Genetic variability and discrimination of low doses of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification assay as a field-friendly molecular tool

Abstract

Abstract:Aim: One of the main diagnostic problems of conventional polymerase chain reaction (PCR) is indiscrimination of low parasitic loads in soil samples. The aim of this study is to determine the genetic diversity and identification of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification (LAMP) assay.Materials and Methods:A total of 180 soil samples were collected from various streets and public parks of northwest Iran. The DNA of recovered Toxocara eggs were extracted and amplified by PCR and LAMP following ZnSO4 flotation technique. The amplicons of internal transcribed spacer-2 gene were sequenced to reveal the heterogeneity traits of Toxocara spp. In addition, Toxocara canis sequences of southwest Iran were directly retrieved to compare gene flow between two distinct populations.Results:Toxocara spp. eggs were found in 57, 14 and 77 of soil samples using the microscopy, PCR and LAMP (detection limit 1-3 eggs/200 g soil), respectively. 7.7% of isolates were identified as T. canis by PCR method, while LAMP was able to detect 27.2%, 15.5% and 12.2% as Toxocara cati, T. canis and mixed infections, respectively. The kappa coefficient between LAMP and microscopy indicated a strong agreement (0.765) but indicated a faint agreement among LAMP-PCR (0.203) and PCR-microscopy (0.308) methods. A pairwise fixation index (Fst) as a degree of gene flow was generally low (0.02156) among Toxocara populations of northwest and southwest Iran.Conclusions:The statistically significant Fst value indicates that the T. canis populations are not genetically well differentiated between northwest and southwest Iran. This shows that here is possibly an epidemiological drift due to the transfer of alleles. The LAMP assay because of its shorter reaction time, more sensitivity, and simultaneous detection of environmental contamination to be appears as valuable field diagnosis compared to PCR. Therefore, the detection of low Toxocara spp. loads from public area soils will help to expand epidemiological understanding of toxocariasis and establishing preventive strategies in resource-limited endemic of Iran.

Molecular evaluation of Toxocara infection in stray cats using loop-mediated isothermal amplification (LAMP) technique as a rapid, sensitive, and simple screening assay

“…Similar to the results of the present study, no difference was found between the results of the microscopic method and 2qPCR technique, a speci c and rapid duplex quantitative real-time PCR on the ITS-2 gene of Toxocara that was used by Durant et al 2012 to detect T. cati and T. canis in soil and feces samples [8]. In a study by Ozlati et al 2016 in Tabriz city, Northwest Iran, 180 samples of suspected contaminated soil were studied using microscopic, PCR and LAMP methods. Based on the results, 57, 14 and 77 samples were detected positive for Toxocara by microscopy, PCR and LAMP methods respectively, of which 49 samples (27.2%) were contaminated with T. cati and 28 samples (15.5%) were contaminated with T. canis.…”

“…Based on the results, 57, 14 and 77 samples were detected positive for Toxocara by microscopy, PCR and LAMP methods respectively, of which 49 samples (27.2%) were contaminated with T. cati and 28 samples (15.5%) were contaminated with T. canis. The results of Ozlati and colleagues showed a higher sensitivity of the LAMP technique than the PCR and microscopy methods [36].…”

Variations in the Rate of Infestations of Dogs with Zoonotic Nematodes and the Contamination of Soil in Different Environments

“…Few authors who detected Toxocara spp. in the environment used molecular methods [ 54 , 55 ]. Ozlati [ 55 ] determined the genetic diversity from soils in public areas, identifying T. canis in 15.5%, T. cati in 27.2%, and mixed infections in 12.2%.…”

“…in the environment used molecular methods [ 54 , 55 ]. Ozlati [ 55 ] determined the genetic diversity from soils in public areas, identifying T. canis in 15.5%, T. cati in 27.2%, and mixed infections in 12.2%. Similarly, Khademvatan [ 53 ] identified T. cati more often than T. canis (28% and 5.7% respectively), and no mixed contamination was observed.…”

Protein-synthesizing function and functional state of the liver of dogs at experimental toxocariasis

“…Значну кількість робіт учених-паразитологів, представлених у літературних джерелах, присвячено поширенню токсокарозу в країнах Європи та в Україні (Rubinsky-Elefant et al, 2011;Bodnia, 2016;Ozlati et al, 2016;Noor et al, 2019). Так, середня інтенсивність контамінації ґрунтів яйцями нематод роду Toxocara в різних областях України становить 1,6-10,7 % (Pryima, 2010;Said et al, 2018).…”