A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp

Introduction: Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp. Leptospirosis is maintained in an environment due to chronic kidney infection of a wide variety of domestic, peridomestic and wild reservoir mammals. In this study the role of pigs in maintenance of leptospires on the Caribbean island of St. Kitts was investigated. Methodology: The condemned kidneys of 60 pigs slaughtered at a St. Kitts abattoir were screened by a quantitative-PCR for the presence of Leptospira spp. Positive samples were genotyped using a six-gene based multilocus sequence typing scheme. Results: Leptospiral DNA was detected in the kidneys of one of the 60 pigs. Multilocus sequence typing identified the infecting species to be L. interrogans. Conclusions: Detection of this zoonotic pathogen in the kidneys of a seemingly healthy pig raises concerns regarding the subclinical carriers of the disease among the island’s swine population.

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Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospira-specific IgM enzyme-linked immunosorbent assay in a clinical environment

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2006.10.004 ◽

2007 ◽

Vol 57 (4) ◽

pp. 361-366 ◽

Cited By ~ 53

Author(s):

Andrew Slack ◽

Meegan Symonds ◽

Michael Dohnt ◽

Christopher Harris ◽

Dianne Brookes ◽

...

Keyword(s):

Immunosorbent Assay ◽

Taqman Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Clinical Environment ◽

Pathogenic Leptospira ◽

Leptospira Spp

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Simultaneous Detection and Differentiation of Pathogenic and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) Assay

Polish Journal of Microbiology ◽

10.33073/pjm-2010-026 ◽

2010 ◽

Vol 59 (3) ◽

pp. 167-173 ◽

Cited By ~ 17

Author(s):

ORHAN BEDIR ◽

ABDULLAH KILIC ◽

ERDINC ATABEK ◽

AHMET MERT KUSKUCU ◽

VEDAT TURHAN ◽

...

Keyword(s):

Real Time ◽

Ribosomal Rna ◽

Real Time Pcr ◽

Molecular Techniques ◽

16S Ribosomal Rna ◽

Taqman Assay ◽

Pathogenic Leptospira ◽

Ribosomal Rna Gene ◽

Leptospira Spp ◽

23S Ribosomal Rna

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.

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Detection and characterization of Leptospira spp. in dogs diagnosed with kidney and/or liver disease in Selangor, Malaysia

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211024575 ◽

2021 ◽

pp. 104063872110245

Author(s):

Sabri A. Rahman ◽

Kuan H. Khor ◽

Siti Khairani-Bejo ◽

Seng F. Lau ◽

Mazlina Mazlan ◽

...

Keyword(s):

Liver Disease ◽

Mortality Rate ◽

Direct Detection ◽

Bacterial Disease ◽

Pathogenic Leptospira ◽

Wide Range ◽

Leptospira Spp ◽

Rrna Sequencing ◽

The Common

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae ( n = 12), Javanica ( n = 10), and Icterohaemorrhagiae ( n = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans, L. borgpetersenii, L. kirschneri, and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira-infected dogs was high (18 of 53; 34%).

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Protein typing of major outer membrane lipoproteins from Chinese pathogenic Leptospira spp. and characterization of their immunogenicity

Vaccine ◽

10.1016/j.vaccine.2009.09.089 ◽

2009 ◽

Vol 28 (1) ◽

pp. 243-255 ◽

Cited By ~ 20

Author(s):

Dongjiao Luo ◽

Feng Xue ◽

David M. Ojcius ◽

Jinfang Zhao ◽

Yafei Mao ◽

...

Keyword(s):

Outer Membrane ◽

Pathogenic Leptospira ◽

Leptospira Spp ◽

Membrane Lipoproteins

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Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Journal of Bacteriology ◽

10.1128/jb.175.13.4225-4234.1993 ◽

1993 ◽

Vol 175 (13) ◽

pp. 4225-4234 ◽

Cited By ~ 68

Author(s):

D A Haake ◽

C I Champion ◽

C Martinich ◽

E S Shang ◽

D R Blanco ◽

...

Keyword(s):

Sequence Analysis ◽

Membrane Protein ◽

Outer Membrane ◽

Molecular Cloning ◽

Outer Membrane Protein ◽

Gene Encoding ◽

Pathogenic Leptospira ◽

Leptospira Spp

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Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

Memórias do Instituto Oswaldo Cruz ◽

10.1590/0074-0276140295 ◽

2014 ◽

Vol 109 (2) ◽

pp. 163-167 ◽

Cited By ~ 8

Author(s):

Sylvia Grune Loffler ◽

Maria Elisa Pavan ◽

Bibiana Vanasco ◽

Luis Samartino ◽

Olga Suarez ◽

...

Keyword(s):

Pathogenic Leptospira ◽

Leptospira Spp

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Association of Rainfall and the Occurrence of Pathogenic Leptospira spp. in Recreational Stream Water, Hulu Langat, Selangor

IFMBE Proceedings - 2nd International Conference for Innovation in Biomedical Engineering and Life Sciences ◽

10.1007/978-981-10-7554-4_20 ◽

2017 ◽

pp. 119-124

Author(s):

K. Dzulaikha ◽

M. Y. Nurul Yuziana ◽

J. J. Maizatulriah ◽

A. W. Marfiah

Keyword(s):

Stream Water ◽

Pathogenic Leptospira ◽

Leptospira Spp

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In Silico Analysis of Genetic VapC Profiles from the Toxin-Antitoxin Type II VapBC Modules among Pathogenic, Intermediate, and Non-Pathogenic Leptospira

Microorganisms ◽

10.3390/microorganisms7020056 ◽

2019 ◽

Vol 7 (2) ◽

pp. 56

Author(s):

Alexandre Lopes ◽

Bruna Azevedo ◽

Rebeca Emídio ◽

Deborah Damiano ◽

Ana Nascimento ◽

...

Keyword(s):

Pathogenic Bacteria ◽

In Silico Analysis ◽

Amino Acid Sequences ◽

Protein Alignment ◽

Pathogenic Leptospira ◽

Leptospira Spp ◽

Unique Species ◽

Silico Analysis ◽

Leptospira Species ◽

Complex Manner

Pathogenic Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.

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