Molecular study to detect the Eimeria species in sheep in Al-Diwaniyah province, Iraq

Molecular study to detect the Eimeria species in sheep

in Al-Diwaniyah province, Iraq

N.M. Majeed¹, N.N. A'aiz² and A.J. Niemah³

^1,2 Department of Microbiology and Parasitology, ³ Department of Zoonotic diseases, College of Veterinary Medicine, University of Al-Qadisiyah, Iraq

Email: ¹[email protected] ²[email protected], ³[email protected]

Abstract

Sheep eimriosis is one of the most important and common disease which infects sheep in all ages but it is more effective in lambs. The diarrhea with or without blood is the main signs of infection. Eimeria protozoan required single host to complete its life cycle which pass in different stages including schizogony, gametogony and sporogony. The study was designed for detection of sheep Eimeria species through the molecular method. This study was conducted in Al-Diwanyah province during the winter months of 2019. In which 200 sheep fecal samples were collected and examined traditionally to investigate the Eimeria oocytsts. Ninety-seven samples of highly intensity infection with Eimeria oocysts were selected to subject for DNA extraction process. The extracted DNAs were tested through amplification of internal transcribed spacer 1 (ITS-1) gene by conventional PCR, and then phylogenetic analysis was made to diagnose the sheep Eimeria species. All samples that examined microscopically were showed positive results of infections with Eimeria protozoan. Out of 97 molecularly examined samples, forty-five (46.39%) were given positive result in conventional PCR technique, where Eimeria spp. detected through succeeded amplification of internal transcribed spacer 1 (ITS-1) gene. Then phylogenetic analysis referred to that there are five species of Eimeria confirmed in sheep in Al-Diwanyah province including 6 (33.33%) samples diagnosed as E. ahsata, 4 (22.22%) samples E. weybridgensis, 3 (16.66%) samples E. ovinoidalis, 3 (16.66%) samples E. bovis and 2 (11.11%) samples E. auburnensis. So, the Eimeria protozoan appears as an endemic parasite and can infect sheep with different species in study area. The sheep can infect with both specific and nonspecific species.

Keywords: Eimeria, Molecular, Sheep, Al-Diwaniyah

DOI: 10.33899/ijvs.2019.126064.1225, ©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/).

دراسة جزیئیة لتحدید أنواع طفیلی الایمیریا فی الأغنام فی محافظة الدیوانیة، العراق

نوره محمد مجید¹، نعمان ناجی عایز¹ و أحمد جاسم نعمه²

¹فرع الأحیاء المجهریة والطفیلیات، ²وحدة الأمراض المشترکة، کلیة الطب البیطری، جامعة القادسیة، القادسیة، العراق

الخلاصة

 تُعد کوکسیدیا الأغنام من أکثر الأمراض شیوعًا حیث تصیب الأغنام فی جمیع الأعمار ولکنها أکثر تأثیراً فی الحملان. یعتبر الإسهال مع أو بدون دم هو أحد أهم العلامات الرئیسیة للعدوى. یحتاج طفیلی الایمیریا مضیفا واحدًا لإکمال دورة حیاته التی تمر بمراحل مختلفة تتضمن التکاثر الانشطاری، تکوین الأمشاج، والتبوغ. تم تصمیم الدراسة لتحدید أنواع الایمیریا فی الأغنام من خلال الطرق الجزیئیة. أجریت هذه الدراسة فی محافظة الدیوانیة خلال أشهر الشتاء 2019. حیث تم جمع 200 عینة برازیة من الأغنام وفحصها تقلیدیًا للکشف عن بویضات الإیمیریا. تم إخضاع سبعة وتسعین عینة ظهرت فیها أکیاس البیضة للایمیریا لعملیة استخلاص الحمض النووی وقد تم اختبار الحمض النووی المستخلص من خلال تضخیم الجین الفاصل الداخلی 1 بواسطة تفاعل سلسلة إنزیم البولمر التقلیدی، ثم تم إجراء التحلیل الوراثی لتشخیص أنواع إیمیریا الأغنام. جمیع العینات المفحوصة أظهرت نتیجة إیجابیة لوجود أکیاس البیضة للایمیریا مع تباین فی شدة الإصابة. ظهر من بین 97 عینة تم اختیارها للفحص الجزیئی على أساس مستوى الإصابة العالیة فیها أن 45 (46.39٪) عینة منها أعطت نتیجة إیجابیة فی تقنیة تفاعل سلسلة إنزیم البولمر التقلیدی، حیث تم تشخیص جنس الایمیریا من خلال التضخیم الناجح للجین الداخلی الفاصل 1. لقد أشار التحلیل الوراثی إلى وجود خمسة أنواع من الإیمیریا فی الأغنام فی محافظة الدیوانیة، منها 6 (33.33%) عینات تم تشخیصها على أنها E. ahsata و 4 (22.22%) عینات E. weybridgensis ، و 3 (16.66%) عینات E. ovinoidalis ، و 3 (16.66%) عینات E. bovis و 2 (11.11%) عینات E. auburnensis. لذلک یظهر طفیلی الایمیریا کطفیلی مستوطن ویمکن أن یصیب الأغنام بأنواع مختلفة فی منطقة الدراسة ویمکن أن تخمج الأغنام بأنواع خاصة وغیر خاصة.

Introduction

Coccidiosis is an importance economic disease in all animals which can be significant problem in the young's members (1). Coccidiosis in sheep can be a serious disease that causes severe diarrhea, emaciation and sometimes death (2). The disease is more serious when sheep are reared in a closed breeding system, in particularly; lambs kept in overcrowded barns or on irrigated pastures during winter months (3).

The parasite has two phases of life cycle, endogenous phase in which the parasite undergoes numerous divisions in the intestinal cells, Where the ingested sporulated oocysts release sporozoites in intestinal lumen (excystation); and the exogenous phase which takes place outside of body in the environment under certain conditions (4).

 Ingestion of contaminated food and water are the main source of infection and the symptoms of the disease begin with diarrhea, sometimes containing mucus or/ and blood, loss appetite, weight loss, anemia, fatigue, wool breaking and finally death of the animal (5).

The morbidity of the disease may be reach 10-40% and the mortality about 10% (5,6) Eimeria highly hosts specific and the disease is usually caused by sporulated oocysts (7,8).

Different species of Eimeria parasitize the sheep intestine and mixed infections with a number of Eimeria spp. are common in natural infections (9).

Different sheep Eimeria species were recorded worldwide including E. ahsata, E. bakuensis, E. parva, E. pallida, E. crandallis, E. weybridgensis, E. ovina, E. ovinoidalis, E. granulosa, E. intricata, E. faurei and E. marsica (2). Among these species only E. ovinoidalis and E. crandallis are appeared to be pathogenic and lead to the most severe infections (10).

Differentiation among these different species is depending mainly upon shape and measurements of oocysts, infection site and sporulation time, but may be unreliable methods due to the large overlapping in size and shape of these different species (11, 12).

Many methods were used in the diagnosis of Eimeria depending upon fecal examination, serological and molecular (13).

Sensitive and specific fecal examination results can get by use of molecular techniques (14). PCR based on amplification of DNA that has been used for the diagnosis of Eimeria in different hosts and have proved to give accurate results in different used samples (15).

As sheep are regarded as the good source of protein for the study area population, therefore the needing to increase the protein sources requires understanding any disease aliment such as coccidian infection which can limit the production of small stocks. So, given that most traditional diagnostic examinations have a significant error rate, we consider studying the diagnosis of Eimeria species based on molecular methods.

Materials and methods

Two hundred sheep fecal samples 5-10 gram were collected directly from animals' rectum and put in clean plastic labeled containers, then transported with ice bags to a Parasitology laboratory in the College of Veterinary Medicine, University of Al-Qadisiya. These samples were getting from different regions of Al-Diwaniyah province during the winter months of 2019.

All samples were examined by flotation method (16), to investigate the Eimeria oocysts. DNA extraction was done for certain positive samples selected based on the density of oocysts existing using fecal DNA extraction kit (Favorgen Biotech Corp^®, Bioneer, Korea) according to manufacturer directives. The purity of extracted DNA was measured by using a Nanodrop spectrophotometer (THERMO. USA), at 260 /280 nm absorbance, then stored at -20 ºCtill used.

Conventional PCR technique was performed to amplification the extracted DNA at 18SrRNA (ITS-1) gene according to a method described by (15), where Eimeria common primers were used (IDT, Canada).

The up-and downstream sequences in internal transcribed spacer 1 (ITS-1) region were: F: 5´- GCA AAA GTC GTA ACA CGG TTT CCG -3´, R: 5´- CTG CAA TTC ACA ATG CGT ATC GC-3´ with expected product sizes of 348-546 bp. PCR construction was prepared by using (AccuPower PCR PreMix Kit), whereby for 50 μl volume of PreMix Kit tube (Taq DNA polymerase, dNTPs, Tris. HCl pH: 9.0, KCl, MgCl₂, stabilizer, and tracking dye) 5 μl of extracted DNA and 1.5 μl (10 pmol) of both forward and reverse primers were added, then the volume completed with nuclease free distilled water.

Reaction conditions set as an initial denaturation at 94ºCfor 30 sec followed by 35 cycles at 94ºC for 10 sec, 55ºCfor 45 sec, and 72ºC for 20sec with final extension at 72ºC for 2min using PCR thermocycler (Thechne/ USA). The PCR products were analyzed by 1.5 % agarose gel electrophoresis.

 To detect the Eimeria species, PCR positive samples were subjected to DNA sequencing (Bioneer / Korea) by AB DNA sequencing system. The Phylogenetic analysis was performed based on NCBI-Blast Alignment identification and Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).

Results

All samples 100% that examined microscopically showed positive results for Eimeria oocysts in different intensity of infections (Figure 1). Ninety-seven samples were selected according to their heavy infection for DNA extraction. Where 45 46.39% extracted DNA showed positive results when succeeded amplification of internal transcribed spacer 1 (ITS-1) gene through conventional PCR technique to produce 348-546 bp product (Figure 2).

Figure 1: Sheep Eimeria oocysts.

For getting perfect sequencing results, 18 positive samples of more specific PCR products were subjected to DNA sequencing to detect sheep Eimeria species. Where the results revealed that 6 (33.33%) samples diagnosed as E. ahsata (accession no. MN269978, MN269980, MN306558, MN269981, MN269982, MN269984), 4 (22.22%) E. weybridgensis (MN306559, MN306560, MN306561, MN306563), 3 (16.66%) E. ovinoidalis (MN306556, MN306565, MN306566) and 3 (16.66%) E. bovis (MN306557, MN306562, MN306564) and 2 (11.11%) E. auburnensis (MN269979, MN269983) (Table 1; Figure 3).

Figure 2: PCR product of agarose gel electrophoresis of Eimeria ITS-1 gene (348- 546 bp), where; M: 1000bp DNA ladder; Lanes 1-9 Positive samples.

Figure 2: Phylogenetic tree analysis based on 18S rRNA gene partial sequence in local Eimera spp. isolates that used for genetic identification. The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version) at total genetic changes (0.10-0.60%).

Discussion

Eimeria protozoan is a principal etiologic agent for coccidiosis in livestock, where the sickness causes economic loss in sheep as a result of the drop in milk and meat production, paleness, and the increment of the prospect to induce mortality (17).

Table1: NCBI-Blast homology sequence identity (%) among local Eimeria spp. and NCBI-BLAST submitted Eimeria spp. isolates