Short- and long-term humoral immune response against Yersinia pestis in plague patients, Madagascar.

BACKGROUND: Plague, a fatal disease caused by the bacillus, Yersinia pestis, still affects resources-limited countries. Information on antibody response to plague infection in human is scarce. Anti-F1 Ig G are among the known protective antibodies against Y. pestis infection. As a vaccine preventable disease, knowledge on antibody response is valuable for the development of an effective vaccine to reduce infection rate among exposed population in plague-endemic regions. In this study, we aim to describe short and long-term humoral immune responses against Y. pestis in plague-confirmed patients from Madagascar, the most affected country in the world. METHODS: Bubonic (BP) and pneumonic plague (PP) patients were recruited from plague- endemic foci in the central highlands of Madagascar between 2005 and 2017. For short-term follow-up, 6 suspected patients were enrolled and prospectively investigated for kinetics of the anti-F1 IgG response, whereas the persistence of antibodies was retrospectively studied in 71 confirmed convalescent patients, using an ELISA which was validated for the detection of plague in human blood samples in Madagascar. RESULTS: Similarly to previous findings, anti-F1 IgG rose quickly during the first week after disease onset and increased up to day 30. In the long-term study, 56% of confirmed cases remained seropositive, amongst which 60 and 40% could be considered as high- and low-antibody responders, respectively. Antibodies persisted for several years and up to 14.8 years for one individual. Antibody titers decreased over time but there was no correlation between titer and time elapsed between the disease onset and serum sampling. In addition, the seroprevalence rate was not significantly different between gender (P = 0.65) nor age (P = 0.096). CONCLUSION: Our study highlighted that the circulating antibody response to F1 antigen, which is specific to Y. pestis, may be attributable to individual immune responsiveness. The finding that a circulating anti-F1 antibody titer could persist for more than a decade in both BP and PP recovered patients, suggests its probable involvement in patients' protection. However, complementary studies including analyses of the cellular immune response to Y. pestis are required for the better understanding of long-lasting protection and development of a potential vaccine against plague.

Trends of Human Plague, Madagascar, 1998-2016.

Madagascar is more seriously affected by plague, a zoonosis caused by Yersinia pestis, than any other country. The Plague National Control Program was established in 1993 and includes human surveillance. During 1998-2016, a total of 13,234 suspected cases were recorded, mainly from the central highlands; 27% were confirmed cases, and 17% were presumptive cases. Patients with bubonic plague (median age 13 years) represented 93% of confirmed and presumptive cases, and patients with pneumonic plague (median age 29 years) represented 7%. Deaths were associated with delay of consultation, pneumonic form, contact with other cases, occurrence after 2009, and not reporting dead rats. A seasonal pattern was observed with recrudescence during September-March. Annual cases peaked in 2004 and decreased to the lowest incidence in 2016. This overall reduction occurred primarily for suspected cases and might be caused by improved adherence to case criteria during widespread implementation of the F1 rapid diagnostic test in 2002.

Lessons learned about pneumonic plague diagnosis from two outbreaks, Democratic Republic of the Congo.

Pneumonic plague is a highly transmissible infectious disease for which fatality rates can be high if untreated; it is considered extremely lethal. Without prompt diagnosis and treatment, disease management can be problematic. In the Democratic Republic of the Congo, 2 outbreaks of pneumonic plague occurred during 2005 and 2006. In 2005, because of limitations in laboratory capabilities, etiology was confirmed only through retrospective serologic studies. This prompted modifications in diagnostic strategies, resulting in isolation of Yersinia pestis during the second outbreak. Results from these outbreaks demonstrate the utility of a rapid diagnostic test detecting F1 antigen for initial diagnosis and public health management, as well as the need for specialized sampling kits and trained personnel for quality specimen collection and appropriate specimen handling and preservation for plague confirmation and Y. pestis isolation. Efficient frontline management and a streamlined diagnostic strategy are essential for confirming plague, especially in remote areas.

Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague.

Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5'-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 (pla gene), and pMT1 (caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5'-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5'-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.

Field assessment of dog as sentinel animal for plague in endemic foci of Madagascar.

The epidemiology of Yersinia pestis, the causative agent of plague, involves vectors and reservoirs in its transmission cycle. The passive plague surveillance in Madagascar targets mainly rodent and fleas. However, carnivores are routinely surveyed as sentinels of local plague activity in some countries. The aim of this study is to assess the use of domestic dog (Canis familiaris) as sentinel animal for field surveillance of plague in a highly endemic area in Madagascar. Cross-sectional surveys of plague antibody prevalence in C. familiaris were conducted in endemic areas with contrasting histories of plague cases in humans, as well as a plague free area. Rodent capture was done in parallel to evaluate evidence for Y. pestis circulation in the primary reservoirs. In 2 sites, dogs were later re-sampled to examine evidence of seroconversion and antibody persistence. Biological samplings were performed between March 2008 and February 2009. Plague antibody detection was assessed using anti-F1 ELISA. Our study showed a significant difference in dog prevalence rates between plague-endemic and plague-free areas, with no seropositive dogs detected in the plague free area. No correlation was found between rodents and dog prevalence rates, with an absence of seropositive rodents in some area where plague circulation was indicated by seropositive dogs. This is consistent with high mortality rates in rodents following infection. Re-sampling dogs identified individuals seropositive on both occasions, indicating high rates of re-exposure and/or persistence of plague antibodies for at least 9 months. Seroconversion or seropositive juvenile dogs indicated recent local plague circulation. In Madagascar, dog surveillance for plague antibody could be useful to identify plague circulation in new areas or quiescent areas within endemic zones. Within active endemic areas, monitoring of dog populations for seroconversion (negative to positive) or seropositive juvenile dogs could be useful for identifying areas at greatest risk of human outbreaks.

Plague.

Bubonic plague is an often fulminant systemic zoonosis, caused by Yersinia pestis. Conventional microbiology, bacterial population genetics, and genome sequence data, all suggest that Y pestis is a recently evolved clone of the enteric pathogen Yersinia pseudotuberculosis. The genetic basis of this organism's rapid adaptation to its insect vector (the flea) with transmission between mammalian hosts by novel subcutaneous and pneumonic routes of infection is becoming clearer. This transition provides a paradigm for the way in which new pathogens could emerge. Plague in humans is controlled by suppression of rodent reservoir hosts and their fleas and by early detection and treatment of cases of disease. Detection systems for plague in non-endemic regions might now be needed because of a bioterrorism threat. Rapid diagnostic tests are available and a subunit vaccine is in clinical trials.

Technical note: a rapid diagnostic test detects plague in ancient human remains: an example of the interaction between archeological and biological approaches (southeastern France, 16th-18th centuries).

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.

[A rapid diagnostic test for plague detects Yersinia pestis F1 antigen in ancient human remains]. / Détection de l'antigène F1 de Yersinia pestis dans les restes humains anciens à l'aide d'un test de diagnostic rapide.

A rapid diagnostic dipstick test (RDT) that detects Yersinia pestis F1 antigen has been recently applied on 18 putative plague victims exhumed from four archaeological burial sites in southeastern France dating back to the 16(th), 17(th) and 18(th) centuries. The Y. pestis antigen F1 was detected in 12 ancient samples out of 18 (67%). Negative controls confirmed their negativity (100%). Our results emphasize that the detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a first retrospective diagnosis of Y. pestis infection in ancient remains, and confirm the high specificity and sensitivity of the assay. Double-blind analyses performed by using two different techniques (RDT and 'suicide PCR') led us to the identification of the Y. pestis F1 antigen and the Y. pestis pla and gplD genes. These data provide clear evidence of the presence of Y. pestis in the examined specimens.

Temporal phylogeography of Yersinia pestis in Madagascar: Insights into the long-term maintenance of plague.

BACKGROUND: Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. METHODOLOGY/PRINCIPAL FINDINGS: We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. CONCLUSIONS/SIGNIFICANCE: Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location.

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague.

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.

A non-stationary relationship between global climate phenomena and human plague incidence in Madagascar.

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.

Plague circulation and population genetics of the reservoir Rattus rattus: the influence of topographic relief on the distribution of the disease within the Madagascan focus.

BACKGROUND: Landscape may affect the distribution of infectious diseases by influencing the population density and dispersal of hosts and vectors. Plague (Yersinia pestis infection) is a highly virulent, re-emerging disease, the ecology of which has been scarcely studied in Africa. Human seroprevalence data for the major plague focus of Madagascar suggest that plague spreads heterogeneously across the landscape as a function of the relief. Plague is primarily a disease of rodents. We therefore investigated the relationship between disease distribution and the population genetic structure of the black rat, Rattus rattus, the main reservoir of plague in Madagascar. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a comparative study of plague seroprevalence and genetic structure (15 microsatellite markers) in rat populations from four geographic areas differing in topology, each covering about 150-200 km(2) within the Madagascan plague focus. The seroprevalence levels in the rat populations mimicked those previously reported for humans. As expected, rat populations clearly displayed a more marked genetic structure with increasing relief. However, the relationship between seroprevalence data and genetic structure differs between areas, suggesting that plague distribution is not related everywhere to the effective dispersal of rats. CONCLUSIONS/SIGNIFICANCE: Genetic diversity estimates suggested that plague epizootics had only a weak impact on rat population sizes. In the highlands of Madagascar, plague dissemination cannot be accounted for solely by the effective dispersal of the reservoir. Human social activities may also be involved in spreading the disease in rat and human populations.

Contrasted patterns of selection on MHC-linked microsatellites in natural populations of the Malagasy plague reservoir.

Plague (Yersinia pestis infection) is a highly virulent rodent disease that persists in many natural ecosystems. The black rat (Rattus rattus) is the main host involved in the plague focus of the central highlands of Madagascar. Black rat populations from this area are highly resistant to plague, whereas those from areas in which the disease is absent (low altitude zones of Madagascar) are susceptible. Various lines of evidence suggest a role for the Major Histocompatibility Complex (MHC) in plague resistance. We therefore used the MHC region as a candidate for detecting signatures of plague-mediated selection in Malagasy black rats, by comparing population genetic structures for five MHC-linked microsatellites and neutral markers in two sampling designs. We first compared four pairs of populations, each pair including one population from the plague focus and one from the disease-free zone. Plague-mediated selection was expected to result in greater genetic differentiation between the two zones than expected under neutrality and this was observed for one MHC-class I-linked locus (D20Img2). For this marker as well as for four other MHC-linked loci, a geographic pattern of genetic structure was found at local scale within the plague focus. This pattern would be expected if plague selection pressures were spatially variable. Finally, another MHC-class I-linked locus (D20Rat21) showed evidences of balancing selection, but it seems more likely that this selection would be related to unknown pathogens more widely distributed in Madagascar than plague.

Immune responses to plague infection in wild Rattus rattus, in Madagascar: a role in foci persistence?

BACKGROUND: Plague is endemic within the central highlands of Madagascar, where its main reservoir is the black rat, Rattus rattus. Typically this species is considered susceptible to plague, rapidly dying after infection inducing the spread of infected fleas and, therefore, dissemination of the disease to humans. However, persistence of transmission foci in the same area from year to year, supposes mechanisms of maintenance among which rat immune responses could play a major role. Immunity against plague and subsequent rat survival could play an important role in the stabilization of the foci. In this study, we aimed to investigate serological responses to plague in wild black rats from endemic areas of Madagascar. In addition, we evaluate the use of a recently developed rapid serological diagnostic test to investigate the immune response of potential reservoir hosts in plague foci. METHODOLOGY/PRINCIPAL FINDINGS: We experimentally infected wild rats with Yersinia pestis to investigate short and long-term antibody responses. Anti-F1 IgM and IgG were detected to evaluate this antibody response. High levels of anti-F1 IgM and IgG were found in rats one and three weeks respectively after challenge, with responses greatly differing between villages. Plateau in anti-F1 IgM and IgG responses were reached for as few as 500 and 1500 colony forming units (cfu) inoculated respectively. More than 10% of rats were able to maintain anti-F1 responses for more than one year. This anti-F1 response was conveniently followed using dipsticks. CONCLUSION/SIGNIFICANCE: Inoculation of very few bacteria is sufficient to induce high immune response in wild rats, allowing their survival after infection. A great heterogeneity of rat immune responses was found within and between villages which could heavily impact on plague epidemiology. In addition, results indicate that, in the field, anti-F1 dipsticks are efficient to investigate plague outbreaks several months after transmission.

Factors associated with negative direct sputum examination in Asian and African HIV-infected patients with tuberculosis (ANRS 1260).

OBJECTIVE: To identify factors associated with negative direct sputum examination among African and Cambodian patients co-infected by Mycobacterium tuberculosis and HIV. DESIGN: Prospective multicenter study (ANRS1260) conducted in Cambodia, Senegal and Central African Republic. METHODS: Univariate and multivariate analyses (logistic regression) were used to identify clinical and radiological features associated with negative direct sputum examination in HIV-infected patients with positive M. tuberculosis culture on Lowenstein-Jensen medium. RESULTS: Between September 2002 and December 2005, 175 co-infected patients were hospitalized with at least one respiratory symptom and pulmonary radiographic anomaly. Acid-fast bacillus (AFB) examination was positive in sputum samples from 110 subjects (63%) and negative in 65 patients (37%). Most patients were at an advanced stage of HIV disease (92% at stage III or IV of the WHO classification) with a median CD4 cell count of 36/mm³. In this context, we found that sputum AFB negativity was more frequent in co-infected subjects with associated respiratory tract infections (OR = 2.8 [95%CI:1.1-7.0]), dyspnea (OR = 2.5 [95%CI:1.1-5.6]), and localized interstitial opacities (OR = 3.1 [95%CI:1.3-7.6]), but was less frequent with CD4 ≤ 50/mm³ (OR = 0.4 [95%CI:0.2-0.90), adenopathies (OR = 0.4 [95%CI:0.2-0.93]) and cavitation (OR = 0.1 [95%CI:0.03-0.6]). CONCLUSIONS: One novel finding of this study is the association between concomitant respiratory tract infection and negative sputum AFB, particularly in Cambodia. This finding suggests that repeating AFB testing in AFB-negative patients should be conducted when broad spectrum antibiotic treatment does not lead to complete recovery from respiratory symptoms. In HIV-infected patients with a CD4 cell count below 50/mm3 without an identified cause of pneumonia, systematic AFB direct sputum examination is justified because of atypical clinical features (without cavitation) and high pulmonary mycobacterial burden.

Phylogeography and molecular epidemiology of Yersinia pestis in Madagascar.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Study on the movement of Rattus rattus and evaluation of the plague dispersion in Madagascar.

Plague affects mainly the rural areas in the central highlands of Madagascar. Rattus rattus is the main rodent host of Yersinia pestis in these localities. Since the introduction of plague, endemic foci have continued to expand, and spatiotemporal variability in the distribution of human plague has been observed. To assess the movements of R. rattus and evaluate the risk of dispersion of the disease, a field study at the scale of the habitats (houses, hedges of sisals, and rice fields) in the plague villages was carried out during high and low seasons of plague transmission to humans. The systemic oral marker Rhodamine B was used to follow rats' movements. Baits were placed in different habitats, and trapping success was carried out once a month for 3 months after the bait distribution. Plague indicators (reservoirs' abundance, flea index, Y. pestis prevalence in fleas, and Y. pestis antibody prevalence in rats) were determined. The highest abundance of rats and marking efficiency were observed in the sisal hedges and the rice fields. Marked rats were captured most commonly near the points where baits were initially placed. The main movements of rats were observed between the houses and sisal hedges. Major differences were observed between the seasons of high and low plague transmission. During the season of low plague transmission, rats were more abundant in the sisal hedges and rice fields, with rats moving from the houses to the rice fields. During the high plague transmission season, rats moved from the hedges of sisal to the rice fields. Important indicators of vector abundance and plague transmission were higher during the high plague transmission season. The three study habitats were the risk areas for plague transmission, but the risk appeared highest in the houses and sisals. Rats' movements according to the season were likely directed by the availability of food.

First isolation and direct evidence for the existence of large small-mammal reservoirs of Leptospira sp. in Madagascar.

BACKGROUND: Leptospirosis has long been a major public health concern in the southwestern Indian Ocean. However, in Madagascar, only a few, old studies have provided indirect serological evidence of the disease in humans or animals. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a large animal study focusing on small-mammal populations. Five field trapping surveys were carried out at five sites, from April 2008 to August 2009. Captures consisted of Rattus norvegicus (35.8%), R. rattus (35.1%), Mus musculus (20.5%) and Suncus murinus (8.6%). We used microbiological culture, serodiagnosis tests (MAT) and real-time PCR to assess Leptospira infection. Leptospira carriage was detected by PCR in 91 (33.9%) of the 268 small mammals, by MAT in 17 of the 151 (11.3%) animals for which serum samples were available and by culture in 9 of the 268 animals (3.3%). Rates of infection based on positive PCR results were significantly higher in Moramanga (54%), Toliara (48%) and Mahajanga (47.4%) than in Antsiranana (8.5%) and Toamasina (14%) (p = 0.001). The prevalence of Leptospira carriage was significantly higher in R. norvegicus (48.9%), S. murinus (43.5%) and R. rattus (30.8%) than in M. musculus (9.1%) (p<0.001). The MAT detected antibodies against the serogroups Canicola and Icterohaemorrhagiae. Isolates were characterized by serology, secY sequence-based phylogeny, partial sequencing of rrs, multi-locus VNTR analysis and pulsed field gel electrophoresis. The 10 isolates obtained from nine rats were all identified as species L. interrogans serogroup Canicola serovar Kuwait and all had identical partial rrs and secY sequences. CONCLUSIONS/SIGNIFICANCE: We present here the first direct evidence of widespread leptospiral carriage in small mammals in Madagascar. Our results strongly suggest a high level of environmental contamination, consistent with probable transmission of the infection to humans. This first isolation of pathogenic Leptospira strains in this country may significantly improve the detection of specific antibodies in human cases.

Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity.

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.