Morpho-Molecular Characterization of Cattle Haemonchus Nematodes From Southeast of Iran

Introduction

Haemonchus species (Trichostrongyloid nematode) are economically important nematodes living in the abomasa of small and large ruminants. The most common species affecting the cattle around the world are Haemonchus placei and H. contortus, respectively (Achi et al., 2003; Fitzpatrick et al., 2013). In Iran, prevalence of haemonchosis was 22% (Eslami and Nabavi, 1976), 9.3% (Tehrani et al., 2012), 16.2% (Rahimi et al., 2020), and 0.22% (Nazarbeigy et al., 2021). However, there is no comprehensive information about the geographical distribution of Haemonchus throughout the country. Many investigations have shown the morpho-molecular diversity of Haemonchus population (Vadlejch et al., 2014; Salle et al., 2019). Knowledge of genetic diversity and morphological analysis can provide a foundation for understanding the drug resistance, epidemiological features, and control strategies in a geographical area (Meshgi et al., 2015; Salle et al., 2019). Based on a morphological study, some parameters including total body length, spicule length, gubernaculum length, spicules, spine conditions, and number of longitudinal ridges can be used for the identification of Haemonchus species (Nabavi, 2017). Many researchers have indicated that Internal Transcript Spacers (ITS) especially ITS2 are the most convenient targets of molecular approach in the identification of the parasite species (Li et al., 2016; Nabavi et al., 2014). Moreover, considering the benzimidazole resistance, the molecular study on beta-tubulin gene is highly recommended (Mohammedsalih et al., 2020). Today benzimidazole resistance in Haemonchus nematodes is highly prevalent and has been noted as a great threat to ruminant production system in many countries worldwide (Von Samson-Himmelstjerna et al., 2009; Nabavi et al., 2011; Mohammedsalih et al., 2020). Sistan and Balouchestan is the widest province in southeast of Iran with very long borders with Pakistan and Afghanistan countries (Masoodian, 2003). Cattle production is prevalent especially in the north of Sistan and Balouchestan province (Nabavi, 2017). The aims of the present study were to evaluate the morphological parameters and molecular targets (ITS1-ITS2 and beta-tubulin) of cattle Haemonchus nematodes and to find species diversity and benzimidazole resistance.

Materials and Methods

The abomasum of 300 slaughtered cattle were inspected for the presence of Haemonchus nematodes. The cattle were collected from Zabol, Zahedan, and Iranshahr slaughterhouses in Sistan and Balouchestan province, southeast of Iran (100 abomasa per each district) from May 2016 to April 2017. A total of 98 adult male nematodes were identified morphologically according to the keys of Lichtenfels et al. (1994) and then stored in 70% ethanol until molecular analysis.

Morphological Analysis

The worms were cleared in phenol-alcohol and morphologically analyzed based on the total body length, gubernaculum length, right and left spicule length, the distance between spicule spine and the spicule posterior end (Left and right) (Achi et al., 2003).

Molecular Analysis

DNA Extraction and PCR

DNA was extracted from 20 male adult worms using the tissue DNA extraction kit (Takapouzist, Tehran, Iran) following the manufacturer’s instructions. The extracted DNA was stored at -20°C until being used. DNA samples (ITS1, 5.8S and ITS2 regions) were amplified individually by PCR using the primer pairs described by Nabavi et al. (2014). For amplification of beta-tubulin fragment (including 198 and 200 codons), the primer pairs described by Nabavi et al. (2011) were used. PCR was performed in a total volume of 50 μL including 1x Mastermix PCR buffer (Pishgam, Tehran, Iran), 30 pmol/50 μL of each primer (Pishgam, Tehran, Iran), and approximately 2 ng per 4 μL of genomic DNA in Mastercycler^® nexus (Eppendorf, Hamburg, Germany) under the following thermal pattern: 5 min incubation at 95°C to denature double-stranded DNA, 35 cycles of 45 s at 58°C (annealing step), 45 s at 72°C (extension step), and 45 s at 94°C (denaturation step). Finally, PCR was completed with an additional post amplification extension step for 10 min at 72°C. For all reactions, samples without DNA served as negative controls.

Sequencing and Data Analysis

Twenty samples (6 from Zabol, 7 from Zahedan, and 7 from Iranshahr) were selected to be sequenced in both directions for ITS and beta-tubulin fragments. Multiple alignments of the ITS1, ITS2, and beta-tubulin sequences for each species were then used to compare and calculate similarity scores between the species. In beta-tubulin sequences, the codons No. 200 and 198 were checked for benzimidazole resistance (Nabavi et al., 2011). ClustalW2 sequence alignment tool (http://www.clustalw.genome.jp) was used for all alignments and calculation of similarity score. The phylogenetic trees were built using the maximum parsimony (MP) and distance methods, namely, neighbor-joining in MEGA 6.0 (Li et al., 2016).

Statistical Analysis

The SPSS software version 18 (SPSS Inc., Chicago, IL., USA) was used for the statistical analysis. A 95% confidence interval was calculated for mean of population.

Results

The overall infection rate was 7.33%. All of the specimens were morphologically identified as H. contortus (Table 1). The mean burden of Haemonchus nematodes was 13 per infected abomasum. DNA amplification of the ITS1-5.8s-ITS2 rDNA produced a single fragment of 975 bp (Figure 1). The length of ITS1 and ITS2 fragments was 400 bp and 231 bp, respectively. The direct sequencing of all nematodes revealed identical genotype (sequence data is available under accession number of KX829170 for 18s, ITS1, 5.8s, ITS2, and 28s). After the edition of sequences and alignment, the sequence identities ranging from 98%- 99% for ITS1 and 97%-100% for ITS2 were detected. The BLAST hit re­sults indicated that our query ITS1 sequences were similar to the sequences of various geographical isolates of Haemonchus species (98-99%).

Figure 1. Amplified total fragment of Haemonchus ITS1-5.8S-ITS2 with 975 bp (Lanes 1-8).

M:100bp marker, NC: negative control.

Table 1. Morphologic and morphometric findings of adults male Haemonchus nematodes from cattle of Sistan and Balouchestan province, Southeast of Iran.

Based on the current analysis on ITS2, the specimens had more similarity to H.contortus. Furthermore, the phylogenetic tree revealed close similarity between the present specimens and H. contortus from other parts of the world including the USA, Turkey, India, Ireland, and Iran (Figure 2).

Figure 2. The phylogenetic tree of H. contortus based on ITS2. Molecular Phylogenetic analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method. The tree with the highest log likelihood (-600.8676) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 18 nucleotide sequences. Codon positions included were 1st+2nd+Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 143 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Amplification of the beta-tubulin fragment including 198 and 200 codons revealed the expected fragment of 403 bp in length (Figure 3). After the comparison of obtained sequences with the available data in GenBank, the specimens were found susceptible to benzimidazole with no mutation at codons 198 and 200 (Figure 4).

Figure 3. PCR product of Haemonchus Beta-tubulin gene (198 and 200 codones, Lanes 1-8).

M:100bp marker, C: negative control.

Figure 4. Alignment of beta-tubulin sequences (Position of codon 198 and 200) of Present study H. contortus with a similar isolate from GenBank (Accession number, JQ342611). Underline reveals the position of codon 198 and 200, indicating no point mutation (Benzimidazole susceptibility).

Discussion

Despite the importance of identification of Haemonchus species, information on its presence in cattle population of Iran is limited (Meshgi et al., 2015). Historically, the accurate morphological identification of Haemonchus species has been complicated and large numbers of specimens have presented intermediate size in body parameters like mean body length, spicule and gubernaculum length, and barb structure (Jacquiet et al., 1995; Rahman and Hamid, 2007; Nabavi, 2017). The morphological analysis in the present study showed similar features between the collected nematodes and H. contortus based on the keys of Lichtenfels et al. (1994). However, some species showed intermediate features common between H. contortus and H. placei. The most useful parameters in species identification were spicule lengths and distances of the barbs from the distal end. In H. contortus, the spicule length varied from 383 to 475 µm (mean 425 µm) and spicule barb length right/left also varied from 37 to 48 µm (Lichtenfels et al., 1994). In the current study, few spicule lengths were out of range. However, other parameters were in the H. contortus measurement ranges. Although the most common species of Haemonchus in the cattle is H. placei, the infection with H. contortus has been mostly reported around the world (Hogg et al., 2010; Kandil et al., 2018). Based on the keys of Lichtenfels et al. (1994), the mean body length, spicule length, and the barb structure may overlap between different species. In addition, worm body length may be influenced by nematode age and even the immunity of host (Coadwell and Ward, 1975; Nabavi, 2017; Hoglund et al., 2019).

Because of morphological difficulties in Haemonchus species identification, molecular tools were considered in the present study. Molecular analysis especially Internal Transcript Spacer 2 (ITS2) was considered as the convenient target of molecular approach in the identification of Haemonchus nematodes (Nabavi et al., 2014; Vadlejch et al., 2014; Meshgi et al., 2015; Dey et al., 2019; Höglund et al., 2019). In this study, the sequence analysis of Haemonchus nematodes showed no nucleotide variation in the ITS2. From the results of our molecular analysis, the collected nematodes were H. contortus that corroborated our morphological results. Additionally, we found some intermediate features common between H. placei and H. contortus; while in molecular analysis, they were identified as H. contortus. Many researchers have presented such differences in morpho-molecular evaluations of Haemonchus species especially in the morphology (Rahman and Hamid, 2007; Nabavi, 2017; Dey et al., 2019). These differences may be due to co-infections as a result of mixed grazing of small ruminants and cattle and accordingly the increased possibility of interspecies hybridization between H. placei and H. contortus in the field (Ali et al., 2015; Salle et al., 2019). Researchers believe that genetic differences in environmental tolerance arise as a consequence of a high level of polymorphism occurring in different climatic areas (Ali et al., 2015). Hence, Haemonchus species are observed in almost all regions where ruminants are raised, with the potential for outbreaks of haemonchosis, regardless of the climatic zone (Ali et al., 2015; Besier et al., 2016). In non-optimal environmental situations like Sistan and Balouchestan province of Iran, genetic diversity and morphological polymorphisms could increase in nematode populations.

Benzimidazole resistance is extremely common in H. contortus in small ruminants but there are only a few well-documented reports of resistance to this anthelmintic drug for Haemonchus nematodes infection (Mohammedsalih et al., 2020). It was demonstrated that single nucleotide polymorphism (SNP) was most commonly associated with resistance at codons 200 and 198 of beta-tubulin isotype1 in Haemonchus species (Shen et al., 2019; Tan et al., 2020). Hence the scientists used different practical and molecular methods to find such mutations in Haemonchus population. In this work, the specimens were susceptible to banzimidazole with no mutation. Nabavi et al. (2011) and Shokrani et al. (2012) reported similar results in H. contortus of small ruminants in Iran.

Conclusion

We believe that in Sistan and Balouchestan province of Iran, due to the harsh climate, ruminants' infection rate to trichostrongyloid nematodes is low (Nabavi, 2017). The prevalent species in cattle was H. contortus with low burden. The consumption of benzimidazole compounds in treatment frequencies is significantly lower in warm rainy areas. It seems that the induction of benzimidazole resistance is less important in many parts of the world.

Acknowledgments

The authors gratefully acknowledge the University of Zabol for financial support.

Conflict of Interest

The authors declared no conflict of interest.