Iraqi Journal of Veterinary Sciences

The genotype of Entamoeba histolytica in bloody diarrhea samples of humans, cows and sheep

H.H. Naser

Zoonotic Diseases Research Unit, College of Veterinary Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq

Email: hassan.naser@qu.edu.iq

Abstract

The present study was carried out to detect the genotype of E. histolytica that found in human fecal specimens and animals feces with Haemorrgic diarrhea by amplifying the SREHP gene, using RT-PCR technique, Cyber ​​green dye and by fusion curve analysis. The study also included molecular detection of amoebic parasite species using Nested-PCR technology. The study recorded presence of parasites E. histolytica; E. dispar; E. bovis with total infection rates 82.9, 26.8, 4.9%, respectively. The study revealed the presence of E. histolytica parasite in five different genotypes (I, II, III, IV, V) with rate presence 9.75, 53.65, 19.5, 9.75, 7.3%, respectively. In conclusion, there are five genotype of E. histolytica, in human and animals, most of these genotypes may be infect any host, E. bovis was recorded in sheep and cows.

Keywords:  Entamoeba spp., Real-Time PCR, SREHP, Genotyping, Melting curve analysis

DOI: 10.33899/ijvs.2020.126135.1242, ©2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

النمط الجینی للأمیبا الحالة للنسجفی عینات الإسهال الدموی للإنسان والأبقار والأغنام

حسن حاجم ناصر

وحدة بحوث الأمراض الانتقالیة، کلیة الطب البیطری، جامعة القادسیة، القادسیة، العراق

الخلاصة

صممت الدراسة لتحدید النمط الجینی لطفیلی الامیبیا الحالة للنسیج E.hitolyticaالموجودة فی عینات الاسهال الدموی للإنسان والحیوان (الابقار والاغنام) باستخدام تقنیة تفاعل السلسلة المتبلمرة فی الوقت الحقیقی عن طریق تضخیم الجین SREHP واستعمال صبغة السایبر الخضراء. شملت الدراســة ایضا تقنیة  Nested-PCR لتحدید انواع طفیلی الامیبیا. سـجلت الانواع E. histolyticaوE. Dispar و E. bovisمن مجموع الاصابات بنسبة 82,9 و 26,8 و 4,9% على التوالی. وجدت خمس انماط جینیة للامیبیا الحالة للنسیج I وII و III و IV و V بنسبة 9,75 و 53,65 و 19,5 و 9,75 و 7,3 على التوالی. استنتجت الدراسة هناک خمس انماط جینیة للامیبیا الحالة للنسیج فی الانسان والحیوان معظمها قد تصیب ای مضیف، تسجیل E.bovis  فی الابقار والاغنام.

Introduction

Many species of amoeba genus were identified in the first quarter of the twentieth century (1) and are spread in various regions of the world in the developing countries of the Indian subcontinent, Central and South America, and in the tropics of Africa (2), as well as many species that parasitize many hosts, some of which are common to more than one host (3).

The parasite has the ability to devour red blood cells by Erythrophagocytosis through the process of analyzing its plasma membrane and then digesting the base material, as the ability of the feeding phases to digest erythrocyte may be due to the mechanism of adhesion to the surfaces of the cells and inhibit the adhesion of the parasite decreases phagocytosis and digestion of the erythrocytes during the parasite invasion of the large intestine layers (4), attributed (5) to (Hemoglobinase) and for the need of iron for the survival of the parasite, hence, the digestion of blood hemoglobin to get iron (4,5). The ability of (E. dispar) to cause liver injury, and some studies taken from the comparison of the genetic structure of both species (dispar and histolytica) have concluded that some sites of the gene responsible for the encoding of certain proteins of pathogenicity in the E. histolytica are the same in the E. dispar such genes as the amoebic hole Gal / GalNac-inhibitable lectin. (6,7).

In the presence of E. histolytica and E. bovis as single infection indicating that these parasites can shift from a coexistence with the host to pathogenicity (5), many studies have returned (and until recently) that have attempted to differentiate between E. histolytica infection and its cause, from invasions of tissues into and out of the intestine and between the infectious of E. dispar and other parasites, even those studies were considered unsatisfactory (8). This hypothesis is offset by recent studies that, using experimental animals, have demonstrated the events of the parasite E. dispar for focal ulcers and have an analytical capacity of the epithelial layer (9). performance development of a parasite which isolated from a person with no symptoms of amoebiasis in a medium containing intestinal flora, it was observed endemicity of amoebiasis taking into account the overlap between the two parasites and pathogenic bacteria or intestinal flora, which may alter the behavior of these organisms into a nurse, since some pathogenic bacteria have genes that directly or indirectly encoded molecules activated by the inflammatory response (10).

Materials and methods

Samples collection

A 34 human samples from Al-Hamza General Hospital and twenty eight animals samples were collected from different areas of Al-Hamza district, the presence of the parasite was confirmed in all samples by general stool examination via microscopic examination in the laboratories of the parasitology and microbiology branch in the College of Veterinary Medicine - Al-Qadisiyah University.

Stool DNA extraction

DNA was extracted from fecal samples using the Genomic DNA stool (AccuPrep® Stool DNA Extraction Kit) supplied by Bioneer Korean company according to the manufacturer instructions. This kit was designed to quickly and conveniently extract of DNA from genomic DNA from microorganisms in stool samples. In the presence of chaotropic salt, DNA is bound to glass fibers fixed in a column. Proteins and other contaminants are removed through washing stems, and the DNA isolated and eluted in the final elution step.

Nested PCR method

Nested PCR assay was performed detection E. histolytica and E. dispar according to (11), and E. bovis according to (12), the Nested PCR method was including using primary PCR primers for detection Entamoeba sp. Followed by Nested PCR primers that used for detection of E. histolytica, E. dispar, and E. bovis (Table 1).

The first round of Nested PCR master mix that include Entamoeba sp. Primary primers were prepared by using (AccuPower® PCR PreMix Kit, Bioneer, Korea) (Table 2).

Table 1: Nested PCR primers

After that, these PCR master mix components that mentioned in table above placed in standard PCR PreMix Kit that containing all other components which needed to PCR reaction such as (Taq DNA polymerase, dNTPs, Tris-HCl pH: 9.0, KCl, MgCl₂, stabilizer, and tracking dye). Then, all the PCR tubes transferred into Exispin vortex centrifuge at 3000rpm for 3 minutes. The reaction was performed in PCR thermocycler (T100 thermal cycler BioRad. USA) by set up the following thermocycler conditions; Reactions were performed using the following protocol, initial denaturation at 95ºC for 4 min, and 35 cycles at 95ºC for 1 min, 47ºC for 1 min and 72ºC for 2 min, and a final incubation at 72ºC for 7 min.

The second round Nested PCR master mix that include E. histolytica, E. dispar, and E. bovis primers were prepared by using (AccuPower® PCR PreMix Kit, Bioneer, Korea) (Table 3).

Table 2: PCR master mix

Table 3: Nested PCR master mix

After that, these Nested PCR master mix component that mentioned in table above placed in same PCR PreMix Kit Then, all the PCR tubes transferred into Exispin vortex centrifuge at 3000rpm for 3 minutes.

The reaction was performed in PCR thermocycler (T100 thermal cycler BioRad. USA) by set up the following thermocycler conditions; Reactions were performed using the following Nested PCR protocol, initial denaturation at 95ºC for 4 min, and 35 cycles at 95ºC for 1 min, (50ºC for E. histolytica, 60ºC for E. dispar, 57ºC for E. bovis) for 1 min and 72ºC for 1 min, and a final incubation at 72ºC for 1 min.

After that PCR products were examined by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and visualized under UV Transilluminator.

Real-Time PCR method

Real-Time PCR was performed genotyping of E. histolytica based on allelic discrimination during melting curve of SREHP gene in positive E. histolytica according to (13) the method was firstly included PCR reaction for amplification of SREHP gene based on SREHP primer (Table 4).

The Real-Time PCR amplification reaction was done by using (AccuPower® Green Star^TM qPCR PreMix kit, Bioneer. Korea) and the qPCR master mix were prepared for each sample according to company instruction (Table 5).

After that, these RT PCR master mix components that mentioned in table above was transferred into Green star qPCR premix standard plate tubes that contain the SYBER green dye and other PCR amplification components.

Then the plate mixed by Exispin vortex centrifuge for 3 minutes, then placed in MiniOpticon Real-Time PCR system and applied the following thermocycler conditions (Table 6).

Table 4: PCR and RT-PCR primers (SREHP) for E. histolytica as (12)