The Challenge of Bacterial Strain Identification: Leptospira interrogans Serovars Australis in a Dog and Long-Term Clinical Follow-up
Tommaso FurlanelloElisa MazzottaCristina BertasioMario D’IncauLaura BellinatiLaura LuccheseAlda Natale
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Abstract:
Leptospirosis is a widespread disease throughout the world, presenting in severe clinical forms in dogs. The pathogenicity of the different serovars in field infections is not fully documented, and clinical diagnosis is often limited to a combination of serological tests and molecular analyses. The latter, although a fundamental tool, cannot identify the infecting strain without further analysis. This study reports the use of various indirect (microscopic agglutination test, MAT) and direct (microbiological culture, real-time PCR) laboratory techniques, followed by typing protocols (Multi-locus Sequence Typing (MLST), Multiple Loci Variable number tandem repeat Analysis (MLVA), serotyping) that allowed for the identification of the Leptospira serovar Australis in a symptomatic and previously vaccinated dog (vaccine containing heterologous strains). This study reports long-term clinical follow-up (0–640 days) and describes the possible role of the infection in the development of chronic renal failure. This study aims to highlight how a combination of different techniques can be useful to better characterise the environmental circulation of zoonotic agents. Therefore, the identification and isolation of circulating L. strains would facilitate the updating of epidemiological data, enhance the knowledge of pathogenicity and long-term clinical effects, and provide a valuable resource for improving the efficacy of a specific serovar vaccination.Keywords:
Multilocus sequence typing
Leptospira interrogans
Direct agglutination test
Leptospirosis is a re-emerging zoonosis of the tropical and subtropical areas of the world. The causative agent, Leptospira spp., can be classified into more than 300 serovars based on a serological technique and 15 genomospecies according to DNA-DNA hybridization. To date, the Microscopic Agglutination Test (MAT) is the only major means to identify Leptospira serovars for epidemiological tracing. MAT is a sophisticated test involving a large panel of serovar-specific antisera. The laboratory personnel is endangered by infection from the requirement of a large number of living leptospires. Therefore, the development of a new simpler technique is essential. To date, whole genome sequences of four Leptospira serovars have been released and over 60 variable number of tandem repeats (VNTR) with difference in length and copy numbers have been identified. Thus, the objective of this study is to evaluate the efficacy of multiple-locus of VNTR analysis (MLVA) in Leptospira serovars identification. A total of 30 Leptospira reference serovars and 55 Leptospira spp. isolated from patients with confirmed leptospirosis in Thailand during 2002 to 2006, were analyzed for the presence of five VNTR loci namely V27, V29, V30, V36 and V50 by PCR. A total of 38 MLVA patterns were identified in this study. Twenty-three MLVA patterns were identified from 30 reference samovars. Three serovars provided similar MLVA patterns and six serovars provided no amplification products. Some of the Leptospira patient isolates yielded identical MLVA patterns to that of the reference serovars, and they provided an extra 15 MLVA patterns. The MLVA results in this study completely conformed to the serovar identification from either MAT or latex agglutination. In conclusion, our data further emphasize the discrimination power of MLVA in Leptospira serovar identification. Nevertheless, more VNTR loci and a larger panel of reference serovars need to be investigated to obtain the complete Thai MLVA pattern database for all Leptospira serovars.
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Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA) to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative.
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钩端螺旋体(钩体)病是一种自然疫源性疾病.近年来以可变数目串联重复序列(variable number of tandemrepeats,VNTR)为基础的分型方法,逐渐被应用于分子流行病学研究中.本研究选取我国致病性钩端螺旋体15群15型参考菌株,初步探讨多位点可变数目串联重复序列分析(MLVA)在钩体病分子流行病学研究中的应用价值。
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Leptospirosis is recognized as a re-emerging infectious disease; therefore, understanding the epidemiology of the disease is vital for designing intervention programs and diminishing its transmission. Recently, Multilocus variable number tandem repeat analysis (MLVA) is used for segregating and identifying Leptospira serovars. The method has potential application in investigating the molecular epidemiology of Leptospira.The propose of this study was genomic identification of pathogenic Leptospires in Iran by MLVA.Leptospira serovars were obtained from National Reference Laboratory of Leptospira at Razi Vaccine and Serum Research Institute, Karaj, Iran. Serovars were cultured into the liquid EMJH medium and incubated at 28˚C for 7 days. DNA of serovars was extracted using the phenol-chloroform method. PCR was performed with 5 selected variable number tandem repeat analysis (VNTR) loci. The amplified products were analyzed by agarose gel electrophoresis. The size of the amplified products was estimated by 100 bp ladder and sequencing analysis.The saprophytic serovar showed no amplified fragments. PCR products in all pathogenic serovars were observed. The 12 reference serovars used for the development of technique displayed distinct patterns.Results showed that MLVA technique with its range of polymorphism is a good marker for identification of pathogenic serovars. Some VNTR loci are more powerful than the other ones with regard to differentiation. Serovars from the same geographical area have more genetic similarity than same serovars from different places. MLVA is a suitable technique for epidemiological survey.
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Objective To establish the method of multiple loci VNTR(variable numbers tandem-repeats) analysis (MLVA) for genotyping Leptospira interrogans serogroup ieterohaemorrhagiae . Methods Seven VNTR loci were chosen for genotyping 117 strains of L. interrogans serogroup Icterohaemorrhagiae by PCR-electrophoresis-based VNTR analysis and the results were analyzed by software BioNumerics( Version 4.0). Results One hundred and seventeen isolates of L. interrogans serogroup Icterohaemorrhagiae detec-ted with 7 VNTR loci were classified into three clusters(A,B,C), twenty-eight types were found, type A 11.97% (14/117), type B 0.85% (1/1 17), type C 87.18% (102/117). Diversity Indexes for the loci varied between 0.0831 and 0.8005. Clinical strains isolated from the same geographic area and belonging to the same serogroup shared a common VNTR pattern. Conclusion MLVA could be used to classify and identify Leptospira interrogans preliminarily. With the improvement of technology, this rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.
Key words:
Leptospira interrogate; Serogroup Icterohaemorrhagiae; Genotyping; MLVA
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ABSTRACT Leptospirosis is a worldwide-distributed zoonosis, endemic in tropical areas. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests. Recently, molecular typing systems based on variable-number tandem-repeat (VNTR) analysis have been described and have been used to identify Leptospira interrogans strains. Although L. interrogans is the most common Leptospira species encountered in human infections around the world, other pathogenic species, such as Leptospira kirschneri and Leptospira borgpetersenii , are also frequently associated with human leptospirosis. In this study, we aimed to extend multilocus VNTR analysis (MLVA) identification of strains to species other than L. interrogans . We designed primers for VNTR loci found in L. interrogans , L. kirschneri , and L. borgpetersenii . The discriminatory power of the redefined primers was evaluated on collection strains and then on clinical strains. We also carried out a retrospective study on 156 strains isolated from patients and animals from New Caledonia, an area of high endemicity in the South Pacific. Our results show that this simple PCR-based MLVA typing technique is a powerful methodology for the epidemiology of leptospirosis.
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Deoxyribonucleic acid hybridization (hydroxyapatite method, 55 and 70°C) was used to characterize 38 serovars from 22 named serogroups of Leptospira interrogans and Leptospira biflexa, 6 serovars from 4 new unnamed serogroups of Leptospira interrogans, and single serovars of the proposed species Leptospira parva and Leptonema illini. Deoxyribonucleic acid relatedness confirmed the validity of Leptospira parva and Leptonema illini. The well-accepted species Leptospira interrogans and Leptospira biflexa, as currently defined, were extremely heterogeneous. Relatedness results revealed at least five new species among the parasitic serovars formerly included in Leptospira interrogans and two new species among the saprophytic serovars formerly included in Leptospira biflexa. Serogrouping did not equate with species identification, as serovars from several different subserogroups belonged to different species. The new species named in this paper are Leptospira, noguchii, Leptospira weilii, Leptospira santarosai, Leptospira borgpetersenii, Leptospira meyeri, Leptospira wolbachii, and Leptospira inadai.
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Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.
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In this study, an improved multiple-locus variable number of tandem repeats analysis (MLVA) method based upon a previously published method is described. Improvements to the method included redesigned primers and PCR conditions, combined with pooled capillary electrophoresis using multicolored dyes. Allele sizes were converted into an allele string, and each unique allele string was assigned a numerical MLVA type (MVT). The improved MLVA method was then applied to 96 previously characterized Leptospira interrogans serovar Australis isolates from human and animal sources. The improved MLVA was found to have between six and 13 alleles at each locus, compared with three to eight in the original. The mean Hunter-Gaston diversity index (HGDI) for the improved MLVA method was 0.654, compared with 0.599 in the original; this increase in diversity was largely due to changes in the analysis of the variable number of tandem repeat (VNTR) data. When the improved MLVA method was compared with the fluorescent amplified fragment length polymorphism (FAFLP) method, there was a high level of concordance between the profiles; however, the MLVA method produced an additional four unique profiles amongst the subset of 30 isolates tested. Given that the improved MLVA method was found to be superior to the original MLVA method, it was subsequently used to redefine the molecular epidemiology of L. interrogans serovar Australis in Queensland, Australia. Using cluster analysis, the authors were able to demonstrate clonal links amongst rodent isolates, rodent and human isolates, and rodent and canine isolates. These results highlight the role of rodents in the disease, and also the potential role of MLVA in defining the molecular epidemiology of L. interrogans.
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ABSTRACT We identified many variable-number tandem repeat (VNTR) loci in the genomes of Neisseria meningitidis serogroups A, B, and C and utilized a number of these loci to develop a multiple-locus variable-number tandem repeat analysis (MLVA). Eighty-five N. meningitidis serogroup B and C isolates obtained from Dutch patients with invasive meningococcal disease and seven reference strains were analyzed using MLVA and multilocus sequence typing (MLST). MLVA, based on eight VNTR loci with limited variability in the number of repeats, yielded clustering of the strains similar to that obtained by MLST, with congruence between both methods amounting to 69%. The ability to recognize clonal complexes makes MLVA a valuable high-throughput method to serve as a tool complementary to MLST. Four highly variable VNTR loci were used in a second assay to analyze N. meningitidis serogroup C strains collected during an outbreak of meningococcal disease in The Netherlands. Typing based on the latter VNTR loci enabled differentiation of isolates with identical MLST sequence types and grouped epidemiologically related strains.
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