Variable Number Tandem Repeat Profiles Research Articles

Identification of Pathogenic Leptospira kirschneri Serogroup Grippotyphosa in Water Voles (Arvicola terrestris) from Ruminant Pastures in Puy-de-Dôme, Central France

Rodents are the primary reservoirs for pathogenic Leptospira species, which cause leptospirosis. Among the key potential carriers are water voles, whose population outbreaks can consequently pose a major threat to human and animal health. We studied the prevalence, prominence, and epidemiology of pathogenic Leptospira species in water voles in central France. First, 46 voles were captured, and DNA was extracted from kidney, lung, liver, blood, and urine and tested for the presence of Leptospira using three molecular methods: PCR, O-antigen typing, and variable number tandem repeat (VNTR) typing. We also attempted to culture leptospires from kidney and urine samples. In addition, we investigated leptospiral antibodies in serum samples from 60 sheep using microscopic agglutination testing. These animals co-occurred with the voles, so we sought to assess their degree of exposure and involvement in pathogen dynamics. The overall prevalence of infection was 76.1% (CI95% [61.2%, 87.4%]). The only strain found was L. kirschneri serogroup Grippotyphosa and a similar VNTR profile was acquired. Leptospires were successfully cultured from kidney and urine samples for four voles. Three sheep had low antibody titers against the Leptospira serogroup Grippotyphosa. Taken together, our results suggest the exclusive carriage of L. kirschneri serogroup Grippotyphosa among water voles in central France. Nevertheless, their ability to act as reservoir hosts that transmit the pathogen to co-occurring livestock remains unclear and merits further research.

Variable Number Tandem Repeat Profiling of Mycobacterium ulcerans Reveals New Genotypes in Buruli Ulcer Endemic Communities in Ghana and Côte d'Ivoire.

Buruli ulcer (BU), a necrotic skin disease caused by Mycobacterium ulcerans, is mainly prevalent in West Africa, but cases have also been reported in other tropical parts of the world. It is the second most common mycobacterial disease after tuberculosis in Ghana and Côte d’Ivoire. Heterogeneity among M. ulcerans from different geographical locations has not been clearly elucidated, and some studies seem to suggest genetic differences between M. ulcerans in humans and in the environment. This study aimed at identifying genetic differences among M. ulcerans strains between two BU endemic countries: Ghana and Côte d’Ivoire. Clinical samples consisting of swabs, fine needle aspirates, and tissue biopsies of suspected BU lesions and environmental samples (e.g., water, biofilms from plants, soil, and detrital material) were analyzed. BU cases were confirmed via acid fast staining and PCR targeting the 16S rRNA, IS2404, IS2606, and ER domain genes present on M. ulcerans. Heterogeneity among M. ulcerans was determined through VNTR profiling targeting 10 loci. Eleven M. ulcerans genotypes were identified within the clinical samples in both Ghana and Côte d’Ivoire, whiles six M. ulcerans genotypes were found among the environmental samples. Clinical M. ulcerans genotypes C, D, F, and G were common in both countries. Genotype E was unique among the Ghanaian samples, whiles genotypes A, Z, J, and K were unique to the Ivorian samples. Environmental isolates were found to be more conserved compared with the clinical isolates. Genotype W was observed only among the Ghanaian environmental samples. Genotype D was found to be prominent in both clinical and environmental samples, suggesting evidence of possible transmission of M. ulcerans from the environment, particularly water bodies and biofilms from aquatic plants, to humans through open lesions on the skin.

Genotyping of Mycobacterium leprae for understanding the distribution and transmission of leprosy in endemic provinces of China

ObjectivesUnderstanding the nature of Mycobacterium leprae transmission is vital to implement better control strategies for leprosy elimination. The present study expands the knowledge of county-level strain diversity, distribution, and transmission patterns of leprosy in endemic provinces of China. MethodsWe genetically characterized 290 clinical isolates of M. leprae from four endemic provinces using variable number tandem repeats (VNTR) and single nucleotide polymorphisms (SNPs). Attained genetic profiles and cluster consequences were contrasted with geographical and migration features of leprosy at county levels. ResultsConsidering the allelic variability of 17 VNTR loci by the discriminatory index, (GTA)9, (AT)17, (AT)15, (TA)18, (TTC)21, and (TA)10 are reported to be more highly polymorphic than other loci. The VNTR profile generated the low-density clustering pattern in the counties of Sichuan and Yunnan, whereas clusters have been observed from the isolates from Huayuan (N = 6), Yongding (N = 3), Zixing (N = 3), Chenxi (N = 2) and Zhongfang (N = 2) counties of Hunan, and Zhijin (N = 3), Anlong (N = 2), Zhenning (N = 2), and Xixiu (N = 2) counties of Guizhou. In some clusters, people’s social relations have been observed between villages. From the 290 clinical isolates, the most predominantly reported SNP was 3K (278, 95.8%), followed by SNP 1D (10, 3.4%), which are typically observed to be predominant in China. We also detected the novel SNP 3J (2, 0.8%), which has not yet been reported in China. ConclusionThe clustering pattern of M. leprae indicates the transmission of leprosy still persists at county levels, suggesting that there is a need to implement better approaches for tracing the close contacts of leprosy patients.

Outcome according to subspecies following lung transplantation in cystic fibrosis pediatric patients infected with Mycobacterium abscessus.

Mycobacterium abscessus infection has been associated with variable outcomes following lung transplantation. Mabscessus comprises three subspecies (Mabscessus subsp abscessus, Mabscessus subsp massiliense, and Mabscessus subsp bolletii). We investigated whether lung transplantation outcome in cystic fibrosis (CF) patients in a single center was related to the Mabscessus subspecies and genetic cluster. CF patients with chronic Mabscessus infection transplanted at Great Ormond Street Hospital between 2004 and 2017 were retrospectively examined. All Mabscessus isolates were identified to subspecies level by polymerase chain reaction and sequencing. Genetic cluster was determined by variable number tandem repeat profiling and whole-genome sequencing (WGS), and sequence type inferred from WGS. Thirteen patients with chronic Mabscessus infection underwent heart/lung or lung transplantation. Subspecies identification showed n=1 with Mabscessus bolletii, n=5 with Mabscessus massiliense, and n=7 with Mabscessus abscessus infection. Eight (62%) patients (one with Mabscessus massiliense and seven with Mabscessus abscessus) died post-lung transplant. The patient with Mabscessus bolletii and three patients with Mabscessus massiliense did well post-transplant. One patient with Mabscessus massiliense is receiving ongoing treatment. Dramatically worse outcomes are observed in patients infected with Mabscessus subspecies abscessus, the majority of whom were infected with ST-1 and ST-26 strains. Patients infected with other Mabcsessus strains can have acceptable outcomes.

Morphological and Molecular Characterization of a New Mycobacterium avium Subsp. paratuberculosis S-Type Strain Genotype in Goats.

Paratuberculosis is a chronic bacterial disease of global importance mainly in domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). In goats, paratuberculosis is mostly caused by the “C-type” (cattle) and in a few cases by the “S-type” (sheep) strain of MAP. In 2017, a caprine S-type III isolate with a new VNTR profile was identified in a Swiss alpine region. In 2018, new caprine isolates with the same novel VNTR profile originating from a farm of a close by neighboring valley were analyzed. Here we report on this MAP S-type III outbreak in a Swiss dairy goat farm in which we investigated the pathological changes, distribution and genotype of MAP tissue homogenates. Full necropsy and histological examination were undertaken on two female adult goats with a history of weight loss and intermitting diarrhea. Routine and special stains were applied to characterize the morphological changes. DNA was extracted from 33 different tissue samples and tested for MAP by qPCR targeting IS900 and F57. Subtyping was performed, using the variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units (MIRU) approach. The goats showed moderate to marked emaciation and displayed typical clinical features of paratuberculosis. A moderate granulomatous enteritis and regional lymphadenitis with a small to moderate number of acid-fast bacteria within macrophages was detected. MAP detection was mainly restricted to the gastrointestinal tract, mesenteric and hepatic lymph nodes. Subtyping the S-type isolates using a panel of eight established MIRU-VNTR loci identified a new genotype, INMV 218.

Prevalence of Mycobacterium bovis infection in traditionally managed cattle at the wildlife-livestock interface in South Africa in the absence of control measures.

Cattle are the domestic animal reservoir for Mycobacterium bovis (M. bovis) which also affects other domestic animals, several wildlife species and humans leading to tuberculosis. The study area is in a resource-poor community that is surrounded by several game parks, where M. bovis infection has been previously diagnosed in wildlife. A cross-sectional study was carried out to determine the prevalence of M. bovis infection in 659 cattle from a total of 192 traditionally managed herds using the BOVIGAM® interferon gamma assay (IFN-γ). Infection was confirmed by post mortem examination and M. bovis isolation from three test-positive cattle. Genotyping of the M. bovis isolates was done using spoligotyping and VNTR (variable number of tandem repeats typing). The apparent M. bovis prevalence rate in cattle at animal level was 12% with a true population prevalence of 6% (95% Confidence interval (C.I) 3.8 to 8.1) and a herd prevalence of 28%. Spoligotyping analysis revealed that the M. bovis isolates belonged to spoligotype SB0130 and were shared with wildlife. Three VNTR profiles were identified among the SB0130 isolates from cattle, two of which had previously been detected in buffalo in a game reserve adjacent to the study area. The apparent widespread presence of M. bovis in the cattle population raises a serious public health concern and justifies further investigation into the risk factors for M. bovis transmission to cattle and humans. Moreover, there is an urgent need for effective bTB control measures to reduce infection in the communal cattle and prevent its spread to uninfected herds.

Distribution of Mycobacterium leprae genotypes from Surabaya and Bandung Clinical Isolates by Multiple Locus Variable Number of Tandem Repeat Analysis

Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis has been proposed as a means of genotyping for tracking leprosy transmission due to tandem repeats’ potential as genetic markers to differentiate M. leprae strains. However, characteristics of polymorphism can vary depending on the population. This study aimed to compare the copy number of repeats in four genetic markers: TTC, AC8a, AC9 and 6-7 in leprosy patients from Surabaya and Bandung. Twenty three patients from Dr. Soetomo General Hospital and 21 from Hasan Sadikin Hospital were recruited. Multiple locus VNTR analysis was applied using total DNA extracts from Slit Skin Smear (SSS). From Surabaya, 7 samples showed the same copy number of four genetic markers (TTC=15; AC8a=10; AC9=10 and 6-7=6) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6); as for samples from Bandung, 2 showed the same copy number (TTC=15; AC8a=8; AC9=10 and 6-7=8) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6). The multiple locus VNTR analysis showed two identical M. leprae VNTR profiles from Bandung and Surabaya which supports the use of VNTR loci for transmission studies.

First Molecular Characterisation of Clinical Strains of Mycobacterium ulcerans in Togo (West Africa)

Background: Buruli ulcer is the third most common mycobacterial disease worldwide. Cases most occur in 30 countries but severe cases occur in West Africa countries such as Benin, Cote d’Ivoire and Togo mainly in rural regions. Early diagnosis may prevent severe disability. The molecular technique seems the best solution and new Mycobacterial Interspersed Repetitive Units (MIRU) and variable number tandem repeats (VNTR) typing method are themost reproducible in this regard. They propose geographical, inter and intraspecies differentiation and can be used as a diagnosis tool.
 Objective: The objective of this study was to investigate the molecular diversity by using MIRUVNTR typing in clinical samples of BU patients in Togo.
 Study Design: 64 DNA extracts from clinical samples were collected from BU patients in the two principal endemics districts in Togo (Yoto and Zio) with three less endemic districts (Bas Mono, Lacs and Vo). First, IS2404 and KR real-time PCR plus IS2606 conventional PCR were performed. In a second step, the strains were analysed by PCR typing for five specific and sensitive markers MIRU1, VNTR6, ST1, VNTR19 and VNTR9.
 Results and Conclusion: 71.11% were positive for IS2404, 3.13% were positives for PCR-KR and 31.11% for IS 2606. By MIRU-VNTR typing, 48.86% positive result was found for MIRU1 and 25.00%, 20.31%, 18.75% and 14.06% for VNTR6, ST1, VNTR19 and VNTR9 respectively. One of the samples was negative for all genotyping markers. Two different genetic profiles were identified by MIRU1, ST1 and VNTR loci by gel-analysed of the amplified products. The VNTR profile B (3,1,1,2) corresponding of 3 copies MIRU1, 1 copy VNTR6, 1 copy ST-1 and two copies of VNTR19 was detected in 15.63% of samples and the VNTR profile A (1,1,1,2) corresponding of 1 copy MIRU1, 1 copy VNTR6, 1 copy ST-1 and 2 copies of VNTR19 was detected in 3.13% of samples and confirms the West African genotype (3,1,1) in Togo. Different genetic strains of Mycobacterium ulcerans (M. ulcerans) were co-circulated in the same endemic region in the country. This study has described first the circulating of different genetic strains of M. ulcerans in Togo.

A Predominant Variable-Number Tandem-Repeat Cluster of Mycobacterium tuberculosis Isolates among Asylum Seekers in the Netherlands and Denmark, Deciphered by Whole-Genome Sequencing.

ABSTRACTIn many countries, Mycobacterium tuberculosis isolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigate M. tuberculosis transmission. Unexpectedly, cross-border clusters were identified among African refugees in the Netherlands and Denmark, although transmission in those countries was unlikely. Whole-genome sequencing (WGS) was applied to analyze transmission in depth and to assess the precision of VNTR typing. WGS was applied to 40 M. tuberculosis isolates from refugees in the Netherlands and Denmark (most of whom were from the Horn of Africa) that shared the exact same VNTR profile. Cluster investigations were undertaken to identify in-country epidemiological links. Combining WGS results for the isolates (all members of the central Asian strain [CAS]/Delhi genotype), from both European countries, an average genetic distance of 80 single-nucleotide polymorphisms (SNPs) (maximum, 153 SNPs) was observed. The few pairs of isolates with confirmed epidemiological links, except for one pair, had a maximum distance of 12 SNPs. WGS divided this refugee cluster into several subclusters of patients from the same country of origin. Although the M. tuberculosis cases, mainly originating from African countries, shared the exact same VNTR profile, most were clearly distinguished by WGS. The average genetic distance in this specific VNTR cluster was 2 times greater than that in other VNTR clusters. Thus, identical VNTR profiles did not represent recent direct M. tuberculosis transmission for this group of patients. It appears that either these strains from Africa are extremely conserved genetically or there is ongoing transmission of this genotype among refugees on their long migration routes from Africa to Europe.

Occurrence and Nature of Double Alleles in Variable-Number Tandem-Repeat Patterns of More than 8,000 Mycobacterium tuberculosis Complex Isolates in The Netherlands

ABSTRACTSince 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084; P = 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386; P = 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants.

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa

BackgroundMycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa.ResultsIn the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the ‘One Health’ concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match.ConclusionsThis is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

Molecular typing of Mycobacterium Abscessus isolated from cystic fibrosis patients.

The possibility of inter-human transmission of Mycobacterium abscessus in cystic fibrosis centres has been recently hypothesized suggesting the need for the molecular characterization of strains isolated from such patients. One hundred and forty one isolates of M. abscessus grown from sputum samples of 29 patients with cystic fibrosis were genotyped resorting to variable number of tandem repeats (VNTR) determination and whole genome sequencing (WGS). Out of 29 VNTR profiles, 15 were unique to the same number of patients while seven were shared by multiple patients. WGS showed that only two of the patients sharing common VNTR patterns were indeed infected by the same strain. The shared VNTR patterns were mostly present among the isolates of M. abscessus subsp. abscessus. As expected WGS showed a clearly higher discriminatory power in comparison with VNTR and appeared the only molecular epidemiology tool suitable to effectively discriminate the isolates of M. abscessus subsp. abscessus.

Hedgehogs and Mustelid Species: Major Carriers of Pathogenic Leptospira, a Survey in 28 Animal Species in France (20122015).

Human leptospirosis is a zoonotic and potentially fatal disease that has increasingly been reported in both developing and developed countries, including France. However, our understanding of the basic aspects of the epidemiology of this disease, including the source of Leptospira serogroup Australis infections in humans and domestic animals, remains incomplete. We investigated the genetic diversity of Leptospira in 28 species of wildlife other than rats using variable number tandem repeat (VNTR) and multispacer sequence typing (MST). The DNA of pathogenic Leptospira was detected in the kidney tissues of 201 individuals out of 3,738 tested individuals. A wide diversity, including 50 VNTR profiles and 8 MST profiles, was observed. Hedgehogs and mustelid species had the highest risk of being infected (logistic regression, OR = 66.8, CI95% = 30.9–144 and OR = 16.7, CI95% = 8.7–31.8, respectively). Almost all genetic profiles obtained from the hedgehogs were related to Leptospira interrogans Australis, suggesting the latter as a host-adapted bacterium, whereas mustelid species were infected by various genotypes, suggesting their interaction with Leptospira was different. By providing an inventory of the circulating strains of Leptospira and by pointing to hedgehogs as a potential reservoir of L. interrogans Australis, our study advances current knowledge on Leptospira animal carriers, and this information could serve to enhance epidemiological investigations in the future.

TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing

Whole-genome sequencing (WGS) with next-generation DNA sequencing (NGS) is an increasingly accessible and affordable method for genotyping hundreds of Mycobacterium tuberculosis (Mtb) isolates, leading to more effective epidemiological studies involving single nucleotide variations (SNVs) in core genomic sequences based on molecular evolution. We developed an all-in-one web-based tool for genotyping Mtb, referred to as the Total Genotyping Solution for TB (TGS-TB), to facilitate multiple genotyping platforms using NGS for spoligotyping and the detection of phylogenies with core genomic SNVs, IS6110 insertion sites, and 43 customized loci for variable number tandem repeat (VNTR) through a user-friendly, simple click interface. This methodology is implemented with a KvarQ script to predict MTBC lineages/sublineages and potential antimicrobial resistance. Seven Mtb isolates (JP01 to JP07) in this study showing the same VNTR profile were accurately discriminated through median-joining network analysis using SNVs unique to those isolates. An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs. The results of in silico analyses using TGS-TB are consistent with those obtained using conventional molecular genotyping methods, suggesting that NGS short reads could provide multiple genotypes to discriminate multiple strains of Mtb, although longer NGS reads (≥300-mer) will be required for full genotyping on the TGS-TB web site. Most available short reads (~100-mer) can be utilized to discriminate the isolates based on the core genome phylogeny. TGS-TB provides a more accurate and discriminative strain typing for clinical and epidemiological investigations; NGS strain typing offers a total genotyping solution for Mtb outbreak and surveillance. TGS-TB web site: https://gph.niid.go.jp/tgs-tb/.

Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation

Intra-species phylogeny of Mycobacterium tuberculosis has been regarded as a clue to estimate its potential risk to develop drug-resistance and various epidemiological tendencies. Genotypic characterization of variable number of tandem repeats (VNTR), a standard tool to ascertain transmission routes, has been improving as a public health effort, but determining phylogenetic information from those efforts alone is difficult. We present a platform based on maximum a posteriori (MAP) estimation to estimate phylogenetic information for M. tuberculosis clinical isolates from individual profiles of VNTR types. This study used 1245 M. tuberculosis clinical isolates obtained throughout Japan for construction of an MAP estimation formula. Two MAP estimation formulae, classification of Beijing family and other lineages, and classification of five Beijing sublineages (ST11/26, STK, ST3, and ST25/19 belonging to the ancient Beijing subfamily and modern Beijing subfamily), were created based on 24 loci VNTR (24Beijing-VNTR) profiles and phylogenetic information of the isolates. Recursive estimation based on the formulae showed high concordance with their authentic phylogeny by multi-locus sequence typing (MLST) of the isolates. The formulae might further support phylogenetic estimation of the Beijing lineage M. tuberculosis from the VNTR genotype with various geographic backgrounds. These results suggest that MAP estimation can function as a reliable probabilistic process to append phylogenetic information to VNTR genotypes of M. tuberculosis independently, which might improve the usage of genotyping data for control, understanding, prevention, and treatment of TB.

321 Changes in lung function and airway microbiology following ivacaftor therapy in an adult G551D homozygote

Objectives Ivacaftor has increased lung function and decreased pulmonary exacerbations and intravenous antibiotic use in patients with CF carrying the G551D mutation. This case study describes the effects of ivacaftor on airway microbiology and lung function in a G551D homozygote with chronic Pseudomonas aeruginosa (PA) infection. Methods All sputum culture reports prior to ivacaftor therapy were reviewed. Following ivacaftor initiation sputum samples were sent for standard culture at each clinic visit. 16S gene rRNA pyrosequencing was performed on paired sputum samples collected immediately prior to and after 6 months of ivacaftor therapy. FEV 1 was measured at baseline and at each follow up visit. Sweat chloride was assessed pre-treatment and at 6 months. Results Mucoid PA infection was acquired in 1983. The pre-ivacaftor baseline sputum sample and all preceding 26 samples taken over 8 years were culture positive for PA. 7 consecutive samples collected over 16 months following ivacaftor were culture negative for PA. A later sample was culture positive for the original infecting strain of PA (using variable number tandem repeat profiling). 16S gene rRNA pyrosequencing showed a drop in relative abundance of PA from 98.2% to 4.8% and an increase in microbial diversity over the 6 month sample period. FEV 1 improved from 68% predicted (baseline) to 90% predicted (20 months post ivacaftor). Sweat chloride reduced from 105 mmol/L (baseline) to 52 mmol/L (6months). Conclusion Bacterial diversity and relative abundance of typical CF pathogens may be significantly altered by modulation of both CFTR alleles. Sputum culture clearance of mucoid PA on should be interpreted with caution.

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus.

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil.

Source Tracking Mycobacterium ulcerans Infections in the Ashanti Region, Ghana

Although several studies have associated Mycobacterium ulcerans (MU) infection, Buruli ulcer (BU), with slow moving water bodies, there is still no definite mode of transmission. Ecological and transmission studies suggest Variable Number Tandem Repeat (VNTR) typing as a useful tool to differentiate MU strains from other Mycolactone Producing Mycobacteria (MPM). Deciphering the genetic relatedness of clinical and environmental isolates is seminal to determining reservoirs, vectors and transmission routes. In this study, we attempted to source-track MU infections to specific water bodies by matching VNTR profiles of MU in human samples to those in the environment. Environmental samples were collected from 10 water bodies in four BU endemic communities in the Ashanti region, Ghana. Four VNTR loci in MU Agy99 genome, were used to genotype environmental MU ecovars, and those from 14 confirmed BU patients within the same study area. Length polymorphism was confirmed with sequencing. MU was present in the 3 different types of water bodies, but significantly higher in biofilm samples. Four MU genotypes, designated W, X, Y and Z, were typed in both human and environmental samples. Other reported genotypes were only found in water bodies. Animal trapping identified 1 mouse with lesion characteristic of BU, which was confirmed as MU infection. Our findings suggest that patients may have been infected from community associated water bodies. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections. M. ulcerans transmission could involve several routes where humans have contact with risk environments, which may be further compounded by water bodies acting as vehicles for disseminating strains.

Extent of Mycobacterium bovis transmission among animals of dairy and beef cattle and deer farms in South Korea determined by variable-number tandem repeats typing

Identifying sources of Mycobacterium bovis transmission would be essential for establishing effective control programs of bovine tuberculosis (TB), a major zoonosis threatening human health worldwide. As an effort to determine the extent of M. bovis transmission among dairy and beef cattle and deer populations, a mycobacterial interspersed repetitive units (MIRU)-variable-number tandem repeats (VNTR) typing method was employed for analysis of 131 M. bovis isolates from 59 Holstein dairy cattle, 39 Korean beef cattle, and 33 deer. Of 31 MIRU-VNTR markers, 15 showed allelic diversity. The most discriminatory locus for M. bovis isolates was VNTR 3336 (h=0.59) followed by QUB 26, MIRU 31, VNTR 2401, and VNTR 3171 which showed high discriminatory power (h=0.43). The combined VNTR loci had an allelic diversity of 0.83. On the basis of the VNTR profiles of 30 VNTR loci, 24 genotypes were identified, and two genotypes were highly prevalent among all M. bovis isolates (33.6% and 19.1%, respectively), thus indicating that more than 50% of the isolates shared common molecular characteristics. Six additional genotypes were common in 2 of the 3 animal species, suggesting a wide interspecies transmission of M. bovis. This study thus demonstrates that MIRU-VNTR typing is useful in differentiation of M. bovis isolates and that M. bovis transmission occurs frequently among farmed animal species, highlighting the importance of bovine TB control programs in different animal species which are often raised in the same villages.

Clonality of Mycobacterium ulcerans by Using VNTR-MIRU Typing in Ivory Coast (Côte d’Ivoire), West Africa

Background: Buruli ulcer (BU) is neglected skin disease caused by Mycobacterium ulcerans. The lack of early diagnosis and treatment causes severe disability. In Central and in West Africa, BU is endemic and its control is difficult because the most cases occur in rural regions. The molecular particularity of M. ulcerans was the acquisition of the virulence plasmid pMUM001. Genetic analyses have demonstrated the high diversity with variable number tandem repeats (VNTR) and Mycobacterial Interspersed Repetitive Units (MIRU) in M. ulcerans and in mycolactone producing Mycobacteria (MPMs). Original Research Article Solange et al.; IJTDH, 7(4): 163-171, 2015; Article no.IJTDH.2015.069 164 Objective: The objective of this study was to investigate the molecular diversity by using MIRUVNTR method in clinical samples of BU patients in Cote d’Ivoire. Study Design: 21 clinical samples were collected from BU patients in different sites and were first analyzed in molecular diagnosis of BU using two targets insertion sequence IS2404 and keto reductase-B-domain (KR). In a second step, we have analyzed the strains by PCR typing for four specific and sensitive markers MIRU1, VNTR6, ST-1 and VNTR19. Results and Conclusion: 100% of clinical samples were positive in molecular tests for IS2404 and 95% for KR and confirm M. ulcerans in the samples. By PCR typing, we have found 61.9 % positive for MIRU1 and 52%, 85.7%, and 61.9% for VNTR6, ST-1 and VNTR19 respectively. One of sample was negative for all genotyping markers. Two different genetic profiles were identified by MIRU1 and ST-1 loci by gel-analyzed of the amplified products. The VNTR profile C (3,1,1) corresponding of 3 copies MIRU1, 1 copy VNTR6 and 1 copy ST-1 was detected in 28.5% of samples and confirms the West African genotype in Cote d’Ivoire. Different genetic strains of M. ulcerans were co-circulated in the same endemic region in the country. This study has described first the circulating of different genetic strains of M. ulcerans in Cote d’Ivoire.