DNA databases of an important tropical timber tree species Shorea leprosula (Dipterocarpaceae) for forensic timber identification.

International timber trade communities are increasingly demanding that timber in the wood supply chain be sourced from sustainably harvested forests and certified plantations. This is to combat illegal logging activities to prevent further depletion of our precious forests worldwide. Hence, timber tracking tools are important to support law enforcement officials in ensuring only sustainably harvested timbers are traded in the market. In this study, we developed chloroplast DNA (cpDNA) and simple sequence repeat (SSR) databases as tracking tools for an important tropical timber tree species, Shorea leprosula from Peninsular Malaysia. A total of 1410 individual trees were sampled from 44 natural populations throughout Peninsular Malaysia. Four cpDNA regions were used to generate a cpDNA haplotype database, resulting in a haplotype map comprising 22 unique haplotypes derived from 28 informative intraspecific variable sites. This cpDNA database can be used to trace the origin of an unknown log at the regional level. Ten SSR loci were used to develop the SSR allele frequency database. Bayesian cluster analysis divided the 44 populations into two genetic clusters corresponding to Region A and Region B. Based on conservativeness evaluation of the SSR databases for individual identification, the coancestry coefficients (θ) were adjusted to 0.1900 and 0.1500 for Region A and B, respectively. These databases are useful tools to complement existing timber tracking systems in ensuring only legally sourced timbers are allowed to enter the wood supply chain.

Non-tandem repeat polymorphisms at microsatellite loci in wine yeast species.

Yeast microsatellite loci consist of short tandem-repeated DNA sequences of variable length. The high mutational rate at these loci generates a remarkable repertoire of alleles, useful for strain differentiation and population genetic studies. In this work, we analyze the DNA sequences of thirteen alleles from each of ten microsatellite loci described for the yeast Starmerella bacillaris. Our results show that polymorphic variants of some informative alleles are dependent on SNPs and indels rather than on length variation at their originally defined tandem-repeated motifs. The analysis was extended to 55 previously described hypervariable microsatellite loci from a total of 26 sequenced genomes of yeast species that dominate the microbiota of spontaneously fermenting grape musts (i.e., Hanseniaspora uvarum, Saccharomyces cerevisae, Saccharomyces uvarum, and Torulaspora delbrueckii) or lead to wine spoilage (Brettanomyces bruxellensis and Meyerozyma guilliermondii). We found that allelic variants for some microsatellite loci of these yeast species are also dependent on SNPs and/or indels flanking their tandem-repeated motifs. For some loci, the number of units at their tandem repeats was found to be identical among the various characterized alleles, with allelic differences being dependent exclusively on flanking polymorphisms. Our results indicate that allele sizing of microsatellite loci using PCR, although valid for strain differentiation and population genetic studies, does not necessarily score the number of units at their tandem-repeated motifs. Sequence analysis of microsatellite loci alleles could provide relevant information for evolutionary and phylogeny studies of yeast species.

Multiple strain infection of Mycobacterium leprae in a family having 4 patients: A study employing short tandem repeats.

BACKGROUND: Leprosy is a slow, chronic disorder caused by Mycobacterium leprae. India has achieved elimination of leprosy in December 2005 but new cases are being detected and continue to occur in some endemic pockets. The possible ways of transmission of leprosy is not fully understood and is believed that leprosy is transmitted from person to person in long term contact. Studying the transmission dynamics is further complicated by inability to grow M. leprae in culture medium and lack of animal models. More than one family members were found to be affected by leprosy in some highly endemic pockets. This study reported the transmission pattern of leprosy in a family having 4 patients. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the transmission of leprosy in a single family having 4 patients using microsatellite typing. DNA was isolated from slit skin smear samples taken from the patients and the isolated DNA were amplified using microsatellite loci TA11CA3. The amplified products were sequenced using Sanger's sequencing methods and the copy number variation in the microsatellite loci between strains were elucidated by multiple sequence alignment. The result showed that all the 4 members of the family acquired infection from 3 different strains of M. leprae from 3 different sources. The elder and middle daughters were infected by same types of strains having the repeat unit TA13CA3 and could have acquired the infection from social contacts of leprosy cases while the father and younger daughter were infected by strains with the repeat unit TA12CA3 and TA11CA3 and could have acquired infection from social contacts. CONCLUSIONS/SIGNIFICANCE: The study suggested that three family members viz, elder daughter, father and younger daughter could be infected by M. leprae from 3 different sources and the history of the disease and genetic analysis showed that the middle daughter acquired infection from her elder sister in due course of contact. This study implies that the transmission of leprosy not only occurred amongst the house hold members but also has been transmitted from social and neighborhood contacts in long term association with the them.

Are patterns of fine-scale spatial genetic structure consistent between sites within tropical tree species?

Documenting the scale and intensity of fine-scale spatial genetic structure (FSGS), and the processes that shape it, is relevant to the sustainable management of genetic resources in timber tree species, particularly where logging or fragmentation might disrupt gene flow. In this study we assessed patterns of FSGS in three species of Dipterocarpaceae (Parashorea tomentella, Shorea leprosula and Shorea parvifolia) across four different tropical rain forests in Malaysia using nuclear microsatellite markers. Topographic heterogeneity varied across the sites. We hypothesised that forests with high topographic heterogeneity would display increased FSGS among the adult populations driven by habitat associations. This hypothesis was not supported for S. leprosula and S. parvifolia which displayed little variation in the intensity and scale of FSGS between sites despite substantial variation in topographic heterogeneity. Conversely, the intensity of FSGS for P. tomentella was greater at a more topographically heterogeneous than a homogeneous site, and a significant difference in the overall pattern of FSGS was detected between sites for this species. These results suggest that local patterns of FSGS may in some species be shaped by habitat heterogeneity in addition to limited gene flow by pollen and seed dispersal. Site factors can therefore contribute to the development of FSGS. Confirming consistency in species' FSGS amongst sites is an important step in managing timber tree genetic diversity as it provides confidence that species specific management recommendations based on species reproductive traits can be applied across a species' range. Forest managers should take into account the interaction between reproductive traits and site characteristics, its consequences for maintaining forest genetic resources and how this might influence natural regeneration across species if management is to be sustainable.

Association between STR -794 CATT5-8 and SNP -173 G/C polymorphisms in the MIF gene and Lepromatous Leprosy in Mestizo patients of western Mexico.

Lepromatous Leprosy (LL) is the most common presentation of leprosy in Mexico. LL patients are unable to activate an effective inflammatory response against Mycobacterium leprae probably due to the genetics of the host. Macrophage Migration Inhibitory Factor (MIF) is important to trigger inflammation processes. Two polymorphisms have been reported for human MIF: STR -794 CATT5-8 and SNP -173 G/C. 7-8 CATT repeats at -794 and the C allele at -173 increase the expression of MIF. We aim to determine the association between the polymorphisms in MIF gene and LL. We carried a case and controls study with 100 Mexican LL patients and 100 healthy subjects (HS). PCR was used for genotyping of STR -794 CATT5-8 polymorphism and PCR-RFLP for -173 G/C. We found that LL patients possess high -794 CATT repeats (47.1%) more often than HS (32.7%). In conclusion, a MIF polymorphism is associated with susceptibility to LL in Western Mexican population.

Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India.

Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community.

A historical overview of leprosy epidemiology and control activities in Amazonas, Brazil

Leprosy is an ancient infectious disease caused by Mycobacterium leprae. According to comparative genomics studies, this disease originated in Eastern Africa or the Near East and spread with successive human migrations. The Europeans and North Africans introduced leprosy into West Africa and the Americas within the past 500 years. In Brazil, this disease arrived with the colonizers who disembarked at the first colonies, Rio de Janeiro, Salvador and Recife, at the end of the sixteenth century, after which it was spread to the other states. In 1854, the first leprosy cases were identified in State of Amazonas in the north of Brazil. The increasing number of leprosy cases and the need for treatment and disease control led to the creation of places to isolate patients, known as leprosaria. One of them, Colonia Antônio Aleixo was built in Amazonas in 1956 according to the most advanced recommendations for isolation at that time and was deactivated in 1979. The history of the Alfredo da Matta Center (AMC), which was the first leprosy dispensary created in 1955, parallels the history of leprosy in the state. Over the years, the AMC has become one of the best training centers for leprosy, general dermatology and sexually transmitted diseases in Brazil. In addition to being responsible for leprosy control programs in the state, the AMC has carried out training programs on leprosy diagnosis and treatment for health professionals in Manaus and other municipalities of the state, aiming to increase the coverage of leprosy control activities. This paper provides a historical overview of leprosy in State of Amazonas, which is an endemic state in Brazil.

Tracking climate change in a dispersal-limited species: reduced spatial and genetic connectivity in a montane salamander.

Tropical montane taxa are often locally adapted to very specific climatic conditions, contributing to their lower dispersal potential across complex landscapes. Climate and landscape features in montane regions affect population genetic structure in predictable ways, yet few empirical studies quantify the effects of both factors in shaping genetic structure of montane-adapted taxa. Here, we considered temporal and spatial variability in climate to explain contemporary genetic differentiation between populations of the montane salamander, Pseudoeurycea leprosa. Specifically, we used ecological niche modelling (ENM) and measured spatial connectivity and gene flow (using both mtDNA and microsatellite markers) across extant populations of P. leprosa in the Trans-Mexican Volcanic Belt (TVB). Our results indicate significant spatial and genetic isolation among populations, but we cannot distinguish between isolation by distance over time or current landscape barriers as mechanisms shaping population genetic divergences. Combining ecological niche modelling, spatial connectivity analyses, and historical and contemporary genetic signatures from different classes of genetic markers allows for inference of historical evolutionary processes and predictions of the impacts future climate change will have on the genetic diversity of montane taxa with low dispersal rates. Pseudoeurycea leprosa is one montane species among many endemic to this region and thus is a case study for the continued persistence of spatially and genetically isolated populations in the highly biodiverse TVB of central Mexico.

Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy.

Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.

IFN-γ +875 microsatellite polymorphism as a potential protection marker for leprosy patients from Amazonas state, Brazil.

Polymorphisms present in the first intron of IFN-γ may have an important role in the regulation of the immune response, which could have functional consequences for gene transcription. Leprosy patients are characterized by different immune responses in different clinical forms. We investigated a possible association of the +874 polymorphism and CA repeats present in the first intron of IFN-γ with susceptibility to leprosy and with the manifestation of the different clinical forms. Nucleotide sequencing was performed with samples from 108 leprosy patients and 113 controls subjects, as well as immunophenotyping of CD(4)(+), CD(8)(+) and CD(69)(+) T cells by flow cytometry. The data showed that there were no significant differences between patients and control subjects, as well as according classification of Ridley-Jopling. However, the A/A genotype was significantly increased in paucibacillary patients (p=0.028) and the microsatellite encoding 16 CA repeats were significantly associated with paucibacillary compared to multibacillary patients (p=0.019). Individuals homozygous for the +874 A allele, the mean level of CD(4)(+) and CD(69)(+) T cells was higher. Our data suggest that polymorphisms present in the first intron of IFN-γ are not associated with susceptibility to leprosy, nevertheless, the +874 polymorphism and the CA repeats number encoded in IFN-γ gene may be related to a higher cellular immune response in patients and are consistently more frequently detected in PB patients.

Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy.

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy. OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification. METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene. RESULTS: M.leprae bacillus could be detected in 22 (78.6%) of the 28 samples. 9 (45%) of the 20 biopsy samples and 6 (75%) of the 8 imprints were positive to TTC. 7 (35.5%) skin biopsy specimens and 5 (62.5%) imprints were positive to AGT, and 11 (55%) biopsies and 4 (50%) were positive to AGT. 11 (55%) skin biopsies and 4 (50%) imprints were positive to AT. 8(38%) skin biopsies and 5 (62.5%) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5% of the samples, and the Ml MntH gene in 57.1% of the samples, independent of the clinical material. In the PB group 55.5% of samples were positive to the microsatellite primers, while 22.2% were positive to the Ml MntH gene. CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprints.

Comparação entre microssatélites e o gene Ml MntH como alvos para a identificação do Mycobacterium leprae por PCR na hanseníase / Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy

FUNDAMENTOS: PCR tem sido frequentemente utilizada no diagnóstico molecular da hanseníase. OBJETIVOS: comparar os resultados da PCR com 4 pares de primers específicos para Mycobacterium leprae, bem como os resultados da PCR à classificação operacional, segundo a OMS, de multibacilar (MB) e paucibacilar (PB) da hanseníase. MÉTODO: Vinte e oito amostras de DNA, extraído de biópsias congeladas de pele e de imprint de biópsias em papel de filtro de 23 pacientes (14 MB e 9 PB), foram utilizadas na PCR com primers que amplificam 131pb, 151pb e 168pb de regiões de microssatélites, e um fragmento de 336pb do gene Ml MntH (ML2098) do bacilo. RESULTADOS: O bacilo pôde ser detectado em 22 (78,6 por cento) das 28 amostras. Nove (45 por cento) das 20 amostras de biópsia e 6 (75 por cento) das 8 amostras de imprints foram positivas para TTC. Sete (35,5 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para AGT, e 11 (55 por cento) biópsias e 4 (50 por cento) imprints foram positivos para AT. Oito (38 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para o gene Ml MntH. Dentre o grupo MB, os microssatélites detectaram o bacilo em 78,5 por cento das amostras, e o gene Ml MntH, em 57,1 por cento das amostras, independentemente do material clínico. No grupo PB, 55,5 por cento das amostras foram positivas para os microssatélites, enquanto que 22,2 por cento o foram para o gene Ml MntH. CONCLUSÕES: Estes resultados mostram que, tanto as regiões específicas de microssatélites quanto o gene Ml MntH, podem representar ferramentas úteis na detecção do Ml MntH por PCR em amostras de biópsias e imprint de biópsias.

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy. OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification. METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene. RESULTS: M.leprae bacillus could be detected in 22 (78.6 percent) of the 28 samples. 9 (45 percent) of the 20 biopsy samples and 6 (75 percent) of the 8 imprints were positive to TTC. 7 (35.5 percent) skin biopsy specimens and 5 (62.5 percent) imprints were positive to AGT, and 11 (55 percent) biopsies and 4 (50 percent) were positive to AGT. 11 (55 percent) skin biopsies and 4 (50 percent) imprints were positive to AT. 8(38 percent) skin biopsies and 5 (62.5 percent) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5 percent of the samples, and the Ml MntH gene in 57.1 percent of the samples, independent of the clinical material. In the PB group 55.5 percent of samples were positive to the microsatellite primers, while 22.2 percent were positive to the Ml MntH gene. CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprints.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

BACKGROUND & OBJECTIVES: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. METHODS: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. RESULTS: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. INTERPRETATION & CONCLUSIONS: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Characterisation of short tandem repeats for genotyping Mycobacterium leprae.

OBJECTIVE: Establish a typing system for Mycobacterium leprae based on polymorphic DNA structures known as short tandem repeats (STR). DESIGN: Assess 16 polymorphic STR for sensitivity, specificity and reproducibility in standard assays using reference strains of M. leprae. RESULTS: Primers for 16 STR loci were selected based on PCR product size and for their ability to sequence each STR locus from both directions. All primer pairs produced a visible PCR amplicon of appropriate size from PCR reactions containing 10 M. leprae cells. DNA sequences for each STR locus, except (AT) 15, was correctly identified as M. leprae-specific in replicate samples containing 1000 M. leprae using either the forward or reverse PCR primers. Twelve of 13 M. leprae STR loci were stable during passage in heavily infected armadillo tissues over a 5 year and 7 month infection cycle. CONCLUSIONS: Certain M. leprae STR provide suitable targets for strain typing with the potential for grouping M. leprae with shared genotypes that may prove useful for establishing linkages between leprosy cases within geographical regions.

Tales from scales: old DNA yields insights into contemporary evolutionary processes affecting fishes.

Salmon and trout populations are suffering declines in abundance and diversity over much of their range around the Atlantic and Pacific rims as a consequence of many factors. One method of dealing with the decline has been to produce them in hatcheries but the wisdom of this approach has been hotly debated (e.g. Hilborn & Winton 1993; Waples 1999; Brannon et al. 2004). One concern is that domesticated hatchery strains will interbreed with locally adapted wild fish; but how do we study the genetic effects if the introgression might have occurred in the past? Hansen (2002) used DNA isolated from archived scales from brown trout, Salmo trutta (Fig. 1), to show that domesticated trout had, to varying degrees, genetically introgressed with wild, native trout in two Danish rivers. Extending that study, Hansen et al. (2009) have examined DNA from brown trout scales in six Danish rivers collected during historical (1927-1956) and contemporary (2000-2006) periods and from two hatchery source populations, to assess the effects of stocking nonlocal strains of hatchery trout and declining abundance on genetic diversity. Using 21 microsatellite loci, they revealed that genetic change occurred between the historic and contemporary time periods. Many populations appeared to have some low level of introgression from hatchery stocks and two populations apparently experienced high levels of introgression. Hansen et al. (2009) also showed that population structure persists in contemporary populations despite apparent admixture and migration among populations, providing evidence that the locally adapted populations have struggled against and, to some extent, resisted being overwhelmed by repeated introductions of and interbreeding with non-native, hatchery-produced conspecifics.

Consanguinity and susceptibility to infectious diseases in humans.

Studies of animal populations suggest that low genetic heterozygosity is an important risk factor for infection by a diverse range of pathogens, but relatively little research has looked to see whether similar patterns exist in humans. We have used microsatellite genome screen data for tuberculosis (TB), hepatitis and leprosy to test the hypothesis that inbreeding depression increases risk of infection. Our results indicate that inbred individuals are more common among our infected cases for TB and hepatitis, but only in populations where consanguineous marriages are common. No effect was found either for leprosy, which is thought to be oligogenic, or for hepatitis in Italy where consanguineous marriages are rare. Our results suggest that consanguinity is an important risk factor in susceptibility to infectious diseases in humans.

Is leprosy spreading among nine-banded armadillos in the southeastern United States?

In the United States, nine-banded armadillo (Dasypus novemcinctus) populations are derived from two sources: (1) a continuous range expansion from Mexico led to western populations, some of which, particularly along the western Gulf Coast and west side of the Mississippi River delta, exhibit persistently high rates of leprosy infection, and (2) a small group of animals released from captivity in Florida gave rise to eastern populations that were all considered leprosy free. Given that western and eastern populations have now merged, an important question becomes, to what extent is leprosy spreading into formerly uninfected populations? To answer this question, we sampled 500 animals from populations in Mississippi, Alabama, and Georgia. Analyses of nuclear microsatellite DNA markers confirmed the historic link between source populations from Texas and Florida, but did not permit resolution of the extent to which these intermediate populations represented eastern versus western gene pools. Prevalence of leprosy was determined by screening blood samples for the presence of antibodies against Mycobacterium leprae and via polymerase chain reaction amplification of armadillo tissues to detect M. leprae DNA. The proportion of infected individuals within each population varied from 0% to 10%. Although rare, a number of positive individuals were identified in eastern sites previously considered uninfected. This indicates leprosy may be spreading eastward and calls into question hypotheses proposing leprosy infection is confined because of ecologic constraints to areas west of the Mississippi River.

Use of short tandem repeat sequences to study Mycobacterium leprae in leprosy patients in Malawi and India.

BACKGROUND: Inadequate understanding of the transmission of Mycobacterium leprae makes it difficult to predict the impact of leprosy control interventions. Genotypic tests that allow tracking of individual bacterial strains would strengthen epidemiological studies and contribute to our understanding of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Genotyping assays based on variation in the copy number of short tandem repeat sequences were applied to biopsies collected in population-based epidemiological studies of leprosy in northern Malawi, and from members of multi-case households in Hyderabad, India. In the Malawi series, considerable genotypic variability was observed between patients, and also within patients, when isolates were collected at different times or from different tissues. Less within-patient variability was observed when isolates were collected from similar tissues at the same time. Less genotypic variability was noted amongst the closely related Indian patients than in the Malawi series. CONCLUSIONS/SIGNIFICANCE: Lineages of M. leprae undergo changes in their pattern of short tandem repeat sequences over time. Genetic divergence is particularly likely between bacilli inhabiting different (e.g., skin and nerve) tissues. Such variability makes short tandem repeat sequences unsuitable as a general tool for population-based strain typing of M. leprae, or for distinguishing relapse from reinfection. Careful use of these markers may provide insights into the development of disease within individuals and for tracking of short transmission chains.

Toll-like receptor 2 (TLR2) polymorphisms are associated with reversal reaction in leprosy.

BACKGROUND: Leprosy is characterized by a spectrum of clinical manifestations that depend on the type of immune response against the pathogen. Patients may undergo immunological changes known as "reactional states" (reversal reaction and erythema nodosum leprosum) that result in major clinical deterioration. The goal of the present study was to assess the effect of Toll-like receptor 2 (TLR2) polymorphisms on susceptibility to and clinical presentation of leprosy. METHODS: Three polymorphisms in TLR2 (597C-->T, 1350T-->C, and a microsatellite marker) were analyzed in 431 Ethiopian patients with leprosy and 187 control subjects. The polymorphism-associated risk of developing leprosy, lepromatous (vs. tuberculoid) leprosy, and leprosy reactions was assessed by multivariate logistic regression models. RESULTS: The microsatellite and the 597C-->T polymorphisms both influenced susceptibility to reversal reaction. Although the 597T allele had a protective effect (odds ratio [OR], 0.34 [95% confidence interval {CI}, 0.17-0.68]; P= .002 under the dominant model), homozygosity for the 280-bp allelic length of the microsatellite strongly increased the risk of reversal reaction (OR, 5.83 [95% CI, 1.98-17.15]; P= .001 under the recessive model). These associations were consistent among 3 different ethnic groups. CONCLUSIONS: These data suggest a significant role for TLR-2 in the occurrence of leprosy reversal reaction and provide new insights into the immunogenetics of the disease.

Simple sequence repeats in mycobacterial genomes.

Simple sequence repeats (SSRs) or microsatellites are the repetitive nucleotide sequences of motifs of length 1-6 bp. They are scattered throughout the genomes of all the known organisms ranging from viruses to eukaryotes. Microsatellites undergo mutations in the form of insertions and deletions (INDELS) of their repeat units with some bias towards insertions that lead to microsatellite tract expansion. Although prokaryotic genomes derive some plasticity due to microsatellite mutations they have in-built mechanisms to arrest undue expansions of microsatellites and one such mechanism is constituted by post-replicative DNA repair enzymes MutL, MutH and MutS. The mycobacterial genomes lack these enzymes and as a null hypothesis one could expect these genomes to harbour many long tracts. It is therefore interesting to analyse the mycobacterial genomes for distribution and abundance of microsatellites tracts and to look for potentially polymorphic microsatellites. Available mycobacterial genomes, Mycobacterium avium, M. leprae, M. bovis and the two strains of M. tuberculosis (CDC1551 and H37Rv) were analysed for frequencies and abundance of SSRs. Our analysis revealed that the SSRs are distributed throughout the mycobacterial genomes at an average of 220-230 SSR tracts per kb. All the mycobacterial genomes contain few regions that are conspicuously denser or poorer in microsatellites compared to their expected genome averages. The genomes distinctly show scarcity of long microsatellites despite the absence of a post-replicative DNA repair system. Such severe scarcity of long microsatellites could arise as a result of strong selection pressures operating against long and unstable sequences although influence of GC-content and role of point mutations in arresting microsatellite expansions can not be ruled out. Nonetheless, the long tracts occasionally found in coding as well as non-coding regions may account for limited genome plasticity in these genomes.