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      Evaluation of pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR

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          Abstract

          Background and objectives

          Leptospirosis is an important zoonotic disease caused by Leptospira interrogans. Leptospirosis leads to economical losses in dairy farm industry. The objective of this study was to evaluate the pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR.

          Materials and Methods

          Two hundred samples (100 urine and 100 blood) were collected from 100 cows randomly and delivered to the laboratory. Samples were stored at -20 °C. DNA was extracted and purified from the plasma and urine samples and concentrated on diatoms in the presence of guanidine thiocyanate (GuSCN). PCR products were detected and identified as Leptospira by ilumination of the expected size of DNA bands after staining of the agarose gel with ethidium bromide gels. PCR products were purified and sequenced.

          Results

          The results showed that 28% of urine samples and 23% of plasma samples were contaminated. The major serotypes were Icterohaemorrhagiae (50%) and Pomona (37.5%). The urine samples of 17 cows were positive for Leptospira without positive plasma samples. This indicated that these cows are reservoirs in dairy herds of Shahrekord and dangerous for human health. The plasma samples of twelve cows were positive for Leptospira without positive urine samples.

          Conclusions

          Leptospira serotypes can be maintained in relatively dry regions and must be considered when dealing with leptospirosis in dairy farms of Shahrekord and human health.

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          Most cited references37

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          Sequence type analysis and recombinational tests (START).

          The 32-bit Windows application START is implemented using Visual Basic and C(++) and 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. START is available at http://outbreak.ceid.ox.ac.uk/software.htm. keith.jolley@ceid.ox.ac.uk
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            Detection of pathogenic leptospires by real-time quantitative PCR.

            Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.
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              Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires.

              Leptospirosis is globally important infectious disease affecting almost all mammals. Pathogenic Leptospira encodes immunoglobulin-like protein (Lig) that is found to express only during infection. We report the development of conventional and real time PCR assays targeting lig genes of leptospires for the early diagnosis of leptospirosis. Sensitivity of the newly designed Lig1/Lig2 primers for conventional PCR was compared with previously published primers LP1/LP2 and G1/G2. G1/G2 primers amplified the target DNA from all the serovars including non-pathogenic Leptospira biflexa whereas LP1/LP2 and Lig1/Lig2 primers amplified only pathogenic leptospires. Diagnostic PCR assay was also developed for the detection of pathogenic Leptospira interrogans in urine samples. We obtained the highest sensitivity in PCR using our Lig1/Lig2 primers with a detection of 6 leptospires. A rapid and sensitive lig-based real time PCR assay was also developed with a detection range of 10-10(7) gene copies. To evaluate the early diagnosis for leptospirosis, we compared the culture with conventional and real time PCR for the detection of spirochetes in experimentally infected hamsters during a time-course study. Culture of infected hamster tissues detected the presence of leptospires from Day 2 of infection but not on the day of infection or Day 1, whereas conventional PCR and real time PCR detected the leptospires from the day of infection. Hence, conventional and real time PCR with lig primers would be a sensitive and rapid tool for early diagnosis of leptospirosis.
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                Author and article information

                Journal
                Iran J Microbiol
                IJM
                Iranian Journal of Microbiology
                Tehran University of Medical Sciences
                2008-3289
                2008-4447
                September 2011
                : 3
                : 3
                : 135-139
                Affiliations
                [1 ]Departments of Large Animal Internal Medicine
                [2 ]Biochemistry Veterinary Faculty, Shahrekord University, Iran
                [3 ]Veterinary Faculty, Shahrekord University, Shahrekord, Iran.
                Author notes
                [* ]Corresponding author: A Jafari Dehkordi Address: Department of Large Animal Internal Medicine, Veterinary faculty of Shahrekord University, Iran. Tel: +98-913-1819132. Fax: +98-381-4424427. E-mail: jafari-a@ 123456vet.sku.ac.ir
                Article
                IJM-3-135
                3279815
                22347596
                e17a8b7c-3901-43f4-980e-ec81172d7d43
                © 2011 Iranian Society of Microbiology & Tehran University of Medical Sciences

                This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.

                History
                : June 2011
                : August 2011
                Categories
                Original Artical

                Microbiology & Virology
                pcr,leptospira,shahrekord,cattle
                Microbiology & Virology
                pcr, leptospira, shahrekord, cattle

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