Trichostrongyloid nematodes in ruminants of northern Iran: prevalence and molecular analysis

Background This study was carried out to investigate the prevalence and analyze the molecular characteristics based on the internal transcribed spacer (ITS) 2 region of the ribosomal RNA (RNA) gene of trichostrongylid nematodes in different ruminants from Guilan province, northern of Iran. Methods The gastrointestinal tracts of 144 ruminants including 72 cattle, 59 sheep, and 13 goats were collected from an abattoir in Guilan province during July to September 2018. After isolation the helminths, male specimens were identified based on morphological parameters. PCR and partial sequencing of the ITS2 fragment were conducted. After phylogenetic analysis, the intraspecific and interspecific differences were calculated. Results The prevalence of total infections with the nematodes was 38.9, 74.6 and 84.6% among cattle, sheep and goats, respectively. Eleven species of trichostrongylid nematodes including Haemonchus contortus, Marshallagia marshalli, Trichostrongylus axei, T. colubriformis, T. vitrinus, Ostertagia trifurcata, Teladorsagia circumcincta, Marshallagia occidentalis, O. lyrata, O. ostertagi, and Cooperia punctate were recovered from the ruminants. The most prevalent trichostrongyloid nematodes in cattle, sheep and goats were O. ostertagi (26.4%), M. marshalli (64.4%) and T. circumcincta (69.2%), respectively. Phylogenetic tree was discriminative for Trichostrongylidae family, while phylogenetic analysis of the ITS2 gene represented low variations and no species identification of Haemonchidae and Cooperiidae families. Conclusions This study suggests the high prevalence and species diversity of trichostrongyloid nematodes in different ruminants, indicating the importance of implement antiparasitic strategies in north regions of Iran. As well, this study showed that the ITS2 fragment is not a discriminative marker for Haemonchidae and Cooperiidae families, and investigation of other genetic markers such as mitochondrial genes would be more valuable for better understanding of their phylogenetic relationships.

animals with a worldwide distribution. Trichostrongyloid nematodes such as Trichostrongylus (Trichostrongylidae), Haemonchus (Haemonchidae), Ostertagia (Haemonchidae), Nematodirus (Molineidae), Marshallagia (Haemonchidae) and Cooperia (Cooperiidae), mostly live in abomasum and the first part of the small intestine in ruminants [2]. Actually, infection with these parasites leads to loss of meat, wool, and milk production and decreased feed intake, weight gains and growth rate [3]. These parasites also can occur serious or even fatal manifestations in ruminants and other grazers. Among the super family, H. contortus is one of the most pathogenic blood sucking nematodes, which causes severe anemia, hypoproteinemia, edema, and even death in ruminants [4]. Transmission of these nematodes is mostly through the ingestion of infective larvae in contaminated pasture and water [5]. The epidemiology of gastrointestinal helminths in domestic animals serves to improve prevention and control strategies, evaluation of risk factors, and decrease in production losses of the animals. In addition, these studies help to determine probable sources of animal and human infections [1,[6][7][8].
Trichostrongylus species are the potential zoonotic nematodes, which are prevalent in humans form some regions of the world, particularly in the Middle East, Far East, and some African countries [9][10][11]. Human trichostrongylosis has the highest infection rates and largest variety of species in Iran compared to other countries in the world due to close contact with ruminants in rural and nomadic populations [9]. Recent studies illustrated that Guilan province in north of Iran is one of most important endemic regions of human trichostrongylosis in Iran [7,[12][13][14]. Other genera of this super family including H. contortus, M. marshalli, N. abnormalis, O. ostertagi and T. circumcincta have been rarely reported in humans from different parts of world, especially in Iran [7,9,10,15].
Recently, PCR based methods are used to identify species and phylogenetic analysis of different trichostrongyloid nematodes [16][17][18][19]. These studies have shown that the internal transcribed spacer 2 (ITS2) and the cytochrome c oxidase subunit 1 (Cox1) genes are useful targets for species differentiation and analyzing of the genetic variations [19][20][21]. The aim of this study was to determine the prevalence of the trichostrongyloid nematodes obtained from slaughtered indigenous ruminants in Guilan province, northern Iran, and to investigate of their genetic diversity using the ITS2 region of the rRNA gene.

Study area and samples collection
Guilan province is located in southwest coast of the Caspian Sea in the northern Iran (Fig. 1). It covers an area of 14,711 km 2 (between 36° 36′ and 38° 27′ N, and between 48° 43′ to 50° 34′ E), and has temperature humid climate with mean annual rainfall of 1359 mm. The average seasonal temperature in Guilan province is 7.5 °C in winter, 18.5 °C in spring 24 °C in summer, and 13.5 °C in autumn. The average relative humidity is about 80%,  which reaches to maximum in autumn and winter, and decreases in summer and spring [22]. In current study, a total of 144 ruminant gastrointestinal tracts from 72 cattle, 59 sheep, and 13 goats were collected from Talesh abattoir of Guilan province, northern of Iran, during July to September 2018. Animals all were local breed and samples were randomly collected from 10 to 15 animals in each sampling time (1 day in each week) for 12 weeks. The contents of abomasum and duodenum were washed using tap water runs through 20, 40, and 100 mesh sieves and examined for infectivity with trichostrongyloid nematodes by stereomicroscope. The obtained specimens were preserved in 70% ethanol for parasitological and molecular examinations. For morphological identification, the male nematodes were stained with Azocarmine, and identified at the level of species using valid nematodes systematic keys [23,24].

DNA extraction and polymerase chain reaction (PCR) amplification
Total genomic DNA was extracted from one male worm of each species of trichostrongyloid nematodes in every kind of ruminants (n = 21) using a commercial DNA extraction kit (Yekta Tajhiz Azma, Tehran, Iran), according to the manufacturer's instructions. The ITS2 region was amplified using the primer pairs NC1 (5′-ACG TCT GGT TCA GGG TTG TT − 3′) and NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) [25]. PCR conditions comprised an initial denaturing step of 95 °C for 6 min followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 60 °C for 90 s and extension at 72 °C for 60 s, and 72 °C for 5 min as a final extension. The amplification products were separated on 1.5% agarose gel electrophoresis, and visualized with UV transluminator (UVITEC, Cambridge, UK). Later, the PCR products were sent to a domestic sequencing company (Codon genetic company, Tehran, Iran) for sequence determination via the Sanger method.

Phylogenetic analysis
The sequence results were edited and tr immed using Chromas version 2.01 (Technelysium Pty Ltd., Brisbane, Queensland, Australia), and compared to the GenBank submitted sequences using the BLAST programs and databases (http:// www. ncbi. nlm. nih. gov/). The sequences of the ITS2 gene were submitted to the GenBank database (accession numbers: MN845160-MN845180). Phylogenetic analysis was performed with sequences obtained in the present study along with the reference sequences, which were deposited in the GenBank database, using Maximum-Likelihood algorithm and Tamura-3-parameter model in the MEGA 6.0 software.
Bootstrap value was determined based on 1000 replications for evaluation of the phylogenetic tree reliability.

Statistical analysis
Statistical analyses were performed using SPSS software version 18 (SPSS Inc., Chicago, Illinois, USA), and statistical tests including chi-squared (χ2) and Fisher's exact tests were used to evaluate the association between trichostrongyloid nematode infections with different kinds of ruminants and sex of animals. A value less than 0.05 was considered statistically significant.

Results
Overall, 83 out of the 144 ruminants (57.6%) were found infected with different genera of trichostrongyloid nematodes. The prevalence of total infections with the nematodes was 38 The most prevalent trichostrongyloid nematodes in cattle and sheep were O. ostertagi (26.4%) and M. marshalli (64.4%), respectively. Also, the most predominant nematodes in goats were T. circumcincta (69.2%) and M. marshalli (53.8%). The prevalence rate of these parasites is shown in Table 1. Thirty-one of all ruminants (21.5%) were males and 113 (78.4%) were females. Of these, 18 (58.1%) of the males and 65 (57.5%) of the females were infected with the trichostrongyloid nematodes. No statistically significant difference was seen between total infection and the sex (P > 0.05). Also, there was not any significant difference between sex and the infection rates of each animal (P >0.05) (    (Fig. 5).
The phylogenetic analysis of the Cooperiidae family showed that our C. punctata (MN845167) obtained from cattle grouped with cattle isolates of C. punctata from New Zealand (KC998744 and KC998745) and cattle isolates of C. spatulata from Brazil (MH267786, MH267791 and MH267790) (Fig. 7).

Discussion
This study revealed 38.9% cattle, 74.6% sheep, and 84.6% goats were infected with the trichostrongyloid nematodes. The prevalence of abomasal nematodes were reported 44 and 25% of the slaughtered cows in Tabriz, northwestern Iran [26] and Kerman southeastern Iran [27], respectively. Moreover, the prevalence of trichostrongyloid nematodes was reported 94 and 6.1% of the cattle in Belgium [28] and Ethiopia [29], respectively. Several studies from Iran showed high infection rate of the parasites in sheep and goats. Kordi et al. [27] reported a high infection rate as 100% in sheep and goats  in Kerman. Also, Garedaghi et al. [30] found the infection rates of 81 and 71.2% in sheep and goats of Mazanderan province, respectively. However, on the contrary to the present study, some previous studies in Iran have reported lower infection rate in the small ruminants [31,32]. Furthermore, Fufa et al. [33] reported the prevalence of abomasum nematodes in sheep and goats, slaughtered in Ethiopia to be 83.6 and 77.6%, respectively. Our finding showed that the most predominant trichostrongyloid nematode in cattle in the study area was O. ostertagi (26.4%). O. ostertagi is a common parasitic nematode of cattle in the world, but it can also be found to a lesser extent in sheep, goats, wild ruminants, and horses [34]. Similar to our study, O. ostertagi was reported as predominant nematode in cattle in Canada [35]. However, H. contortus was reported in cattle with highest prevalence (17.6%) in Tabriz, northwestern of Iran [26]. Abuhay et al. [29] reported T. axei (2.86%) was the most predominant abomasal nematodes of cattle slaughtered in Ethiopia.
In present study, M. marshalli was identified as the most predominant nematode in sheep (64.4%). Also, the most prevalent nematodes in goats were T. circumcincta (69.2%) and M. marshalli (53.8%). Similar to our results, Pestechian et al. [36] reported that M. marshalli (57.1%) in sheep and T. circumcincta (47.2%) in goat were dominant species in Isfehan, central part of Iran. M. marshalli was also reported as more prevalent species among both sheep (20.5%) and goats (60%) in Qazvin province, northwestern of the country [37]. In other studies, in Iran, H. contortus in goats and T. circumcincta in sheep were also found as the frequently reported species in northwest and west of the country, respectively [31,38]. Aligned with our findings, M. marshalli occurred with the highest prevalence among sheep in Canada [39]. In other study in Ethiopia, Haemonchus spp. were found as most predominant abomasal nematodes in both sheep and goats [40]. Recently, Ashrafi et al. [7] reported the occurrence of natural human infection by T. circumcincta for the first time in Iran and the second time in the world.
In the current study, Trichostrongylus species including T. colubriformis, T. axei, and T. vitrinus were detected from the animals. Previously, several studies reported these nematodes as the predominant species of Trichostrongylus among different herbivores in most parts of Iran [9]. Recently, infection with some species of Trichostrongylus including T. colubriformis, T. axei, T. vitrinus, and T. longispicularis have been reported from humans in Guilan province from which T. colubriformis was as the most common species [12]. Additionally, Gholami et al. [41] and Sharifdini et al. [17] reported T. colubriformis as the most probable common species and T. axei in inhabitants of Mazandaran province, north of Iran. Also, T. colubriformis was identified as a predominant species among humans in Thailand, France, and Laos [11,16,42].
In this study, O. lyrata was reported from cattle (8.3%), sheep (1.7%) and goats (7.7%). Until now, O. lyrata has not been reported from Iran, and to best of our knowledge this study is the first report of this species in the ruminants. This nematode has mainly reported from cattle in different parts of the world [43][44][45].
In the current study, there was no significant difference between trichostrongyloid infection and the sex (P > 0.05). Our findings are in consistent with Kordi et al. [27], Garedaghi et al. [31] and Amniattalab et al. [46]. Nevertheless, some researchers have observed higher rates of trichostrongyloid nematode infections in female hosts compared to the males [47,48].
The existence of genetic diversity of the ITS2 fragment among different Trichostrongylus species has been confirmed previously [7,12,17,41]. The phylogenetic tree of Trichostrongylidae family represented that all species were clearly separated and they were similar to the species obtained from human and animal subjects in Iran and other countries. Our study also showed that T. colubriformis, T. vitrinus, and T. axei isolates obtained from different animals had 100% similarity. Sequences of Trichostrongylus species isolated in present study had 99-100% homology with those obtained from humans and domestic animals in Iran. This finding confirmed that the ruminant and human Trichostrongylus species have close phylogenetic relationship. This could explain that human infections in the study area may occur due to close contact with domestic animals and application of domestic animal's dung as fertilizer in vegetable farms.
Our phylogenetic analysis of the Cooperiidae family showed that isolates of C. punctata and C. spatulata clustered together in one clade. This finding presented that the ITS2 fragment was unreliable for differentiation at the species level. Recently, Ramünke et al. [53] reported that the ITS2 has minimal nucleotide differences between the Cooperia species and is accurate only up to genus level identification. The authors also showed that, in contrast to the morphological characterization, no molecular distinction between C. punctata and C. spatulata was possible with any of the four gene markers including isotype 1 β-tubulin, the mitochondrial Cox2, ITS, and mitochondrial 12S rRNA genes [53].

Conclusions
The findings of this study showed the high prevalence and species diversity of trichostrongyloid nematodes in different ruminants of northern Iran. Therefore, implement antiparasitic strategies seems to be necessary to increase productivity of the domestic animals. Furthermore, extra epidemiological studies in different seasons and reigns of the country should be conducted to provide more information about the seasonal dynamics of the gastrointestinal helminths. The present molecular analysis of the ITS2 fragment is not sufficient for valid species identification of Haemonchidae and Cooperiidae families and only can differentiate members of Trichostrongylidae family. Providing more genetic markers alongside obtaining specimens from other geographical locations and hosts would be valuable for better understanding of their phylogenetic relationships.