COX1 gene and ITS-2 region: A comparative study of molecular diagnosis of Parabronema skrjabini in camels (Camelus dromedaries), Al-Najaf Province, Iraq

COX1 gene and ITS-2 region: A comparative study of molecular diagnosis of Parabronema skrjabini in camels (Camelus dromedaries), Al-Najaf Province, Iraq

M.A. Alfatlawi and Haider Hamza Alfatlawy

Department of Veterinary Microbiology, College of Veterinary Medicine, University of Al-Qadisiyah, Al-Diwaniyah, Iraq

monyerr.abd@qu.edu.iq, 0000-0002-7874-7783, corresponding

hdr.aldewani@gmail.com, 0000-0002-7023-5812

2020-12-25

2021-03-21

Abstract

The current study was contributed to the analysis of the nucleotide sequence pattern of the nucleotide sequence of the tissue DNA isolates based on the Internal Transcribed Spacer ribosomal DNA (ITS2rDNA) gene and Cytochrome c oxidase subunit 1 mitochondrial DNA (COX1 mtDNA) using the traditional polymerase chain reaction (150 samples of abomasum collected directly from camel carcasses after working in Al-Najaf slaughterhouse from 1/10/2019 to 1/2/2020). ITS2rDNA were well suited for the prepared primer with size783 bp and identical ratio ranged 81.17-99.73% of the same species, as indicated a high similarity of the isolates taken from two-humped camels in China or less related to Parabronema skrjabini in sheep and goat. In addition, the study identified the number of the mutations within the four COX1 gene and ITS-2 region, which were the most conservative region of the host's species, supporting the concept of host specificity with Parabronema skrjabini. The COX1 gene and ITS-2 region applied to confirm the diagnosis using a universal primer, as it included eight isolates with a size of 689 bp, identical values were ranged from 84.99-98.02% depending on the multiple sequence alignment and showed an increase in the substitution level among isolates at an upper taxonomic level. Studying of the COX1 gene and ITS-2 region in Parabronema skrjabini demonstrated a significant relation in the cluster and an early common ancestor with isolates of the two-humped camel (China). As for the COX1 gene and ITS-2 region, the phylogenetic relationship supported the ribosomal gene results, especially with Habronema muscae or related species such as Habronema majus, Dirofilaria repens, Dipetalonema evansi, Setaria tundra, Cercopithifilaria sp towards the root node. Therefore, considering COX1 gene and ITS-2 region as an ideal tool in determining the phylogenetic history of the sequence maps, but less conservative mode than the ITS2 ribosome gene based to a taxonomic species level.

Keywords: ITS2rDNA gene, COX1mt DNA gene, Camels (Camelus dromedaries), Iraq, Parabronema skrjabini

جین COX1 و منطقة ITS-2 : دراسة مقارنة للتشخیص الجزیئی لطفیلی Parabronema skrjabini فی الجمال (Camelus dromedaries) فی محافظة النجف، العراق.

منیر عبد الأمیر الفتلاوی و حیدر غالب الفتلاوی

فرع الأحیاء المجهریة البیطریة، کلیة الطب البیطری، جامعة القادسیة، الدیوانیة، العراق

الخلاصة

ساهمت الدراسة الحالیة فی تحلیل نمط تسلسل النوکلیوتیدات لعزلات الحمض النووی للأنسجة استنادًا إلى جین DNA الریبوزومی المنقسم الداخلی (ITS2rDNA) والجین الفرعی 1 للمیتوکوندریا (COX1 mtDNA) باستخدام تفاعل متسلسل للبولیمیراز التقلیدی (150 عینة من الانفحة تم جمعها مباشرة من جثث الإبل بعد الذبح فی مسلخ النجف من 1/10/2019 إلى 1/2/2020. کان ITS2rDNA مناسبً تمامًا للطبقة الأولیة المحضرة بحجم 783 نقطة أساس ونسبة مماثلة تراوحت 81.17-99.73٪ من نفس النوع، کما یتضح من التشابه الکبیر للعزلات المأخوذة من الإبل ذات السنامین فی الصین أو أقل ارتباطًا بـ Parabronema skrjabini فی الأغنام والماعز. بالإضافة إلى ذلک، حددت الدراسة عدد الطفرات داخل الجینات الأربعة COX1 ومنطقة ITS-2، والتی کانت المنطقة الأکثر تحفظًا لأنواع المضیف، ودعم مفهوم خصوصیة المضیف مع Parabronema skrjabini. تم تطبیق جین COX1 ومنطقة ITS-2 لتأکید التشخیص باستخدام أساس عالمی، حیث اشتمل على ثمانی عزلات بحجم 689 نقطة أساس، وتراوحت القیم المتطابقة من 84.99-98.02٪ اعتمادًا على محاذاة التسلسل المتعدد وأظهرت زیادة فی مستوى الإحلال بین العزلات عند مستوى تصنیفی أعلى. أظهرت دراسة جین COX1 ومنطقة ITS-2 فی Parabronema skrjabini وجود علاقة مهمة فی الترکیب الجینی مع عزلات من الجمل ذی السنامین (الصین). أما بالنسبة لجین COX1 ومنطقة ITS-2، فإن علاقة النشوء والتطور تدعم نتائج الجینات الریبوزومیة، خاصة مع Habronema muscae أو الأنواع ذات الصلة مثل Habronema majus و Dirofilaria repens و Dipetalonema evansi و Setaria tundra و Cercopithifilaria sp تجاه عقدة الجذر. لذلک، بالنظر إلى جین COX1 ومنطقة ITS-2 کأداة مثالیة فی تحدید تاریخ تطور خرائط التسلسل، ولکن وضع أقل تحفظًا من جین الریبوسوم ITS2 على أساس مستوى الأنواع التصنیفیة.

Introduction

Parabronema skrjabini: one of the spirurida nematodes that infect abomasum of the domesticated ruminants such as camel, sheep and goats (1). Sarwar (2) was identified a genus Parabronema, a first time was described from Russia (1924). Most abomasal nematodes are causes diarrhea and lack of absorption, weakness, low weight and histopathological changes in abomasa mucosa which lead to reduce in production or working efficiency of the infected animal (3). P. skrjabiniis known with an indirect biological cycle, which require an intermediate host (flies) such as Stomoxys bengalensis, paregle spp. and Lyperosia spp (4), after 1-2 days fly's egg begin to hatch and feed on the contaminated dung. Finally, Larva3 of the parasite penetrating the intestine wall of the flies by the anterior lancet (5). In small ruminants, P. skrjabini is isolated from sheep and recorded high prevalence rate of the gastrointestinal nematodes during December (100%), however less in March about 44.47% (6). Garedaghi et al (7) recorded infection rate 4.91% in sheep. Most researchers depended on microscopically examination of faeces and adult worms (3). Recent researches used a molecular technique to clarify the genotype of P. skrjabini in many host (8). Different factors effect on the prevalence rate of abomasal worms (age, sex, body condition and immunity status) Borji (9), and he observed high prevalence of gastrointestinal nematode in sheep's females than males, adult sheep with high ability to resistance nematode's invasion compared to the young ages due to development of immunity response, in other hand the seasonal fluctuation associated with the activation of the intermediate host (10). Epidemiological studies of abomasal nematodes are showed that annual temperature changing during year play an important role in larval development or arrested (7). Arabian camels (Camelus dromedaries) are important livestock animal live in harsh environments (11), the number of camels according to FAO, 2003 statistics is about 12.5 million camels about 70% of the world population. There are an increased the economic importance of camels has increased with utilization of their products such as milk, meat and skin (12), due to its geographical distribution, few studies were concentrated on gastrointestinal nematodes in camels and difficulties in taking samples due to the fact that the Bedouin majority is rising (13), Al-Taif (14) was the first study on one-humped camel's infection in Iraq.

Limited information has prompted us to identify this nematode by morphological and molecular distinguishing; and sequencing of ITS2r DNA and COX1mt DNA for determinate a diagnostic level.

Materials and methods

Collecting 150 samples of abomasum directly from camel carcasses after working in Al-Najaf slaughterhouse. Before that, we installed data of the camels for determining the sex, age and periods. Transferring the samples in a cold box to the parasitology lab of the College of Veterinary Medicine, Al-Qadisiyah University, gross examination of the samples grossly for collecting the adults, isolated worms were taken by forceps to diagnose species of parasites, depending on the diagnostic characteristics described by (1,15). Molecular identification of the nematodes uses extracted of 20 rDNA isolates and traditional PCR technique with higher purity of DNA (16-18), PCR master mix prepared by using AccuPower® PCR PreMix Kit and this master mix done according to company instructions included: DNA template 5µl, Forward primer 1.5µl, Reveres primer 1.5µl and PCR water 12µl. The PCR primers used in this analysis to the amplify ITS2rDNA gene of P. skrjabini, it is being designed according to (9), while COX1mtDNA gene was prepared as (non-degenerate primer) according to the molecular study of other Spirurida (19), these primers were produced from Bioneer company, Korea as following table 1, and reaction conditions of PCR for amplification showed as in the table 2.

Table 1: Sequences of primers used for identification of Parabronema skrjabini using traditional PCR

Table 2: Cycling conditions of PCR for amplification of Parabronema skrjabini

Positive products of the PCR ribosomal genes were transferred in an ice bag by DHL to Macrogen Company in Korea for conducting DNA sequencing with DNA sequencing method then the sequencing data submitted into NCBI. The DNA sequencing study was carried out using the local alignment tool in NCBI website (http://www.ncbi.nlm.nih.Gov/ BLAST/), either multiple sequence alignment of the partial small subunit rDNA gene or pairwise sequence model for determining the genetic variation and substitution among the local isolates and other species depending on the nucleotide sequences (20-22). The phylogenetic tree is using the Maximum Composite Likelihood system. Phylogenetic analysis that uses the ITS2 rDNA gene according to Hasheminasab et al. (3) and COX1mtDNA sequences of P. skrjabini, that determined by UPGMA analysis.

Results

Electrophoresis of ITS2rDNA gene products

Results of electrophoresis showed that analysis ITS2rDNA for P. skrjabini was positive at 783bp product size (Figure 1).

Figure 1: Agarose gel electrophoresis image that showed PCR product analysis ITS2-rDNA for Parabronema Skrjabini. M (Marker ladder 1500-100bp). Lane (1-10) positive Parabronema skrjabini isolates at 783bp product size.

Electrophoresis of the COX1mtDNA gene products

Results of electrophoresis showed that analysis COX1mtDNA for P. skrjabini was positive at 689bp product size (Figure 2).

Figure 2. Agarose gel electrophoresis image that showed PCR product analysis COX1-mtDNA for Parabronema skrjabini. M (Marker ladder 2000-100bp). Lane (1-8) positive Parabronema skrjabini isolates at 689 bp product size.

DNA sequencing analysis and phylogenetic tree of ITS2rDNA gene

PCR products are estimated at 783bp in the size and that submitted to the NCBI website (accession number: MT509436, MT509437, MT509438 and MT509439). The current sequences were compared to the previous results documented on the NCBI website, ITS2-rDNA have a higher homogeneity range 98.01-99.73% with Bacterain camel isolates (Accession number:EU375510, EU420132), but less similarity to other isolates from the small ruminant about 81.17-86.29% LC275906, LC275902, LC325445, LC275908, LC275903, LC325447, LC325450 and 84.04% of P. smithi MH445384. Habronema muscae or Habronema majus collected from the equines or donkey with a homogeneity ratio about 83.08-86.99% as indicated in the (Table 3).

Table 3: The NCBI-BLAST homology sequence identity

Phylogenetic tree of Parabronema skrjabini depending on ITS2 rDNA sequences uses the maximum log-likelihood method. The current study showed the overall nucleotide identity among isolates reported in Iraq and other sites of the world as indicated in the (Figure 3-6).

Figure 3: The phylogenetic tree analysis based on ITS2rDNA in local camel's isolates that used for confirmative genetic identification. The phylogenetic tree was constructed using (MEGA 6.0version). The local Parabronema skrjabini IQ camel isolates (No.1 and 4) were showed genetic closed related to NCBI-BLAST Parabronema skrjabini isolate from China at total genetic changes 0-0.40.

 In table 4, the differences in the substitution ratio have a clear divergence among local isolates and Basra isolates from small ruminants, reaching 71point mutation and a number of the gaps on NCBI website (pairwise sequence alignment).

Table 4: The number of the substitution (gaps, point mutation in local isolates included identical isolates of the camel, sheep and related species by Multiple sequence alignment tool

DNA sequencing analysis and phylogenetic tree of COX1mtDNA

The present study provides the first phylogenetic description of the mitochondrial COX1region in P. skrjabini and its role in the phylogenetic rate with other nematodes, then comparing of the substitution level among this nematode and some available sequences for other spirurida by BLAST software on the NCBI website. The mtDNA sequences have an identic level about 84.99-98.02% in eight local isolates of P.skrjabini. The COX1mtDNA gene were 689 bp in length after electrophoresis processing. we were able for comparing the identical ratio in the local sequences of the COX1mtDNA gene and determined 97.39-98.02% for H. muscae isolate KX868085 and Habronema majus 87.82-89.62% KX868083, the current sequences reported on the NCBI website (Accession number: MT664736, MT664737, MT664738, MT664739, MT664740, MT664741, MT664742 andMT664743). Finally, the less identical species were estimated Dirofilaria repens, Dipetalonema evansi, Setaria tundra, Cercopithifilaria sp. the genetic variation ranged 84.99-86.34% as shown in the (Table 5). The phylogenetic tree constructed using the data of strong bootstrap value among the Parabronema isolates and some spirurida, in the (Figure 4) the monophyletic clade involved Habronema sp. and the current isolates at most common ancestor. The related species of China, Poland, Austria and Italy have inserted to the second cluster within same common ancestor by the bootstrap value (polyphyletic group). The number of mutations/ substitutions per isolate (9-12) and the genetic variation (1.98-2.6%) with Habronema muscae KX868085 indicated in the (Table 6). Knowledge of the evolutionary principles as the substitution patterns and variability rate are a necessary step of the phylogenetic studies. We have identified the substitution model of the mitochondrial COX1mtDNA gene with related species. The percentage of transition bias was 79.7% T.i. and 11.9% T.v. of the total number of the substitutions, the most genotype is T/C, then A/G shown in the (Table 8). The results of local isolates with a number of species revealed that spirurida possesses an advanced level of the mutation, the common genotype of each nematode, we can be reflecting the percentage of the substitution about 15.26% and a number of mutations within the eight local isolates 47-71 mutation shown in the (Table 7). The percentage of nucleotides from the COX1 mtDNA gene within the current isolates and other isolates showed the rich A + T content as indicated in the (Figure 5), The ratio of higher A + T content observed within local isolates in three positions.

Table 5: The NCBI-BLAST homology sequence identity

Table 5: The NCBI-BLAST homology sequence identity (continue)

Figure 4: The phylogenetic tree analysis based on COX1mtDNA gene partial sequence in local Parabronema skrjabini isolate Iraqi Camel isolates that used for the genetic variation identification. The phylogenetic tree was constructed using UPGMA method in (MEGA). The local Parabronema skrjabini isolate Iraqi-Camel isolate (No.1: MT664736.1-No.8: MT664743.1) were showed the genetic variation in COX1mtDNA gene among local Parabronema skrjabini isolates and genetic related species at total genetic variation 0-0.80% and relationship included genus Habronema sp related to an early most common ancestor.

Table 6: The mutation analysis of COX1mtDNA gene in Parabronema skrjabini and the substitutions among local isolates with Habronema muscae KX868085, the ratio of mutation range (1.98%-2.6%) by pairwise sequence alignment tool

Table 7: The mutation number of the nucleotides database from COX1mtDNA gene in Parabronema skrjabini and identify the substitutions with other spiurida by multiple sequence alignment tool:

Underline indicated to the most frequency genotype of NO.1-NO.8 isolates

Table 8: The total number of T.i.: T.v. substitutions among Habroenema muscae KX868085 and NO.1-NO.8 local IQD-Camel isolates of cox1mtDNA by Pairwise sequence alignment in NCBI Website

Figure 5: The nucleotide composition of the COX1mtDNA gene in the local isolates (No1-No8) and other isolates inserted to NCBI website by Mega 6.0 software: