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The Effect of Environmental Stresses on lipL32 Gene Expression in Pathogenic Leptospira spp. through Real-Time PCR

SONA ROSTAMPOUR YASOURI
SONA ROSTAMPOUR YASOURI
, 
MONIR DOUDI
MONIR DOUDI
, 
MASOOD GHANE
MASOOD GHANE
, 
NAFISEH SADAT NAGHAVI
NAFISEH SADAT NAGHAVI
 oraz 
ABOLHASAN REZAEI
ABOLHASAN REZAEI
   | 08 wrz 2020
Polish Journal of Microbiology's Cover Image
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Article Category: original-paper
Data publikacji: 08 wrz 2020
Zakres stron: 301 - 310
Otrzymano: 10 kwi 2020
Przyjęty: 10 lip 2020
DOI: https://doi.org/10.33073/pjm-2020-033
Słowa kluczowe
© 2020 Sona Rostampour Yasouri et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Localization of ten environmental sites investigated.
Localization of ten environmental sites investigated.

Fig. 2.

Leptospira colonies.(a) Formation of surface’s black colonies, with round shape, needle tip size (at 30 days of incubation on EMJH agar), wooly margins, and hilly elevation related to Leptospira pathogen species. (b) Formation of sub surface’s white colonies with round shape, small size, and some smaller colonies, size 1–2 mm (after 30 days of incubation on EMJH agar), smooth (entire) margins, flat elevation related to Leptospira perdikensis. (c) Formation of sub surface’s very bright cream colonies with round shape, small size, 1–3 mm, (after 30 days of incubation on EMJH agar), smooth (entire) margins, with small to medium halo, flat elevation related to Leptospira ryugenii. (d) It had the characteristics of a colony (a), except that in this case, rings were seen to the same distance around the surface black colony related to Leptospira pathogen species.
Leptospira colonies.(a) Formation of surface’s black colonies, with round shape, needle tip size (at 30 days of incubation on EMJH agar), wooly margins, and hilly elevation related to Leptospira pathogen species. (b) Formation of sub surface’s white colonies with round shape, small size, and some smaller colonies, size 1–2 mm (after 30 days of incubation on EMJH agar), smooth (entire) margins, flat elevation related to Leptospira perdikensis. (c) Formation of sub surface’s very bright cream colonies with round shape, small size, 1–3 mm, (after 30 days of incubation on EMJH agar), smooth (entire) margins, with small to medium halo, flat elevation related to Leptospira ryugenii. (d) It had the characteristics of a colony (a), except that in this case, rings were seen to the same distance around the surface black colony related to Leptospira pathogen species.

Fig. 3.

Microscopic observation of Leptospira with a 100× magnification optical microscopeA: Multiplying Leptospira; the image relates to pathogenic Leptospira spp., B: The Leptospira bacterium that is twisted over itself is located on the right. In the lower-left a shorter Leptospira is visible; the image related to Leptospira ryugenii, C and E: Long Leptospira, D: The hook area of the Leptospira bacterium was clearly observed in a microscopic view; the image related to Leptospira perdikensis, F: Two Leptospira bacteria that are interconnected from one end and form a long separable leptospiral chain; the image related to Leptospira sp.
Microscopic observation of Leptospira with a 100× magnification optical microscopeA: Multiplying Leptospira; the image relates to pathogenic Leptospira spp., B: The Leptospira bacterium that is twisted over itself is located on the right. In the lower-left a shorter Leptospira is visible; the image related to Leptospira ryugenii, C and E: Long Leptospira, D: The hook area of the Leptospira bacterium was clearly observed in a microscopic view; the image related to Leptospira perdikensis, F: Two Leptospira bacteria that are interconnected from one end and form a long separable leptospiral chain; the image related to Leptospira sp.

Fig. 4.

Agarose gel electrophoresis (1.5%) of PCR products of environmental samples.Columns 1, 2, and 3, pathogenic Leptospira spp.; Column 4 – a reference strain; Column 5 – a negative control; Column 6 – E. coli; Column M – 100 bp DNA ladder.
Agarose gel electrophoresis (1.5%) of PCR products of environmental samples.Columns 1, 2, and 3, pathogenic Leptospira spp.; Column 4 – a reference strain; Column 5 – a negative control; Column 6 – E. coli; Column M – 100 bp DNA ladder.

Fig. 5.

Agarose gel (1.5%) analysis of a PCR products of environmental samples; Columns 1–9 – species of Leptospira; Column 10 – a reference strain; column 11 – a negative control; Column 12 – E. coli E. coli; Column M – 100 bp DNA ladder. Sampling sites that were positive for the presence of pathogenic Leptospira by both culture and PCR were reported in Fig. 1 and Table II.
Agarose gel (1.5%) analysis of a PCR products of environmental samples; Columns 1–9 – species of Leptospira; Column 10 – a reference strain; column 11 – a negative control; Column 12 – E. coli E. coli; Column M – 100 bp DNA ladder. Sampling sites that were positive for the presence of pathogenic Leptospira by both culture and PCR were reported in Fig. 1 and Table II.

Fig. 6.

Comparison of the lipL32 gene expression in pathogenic Leptospira under the influence of temperature, pH, and Ph. amarus plant extract.
Comparison of the lipL32 gene expression in pathogenic Leptospira under the influence of temperature, pH, and Ph. amarus plant extract.

The specific primers of Leptospira.

Name of primerTypeSequence of primer
16S rRNAForward5’-GAACTGAGACACGGTCCAT-3’
16S rRNAReverse5’-GCCTCAGCGTCAGTTTTAGG-3’
LipL32Forward5’-ATCTCCGTTGCACTCTTTGC-3’
LipL32Reverse5’-ACCATCATCATCATCGTCCA-3’

The results of the sequencing.

Molecular detectionMax ScoreTotal ScoreQuery CoverE ValuePer IdentAccession
Leptospira perdikensis strain CES 16S ribosomal RNA gene612612    93%  3e-171        99.41%MN086353.1
Leptospira ryugenii strain CES 16S ribosomal RNA gene176176    26%5e-40100%MN086356.1
Leptospira sp. MS336 gene for 16S ribosomal RNA gene593593100%  2e-166100%AB758752.1

The sampling sites and positive cases reported in the study.

Number of positive cases of saprophytic LeptospiraNumber of positive cases of pathogenic LeptospiraNumber of samplesSite
32  5Valiabad
22  5Bagh e nazar
01  5Mir shams-ol
11  5Rud e posht
10  5Alkaleh
10  5Abkele sar
01  5Lashkarak
11  5Nematabad
11  5Shiraj mahalleh-ye bozorg
11  5Tilpordehsar
         11 (22%)         10 (20%)50Total
eISSN:
2544-4646
Język:
Angielski
Częstotliwość wydawania:
4 razy w roku
Dziedziny czasopisma:
Life Sciences, Microbiology and Virology
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