Leptospirosis is a contagious zoonotic disease spread worldwide and is caused by the bacterium
The natural reservoirs of
Many virulence factors are involved in the pathogenesis and infection of this bacterium, including lipopolysaccharide (LPS), hemolysin, surface proteins, adhesion molecules, and the ability to move and swim in liquid environments (Fraga et al. 2011). The 32-kDa lipL32 lipoprotein, a type of outer membrane protein of
Bacteria rely on the ability to sense and respond to environmental stressors, including changes in temperature, pH, osmolarity, oxygen availability, and nutrient conditions. Environmental signals, such as increased oxidative stress or temperature, can stimulate the response of regulators of virulence genes to pathogenic
The aim of this research was to isolate and identify the leptospires from the farming fields of Tonekabon town situated in northern Iran as well as to study the effect of different environmental stresses, including pH, temperature, and
Localization of ten environmental sites investigated.
Samples were centrifuged at 10,000 × g for 10 min. The supernatant was discarded, and the remaining contents were passed through 0.45 µm and 0.2 µm filters, respectively, and transferred to liquid EMJH medium (Ellinghausen and Mccullough 1965; Johnson and Harris 1967). All the samples were incubated at 30°C for 30 days. Aliquots of the medium were transferred to the solid EMJH medium and cultured. Plates were incubated at 30°C for 30 days.
The specific primers of
Name of primer | Type | Sequence of primer |
---|---|---|
16S rRNA | Forward | 5’-GAACTGAGACACGGTCCAT-3’ |
16S rRNA | Reverse | 5’-GCCTCAGCGTCAGTTTTAGG-3’ |
LipL32 | Forward | 5’-ATCTCCGTTGCACTCTTTGC-3’ |
LipL32 | Reverse | 5’-ACCATCATCATCATCGTCCA-3’ |
PCR amplification conditions using a thermocycler (Sensoquest, Germany) were as follows: one denaturation cycle at 95°C for 5 min, 40 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 7 min.
(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
Microscopic observation of
A: Multiplying
Agarose gel electrophoresis (1.5%) of PCR products of environmental samples.
Columns 1, 2, and 3, pathogenic
Agarose gel (1.5%) analysis of a PCR products of environmental samples; Columns 1–9 – species of
The sampling sites and positive cases reported in the study.
Number of positive cases of saprophytic | Number of positive cases of pathogenic | Number of samples | Site |
---|---|---|---|
3 | 2 | 5 | Valiabad |
2 | 2 | 5 | Bagh e nazar |
0 | 1 | 5 | Mir shams-ol |
1 | 1 | 5 | Rud e posht |
1 | 0 | 5 | Alkaleh |
1 | 0 | 5 | Abkele sar |
0 | 1 | 5 | Lashkarak |
1 | 1 | 5 | Nematabad |
1 | 1 | 5 | Shiraj mahalleh-ye bozorg |
1 | 1 | 5 | Tilpordehsar |
11 (22%) | 10 (20%) | 50 | Total |
The results of the sequencing.
Molecular detection | Max Score | Total Score | Query Cover | E Value | Per Ident | Accession |
---|---|---|---|---|---|---|
612 | 612 | 93% | 3e-171 | 99.41% | MN086353.1 | |
176 | 176 | 26% | 5e-40 | 100% | MN086356.1 | |
593 | 593 | 100% | 2e-166 | 100% | AB758752.1 |
Fold change associated to gene
Fold change associated with the
Comparison of the
Fold change associated with the
Fold change associated with the
Due to the temperate climate of the northern regions of Iran, the prevalence of leptospirosis is high in these areas. In the study of the pollution status of stagnant waters and wetlands, more than 7% of the evaluated wetlands in Mazandaran were contaminated with pathogenic
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