patients thought to have plague were tested with the RDT in the laboratory and by health workers in 26 pilot sites in Madagascar. The RDT detected concentrations of F1 antigen as low as 0·5 ng/mL in up to 15 min, and had a shelf life of 21 days at 60°C. Its sensitivity and specificity were both 100%. RDT detected 41·6% and 31% more positive clinical specimens than did bacteriological methods and ELISA, respectively. The agreement rate between tests done at remote centres and in the laboratory was 89·8%. With the combination of bacteriological methods and F1 ELISA as reference standard, the positive and negative predictive values of the RDT were 90·6% and 86·7%, respectively. Our RDT is a specific, sensitive, and reliable test that can easily be done by health workers at the patient's bedside, for the rapid diagnosis of pneumonic and bubonic plague. This test will be of key importance for the control of plague in endemic countries.

Aim To describe the phylogeographic patterns of the black rat, Rattus rattus, from islands in the western Indian Ocean where the species has been introduced (Madagascar and the neighbouring islands of Réunion, Mayotte and Grande Comore), in comparison with the postulated source area (India). Location Western Indian Ocean: India, Arabian Peninsula, East Africa and the islands of Madagascar, Réunion, Grande Comore and Mayotte. Methods Mitochondrial DNA (cytochrome b, tRNA and D-loop, 1762 bp) was sequenced for 71 individuals from 11 countries in the western Indian Ocean. A partial D-loop (419 bp) was also sequenced for eight populations from Madagascar (97 individuals), which were analysed in addition to six previously published populations from southern Madagascar. Results Haplotypes from India and the Arabian Peninsula occupied a basal position in the phylogenetic tree, whereas those from islands were distributed in different monophyletic clusters: Madagascar grouped with Mayotte, while Réunion and Grand Comore were present in two other separate groups. The only exception was one individual from Madagascar (out of 190) carrying a haplotype that clustered with those from Réunion and South Africa. 'Isolation with migration' simulations favoured a model with no recurrent migration between Oman and Madagascar. Mismatch distribution analyses dated the expansion of Malagasy populations on a time-scale compatible with human colonization history. Higher haplotype diversity and older expansion times were found on the east coast of Madagascar compared with the central highlands. Main conclusions Phylogeographic patterns supported the hypothesis of human-mediated colonization of R. rattus from source populations in either the native area (India) or anciently colonized regions (the Arabian Peninsula) to islands of the western Indian Ocean. Despite their proximity, each island has a distinct colonization history. Independent colonization events may have occurred simultaneously in Madagascar and Grande Comore, whereas Mayotte would have been colonized from Madagascar. Réunion was colonized independently, presumably from Europe. Malagasy populations may have originated from a single successful colonization event, followed by rapid expansion, first in coastal zones and then in the central highlands. The congruence of the observed phylogeographic pattern with human colonization events and pathways supports the potential relevance of the black rat in tracing human history.

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. 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. 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). 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.

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.

In summary, PCR was successful in only two cases, suggesting that in 83% of samples the PCR reactions started from less template molecules than is needed for positive identification.5,6 This finding is probably the result of several factors: PCR inhibition by co-extracted soil components or degraded collagen, the minute amounts of nucleic acids and high degree of fragmentation of the old DNA in the samples (our attempts to amplify larger caf1 PCR products failed), chemical modification of the nucleic acids, and severe DNA denaturation by alkaline treatment with lime.5,10,13 The poor state of sample preservation is confirmed by the high degree of racemisation of aspartic acid (D/L ratio higher than 0·08)14 and depleted aminoacid content (14% compared with modern bone). [...]this approach is hampered by the poor yields of nucleic acids and proteins.4,5,15 The soil in which the individuals were buried should be assayed for dipstickreacting substances in the same way as the bone samples. Because the skeletons were from a charnel house, no soil sample from the former graveyard was available for analysis. Furthermore, even with the recent statement that the aetiology of Black Death remains unproven by molecular techniques,2 we feel encouraged to propose inclusion of the dipstick assay in research on putative yersinial plague. Since almost all Black Death studies focused on DNA and PCR, which we showed to be less promising, the nature and complexity of Black Death is not properly understood.

R. novegicus has gradually replaced R. rattus in Antananarivo city after its introduction in the 50′s. In 2001, all the rats trapped were R. norvegicus suggesting that this species is nowadays the main reservoir of plague in Antananarivo city. Experimentally, we have demonstrated the natural resistance to Y. pestis of the two populations of rats in Antananarivo (R. norvegicus and R. rattus), as compared to the white laboratory rats and to the rats from non endemic areas. The longitudinal surveillance of the rodents during 4 years, has showed their high seroprevalence for the anti-F1 IgG antibody and a high flea index. The survey has also evidenced Y. pestis in pools of fleas confirming the transmission of the plague bacillus among these rodents. It is likely that many of the rodents get infected but survive and further develop anti-F1 antibodies or remain carriers of the plague bacillus. Indeed rat mortality phenomenon is not observed in the capital, conversely to what is seen in the rural villages or in the harbor of Mahajanga. This natural resistance may explain the maintenance of plague in this city. The absence of epizootic and subsequently the lack of free fleas probably explain the sporadic transmission of plague to human.

Reports the pattern of spread of an exceptional outbreak of pneumonic plague and the biological features and characteristics of the diagnosis by conventional and modern tests. Concludes that 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 in Madagascar in 1997. (Original abstract - amended)