Prevalence and molecular identification of Nematodirus helvetianus in camels in Iraq

Background and Aim: Camels from the central part of Iraq are infected with multiple parasitic diseases that have an economic impact by decreasing meat and milk production. This study aimed to evaluate Nematodirus spp. in camels (Camelus dromedarius). Materials and Methods: The study animals consisted of camels slaughtered in the central area of Iraq at the Al-Najaf slaughterhouse. All ages and sexes of camels were examined. Worms were recovered and identified microscopically. For molecular characterization, two Iraqi Nematodirus spp. partial ribosomal genes (ITS1 and ITS2) were sequenced and submitted to the NCBI database. Results: Of 160 camels tested, 29 were infected with Nematodirus spp. (18.13%). Twenty-one nematodes containing the Nematodirus genes were identified in the small intestines of naturally infected camels. BLAST analysis revealed 88.1% sequence similarity with that of Nematodirus helvetianus isolated in China and 87.2% similarity with N. helvetianus isolated in the United States. Conclusion: The prevalence of N. helvetianus warrants the use of anti-helminthic drugs for these animals and a rationale for future control strategies to prevent the transmission of this infection to other livestock.


Introduction
Camels are generally used for transportation, but are also considered a source of meat, milk, leather, and wool since ancient times [1]. Their biological and anatomical features as well as their demeanor make them well adapted for dry climates [2].
Parasites represent a significant challenge to animal health. They primarily affect normal physiology and productivity because they decrease food consumption and cause clinical symptoms such as diarrhea. However, most helminthiases are asymptomatic or without clinical manifestation, thus the herd may appear normal, despite performing below its full potential [3,4]. The genus Nematodirus belongs to trichostrongylids which are the most common helminths and are considered the most important nematodes that have an economic impact by afflicting ruminates, including camels and camelids [5,6]. Nematodirus is a nematode of the small intestine of large and small ruminants and exhibits a direct life cycle. The genus Nematodirus consists of more than 45 species distributed in domestic ruminants worldwide [6,7]. Nematodirus helvetianus is frequently a parasite of cattle (generally named the twisted wireworm). The adults are 10-25 mm in length and coiled in a spiral-like fashion. The eggs are characterized by a large thick shell and contain 4-8 cells (blastomeres). They are shed into the feces and do not hatch until the larva 3 has developed inside. These parasites are found in dry or frigid climates where other genera die because of desiccation [8].
In Iraq, a few studies have established camelid gastrointestinal nematodes, in particular, Nematodirus spp. Altaif [9] identified the infection with helminths in the gastrointestinal (GI) tract. This study aimed to evaluate Nematodirus spp. in camels (Camelus dromedarius).

Ethical approval
Samples collection and transformation were permitted by the Iraqi Ministry of Agriculture Regulation, and the study research protocols were approved by the College of Veterinary Medicine, University of Baghdad laboratory standards.

Study period and location
The study started from early September till the end of December 2018. Samples were procured from the slaughterhouse and processed at the Parasitology Department in the College of Veterinary Medicine at Baghdad University.

Samples collection
Small intestine samples were obtained directly from animal carcasses after slaughtering in Al-Najaf Available at www.veterinaryworld.org/Vol.14/May-2021/31.pdf abattoir (in the central province of Iraq). A total of 160 small intestine samples of camel were included (130 males and 30 females) in the study.

Morphological examination
Samples were sheared longitudinally and examined directly for any nematodes attached to the mucosal surface or with digested contents. Initially, the isolated worms were washed with distilled water to prevent damage and directly examined under a light microscope to maintain its integrity, and lactophenol was used to observe the structures. Captured images, scanning, and morphometric data were obtained using an Olympus L × 70 microscope (Olympus America, Inc., Center Valley, PA) using Spot Software 5.2 Macro photography © 2016. For further examinations, the nematodes were preserved in 70% ethanol. The morphological identification of the adult male nematodes was made based on Soulsby [8].

DNA extraction
A teen adult female Nematodirus spp. (morphologically identified) was randomly selected for DNA extraction [WizPrep™ gDNA Mini Kit (Cell/Tissue), Wizbio Co., Korea)]. Electrophoresis on 2% agarose gel was done to confirm the integrity and purity of the extracted DNA. The DNA concentration was determined by spectrophotometry (NanoDropND-1000 Spectrophotometer, Wilmington, DE, USA). The DNA concentrations were stored at −20℃.

Primer selection
Ribosomal DNA is a common target for the design of probes or selected markers. For the identification of different types of bursate nematodes, primers were synthesized to target the ITS1-5.8s-ITS2 region of the ribosomal RNA gene of Nematodirus oiratianus based on the GenBank sequence. The forward primer was 5′-GTAGGTGAACTGCGGAAGGATCATT-3′ and the reverse primer was 5′-TTAGTTTT CCTCCGCT-3′ [10].

Polymerase chain reaction (PCR)
The amplification reactions (PCR mix and amplification profile) were performed according to the manufacturer's instructions (GoTaq® G2 Hot Start Green Master Mix Co., USA)

Sequencing
Amplicons of approximately 870 bp from five worm samples were submitted for Sanger sequencing (Sanger Sequencer, NICEM, South Korea). The resulting sequences were analyzed by MEGA6.0 (https:// www.megasoftware.net/) [11] and adjusted to a corresponding fragment length. Similarities to the ITS1-ITS2 gene sequences in the GenBank database were determined using the NCBI BLAST program [12]. The sequences were deposited into the GenBank database and accession numbers (MT850129 and MT705244) were assigned. The sequences were aligned using Clustal W2 software (https://www.ebi.ac.uk/Tools/ msa/clustalw2). The final sequence alignment with the isolates found in GenBank was used to construct a maximum likelihood tree in DIVEN (https://indra. mullins.microbiol.washington.edu/DIVEIN/diver. html) using the default setting.

Prevalence finding
This study revealed the presence of the adult male and female nematodes in the small intestine of the camel, Figure-1 [13]. Nematodirus spp. is usually listed as a cattle nematode, though it has been found in many other domestic and wild ruminants, including sheep and camels [3,[14][15][16][17]. There are few studies on the adult helminths in camels and the previous studies have focused on the eggs or the whole larva [18][19][20]. The data showed a total Nematodirus spp. infection rate of 18.13% (Table-3). A previous morphometric analysis recorded a measurement of 14 mm for the adult male body and a 21.5 mm length for the female adult parasite [14]. Male camels showed a higher infection rate (24/130; 18.46%) compared with females (5/30; 16.67%) ( Table-3).

Molecular findings
DNA amplification of the large subunit of the ribosomal RNA gene yielded a single fragment of approximately 870 bp in 15 samples (Figure-2). Unfortunately, of the submitted PCR amplicons, sequencing data for only two samples were obtained, however, they consisted of short nucleotide fragments. Both sequences (MT705244.1; 448 bp and MT850129.1; 207 bp) exhibited high similarity to N. helvetianus gene sequences.

Sequencing analyses
A part of the large subunit of the ribosomal RNA gene of Nematodirus spp. was sequenced. The sequences obtained in this study were compared with those in the GenBank database using BLAST analysis. The results revealed 88.1% similarity with N. helvetianus isolated in China (KC580751.1) and 87.2% similarity with N. helvetianus isolate in the USA (AF194141.1).
The two sequences were aligned with 22 corresponding ITS-ITS2 gene sequence and were closely related taxa. The final aligned sequence was 206 bp after trimming the sequence as required for continual fragmentation. A maximum-likelihood phylogenetic tree was generated from the final alignment of the sequences and showed three main clades of Nematodirus infecting a variety of host ruminants (cattle, sheep, and camels). The first clade contained the Iraqi sequences and two isolates of N. helvetianus identified in China. The second consisted of three clusters, but branched into N. helvetianus (from the USA), N. spathiger, and N. oiratianus. The second Iraqi isolates grouped with the three main clades. The comparison revealed variations among and within geographic regions. The phylogenetic tree topology shows similarities and differences among the globally registered sequences of N. helvetianus and our registered isolates (MT850129 and MT705244) (Figure-3).
A general analysis of the genetic similarity using rRNA sequences data showed the greatest pairwise similarity between N. spathiger and N. helvetianus, Nematodirus battus, and the nucleotide sequence of ITS-2 rRNA (3.9-24.7%) [21]. The previous studies used the ITS1/ITS2 genes (ribosomal) as well as the cytochrome b gene (mitochondrial) as reliable molecular markers for different nematode and protozoan parasites in Iraqi camels and cattle [22]. This is the first morphological (male) and molecular (female) study to identify N. helvetianus in the Iraqi camel [23,24]. However, the lack of data within the geographic locations renders the comparison with the corresponding references insufficient to address    genetic variation. Therefore, future studies with similar or different genetic markers such as mitochondrial DNA are needed.

Conclusion
Camels can be infected with gastrointestinal tract helminths including N. helvetianus which known to infect cattle, sheep, and other ruminants. General protection programs and control strategies are necessary to enhance animal health and productivity as camels may transmit diseases to other livestock and humans.