Introduction

Haemonchus contortus (H.contortus) belongs to Haemonchus, Trichostrongylidae, Secernentea (Phasmidia), Nematoda. It is a blood-sucking parasitic nematode that parasitizes in the abomasums of ruminants such as cattle and sheep1. Since its digestive tract will turn red after sucking blood, and intertwined with the white reproductive tract, so it looks like “fried dough twist”2. The life history of H.contortus is divided into free life period and parasitic life period. The free-living stage included eggs, first-stage larvae ( L1 ), second-stage larvae ( L2 ), and infectious third-stage larvae ( L3 ). The parasitic life stage includes fourth stage larvae ( L4 ) and adults ( adults )3. Females can lay up to 10,000 eggs per day, and the number of larvae / eggs on the pasture is usually greater than the number of adults in the host4. Adults suck blood from the host ‘s abomasum, leading to anemia, edema, diarrhea and even death5. The parasite is widely distributed worldwide, usually in tropical, subtropical and temperate regions6. It is seriously harmful to the host and has become an important factor restricting the development of small ruminant animal breeding industry7, causing serious capacity decline and economic losses. So for the prevention and treatment of such nematodes, most of the chemical drugs are used for deworming, mainly including Ivermectin (IVM), Albendazole, Levamisole, Closantel, Nitroxynil and Monepantel. From the beginning, the anthelmintics had the advantages of quick effect and convenient prevention and control, so herdsmen gradually relied on anthelmintics for prevention and control. Due to their non-standard use of drugs and even abuse of drugs, the problem of anthelmintic resistance(AR)has also followed over time. As early as 1980s, there were reports on AR of IVM in Australia8.With the development of recent years, AR seems to become more serious. Common anthelmintics had been found in the world to produce different degrees of AR, even including the emerging Monepantel9. At this point in time, researchers must learn from the past and not allow AR to continue to develop. Once a new anthelmintic is marketed, how to proper use and avoid rapidly occurring AR of new drugs are also the key. Survival of the fittest animals in natural selection. While humans want to remove the worm, the worm is also resisting the impact of anthelmintics. From the perspective of genetic evolution, the species may change in order to survive over time. Therefore, it is very important to correctly identify genetic variation and understand the epidemiological and genetic characteristics for the development of sustainable control strategies.

Genetic Diversity refers to the sum of genetic variation among different populations in the same species or among different individuals in the same population. Generally speaking, the degree of genetic variation is the most direct expression of genetic diversity10. In nature, population is the basic unit of biological evolution, and the difference of population genetic structure also reflects the degree of genetic diversity. Therefore, the size of genetic variation and the difference of population genetic structure simultaneously determine the evolutionary potential and resistance to adverse environment of a species11. The study of population genetics of parasitic nematodes can be carried out between or within populations. Polymorphism depends on the mutation rate of bases, the effective size of the population and the migration rate of the population. The study of genetic diversity is of great significance for understanding the epidemic mode of parasitic nematodes, the diffusion of drug-resistant alleles, and the prevention and control of diseases4. There had reported that compared with other sympatric species, H.contortus had relatively high fecundity, short latency, and high genetic variation of AR6,12. In addition to the differences at the species level, within the species had significant levels of genetic diversity. The genetic structure of H.contortus population may be affected by many factors, including geographical barriers, effective population size, host movement, and multiple host species co-reared on pastures. It has been reported that gene flow rates are high among populations of Haemonchus, especially in geographic areas consistent with host movement13,14.

Ribosomal and mitochondrial DNA markers of nematodes have been used for species identification and genetic diversity studies. Ribosome exists in all life history stages of parasites, and due to its very conserved during evolution, Ribosome is a common tool for inter- and intraspecific identification classification and evolutionary studies of parasites15.The internal transcribed spacer (ITS) sequences have the characteristics of large inter-specific differences and small intra-specific differences, and the ITS1 and ITS2 sequences of most parasites have high variability. This feature makes the ITS gene sequence important in the discovery of new parasite species, inter-species identification and population relationship. Among them, the IST2 gene located in 5.8 S rDNA and 28 S rDNA is one of the most commonly used target genes for nematode-specific detection. The variation of its sequence can distinguish the sympatric species of Haemonchus5,6,16. Mitochondrial genome is independent of the nucleus. Compared with the nuclear genome, it has a fast evolution rate, maternal genetic characteristics and can be stably inherited, and is relatively conserved. It is often used in the study of population genetics. Among them, the variation of mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad4) and cytochrome oxidase (COI) gene are good candidate genes for genetic diversity and population structure analysis6. Using mitochondrial genes as genetic markers, revealed high gene flow among nematode populations parasitizing cattle and sheep, and nematodes parasitizing deer showed significant differentiation17,18. Yin et al.collected 152 H.contortus from 7 different regions of China in 2013. Using ITS2 and nad4 analysis, it was found that the variation within the H.contortus population was not high and the genetic differentiation was low14. With the increasingly serious AR, some studies have suggested that AR is closely related to genetic factors. Under the action of anthelmintic, the worms may have genetic evolution about the part of resisting anthelmintic19,20. However, few studies have analyzed the genetic variation of IVM-resistant strains by genetic markers.Therefore, in order to grasp the current situation of AR of H. contortus in sheep in Hohhot, Inner Mongolia, China, this study obtained the IVM-resistant strain of H. contortus, amplified analyzed the full-length ITS sequence, ITS2 and nad4 part gene sequence, also analyzed against reported H.contortus sequences in the NCBI database. Preliminary indication of the genetic structure of IVM-resistant H. contortus, analysis of its genetic variation and diversity, providing new ideas and directions for solving the AR mechanism, aiming to provide reference data for the prevention and control of H. contortus.

Methods

IVM-resistant H.contortus determination and collection

H. contortus IVM-resistant strain(IVM-RR)L3 was provided by the Veterinary College of Inner Mongolia Agricultural University. The experimental sheep (2–3 months old) purchased from Inner Mongolia Agricultural University Science and Technology Base. Sheep were raised in captivity alone, with free access to food and water. Fecal samples were collected regularly and examined using McMaster counting to ensure that the egg count of sheep was negative (average fecal egg count = 0 eggs per gram). Select three sheep (as 3 groups) without any parasitic infection, and each sheep was oral gavage with 4000 L3, which met the requirements of WAAVP guidelines for the number of infected L321,22. The infected animals were fed and closely observed by professional personnel. After 21 days of artificial infection, detected sheep feces eggs per gram (EPG) to ensure the success of infection, and carried out three rounds of backcross21,22. When EPG > 1000, the animals were weighed separately, and IVM (oral, 0.2 mg/kg Body Weight) was administered to deworm according to the drug specifications. After 14 days, the compared EPG before and after administration, and calculated fecal eggs count reduction rate (FECR)21. Under the microscope, the eggs were identified with reference to the egg map in the parasite-related books23,24, finally, the male/female adults of H.contortus were straind from the sheep abomasums and thoroughly washed in PBS. In order to better analyze the genetic relationship, placing the straind females in 37℃ warm saline to maintain the temperature, so that the females remained active and laid eggs, picked out the females and centrifuged to collect the eggs (3000 rpm, 3 min). In addition, in 2024, our laboratory conducted an investigation on the AR of H. contortus in Hinggan League area25, and cultivated the IVM-resistant L3, which was also used for the follow-up control analysis of this experiment. The H. contortus in each stage of each sheep all were repeated in 3 groups, and 30 samples were transferred to liquid nitrogen for storage until use.

Larval development test

By standard sugar gradient purification method26,27 collected H.contortus eggs from sheep feces and rinsed them with dd water. The washed eggs were diluted to 10,000/mL. 48-well cell culture plates were supplemented with 1640 medium 100µL, eggs 10µL (100 eggs/well), normal saline 62µL, amphotericin B (1X) 4µL and penicillium streptomycin (1X) 4µL. Recorded the number of eggs per well and cultured in a constant temperature and humidity incubator at 27℃ for 24 h. After 24 h, added 20 µL of each gradient concentration of IVM each well, and 20µL of 0.2% dimethyl sulfoxide (DMSO) was used as the control group. Each drug concentration was set up with three replicates, and continued to be cultured at 24℃ in a constant temperature and humidity incubator. After 6 ~ 7 days, observed and counted the number of developed larvae, undeveloped L3 and developed to L3 by inverted microscope28,29.

IVM standard from the China National Institutes for Food and Drug Control. Preparation of IVM solution: accurately weighed the 0.01 g of IVM standardand and dissolved in 1 mL DMSO. After blowing and mixing, it was diluted to 11 gradient concentrations: 10 µg/mL, 5 µg/mL, 2.5 µg/mL, 1.25 µg/mL, 625ng/mL, 312.5 ng/mL, 156.25 ng/mL, 78 ng/mL, 39 ng/mL, 19.53 ng/mL, 9.76 ng/mL29,30.

Counted the number of H.contortus developing to the L3 and non-developing to the L3 per well, and calculated the inhibition rate of larval development. established the linear regression equation (y = ax + b) with the logarithm of IVM concentration as the independent variable x value and the larval development inhibition rate as the dependent variable y value, and calculated the median lethal dose LD50. The formula of larval development inhibition rate is as follows:

$${\text{larval developmental Inhibition rate}}\left( {\text{\% }} \right){\text{ = }}\left( {{\text{ number of undeveloped to L3 / total number of larvae}}} \right){\text{ }} \times {\text{100\% }}.$$
$$LD50 = EXP\left( {\left( {5 - b} \right)/a} \right).$$

Detection of larval locomotor behaviour

Collected L3 according to the Baermann ‘s technique, washed excess feces and impurities with PBS and ddH2O multiple times until the liquid was clear and stored at − 4°C. The 24-well cell culture plate contained 80–100 pieces of L3 per well, 1640 medium 200 µL, DEPC-treated Water 380 µL, amphotericin B ( 1X ) 8 µL and penicillium streptomycin ( 1X ) 8 µL. After recording the number of L3 per well, respectively added 20 µL IVM gradient concentration each well, and the concentrations of IVM were : 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5µg/mL, 31.25µg/mL, 15.625 µg/mL, 7.8 µg/mL, 3.9 µg/mL, 1.953 µg/mL, 0.976 µg/mL. 0.2% DMSO (20µL) was used as the control group, and set blank control at the same time29]– [30. Set three replicates for each well. And cultured in a constant temperature and humidity incubator at 27°C for 48 h. During this period, respectively recorded pre-treament, post treament (30 min, 15 h and 30 h) the number of viable L3 ( head swing frequency ≥ 50/min), and compared with different IVM gradient concentrations and control groups to observe the concentration and effective time of IVM.

Genomic DNA isolation and species confirmation

We collected total 30 samples of IVM-RR, including L3, adult, female, male and egg, and extracted the genomic DNA of H.contortus using the tissue DNA extraction kit (TIANGEN®) and labeled separately. DNA samples were stored at − 20 °C until use.

PCR amplification and sequencing

In order to obtain the complete rDNA ITS gene fragment of H. contortus, the ITS gene loci were amplified by using the universal primers of nematode ITS : NC5 and NC231. Primer information is shown in Table 1. Referred to the 5.8s-ITS2-28s and ITS2-28 S gene sequence fragments of H.contortus in GenBank, we screened the intraspecies conserved and interspecies highly variable base sequences(accession no. KX829170.1.)by ClustalX1.83 biological software. Refer to the sequence, used Primer-blast in NCBI, oligo7 and Primer Premier 5 etc. relevant biological software to design specific primers of H. contortus ITS2. In addition, nad4 gene was amplified using reported primers14,32. The related primer sequences are shown in Table 2.

Table 1 Universal primers of nematode ITS.
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Table 2 Specific primers of ITS/nad4.
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The total PCR amplification reaction system was 25 µL, and the ratio was as follows: Premix Taq (TaKaRa Taq™ Version 2.0 plus dye)12.5 µL, 1 µL of each primer(10 µmol), 1 µL DNA, and 9.5 µL DEPC H2O. The PCR reaction of each group used DEPC H2O as a negative control. The PCR reaction conditions of universal primers NC5 / 2 were as follows : 94 °C 5 min ; 94 °C 30 s, 50 °C 30 s, 72 °C 30 s, a total of 35 cycles ; 72 °C for 7 min. Amplicons (5 µL) were identified on 1.0% agarose gels to verify that they represented about 850 bp single bands, purified using the TIANgel Midi Purification Kit (Tiangen®; China) and sent to the BGI.Write Company (China) for sequencing. The PCR reaction conditions for the specific primers of 5.8s-ITS2-28s, ITS2-28s and mitochondrial Nad4 were : 94 °C 5 min ; 94 °C 30 s, 52 °C 30 s, 72 °C 30 s, a total of 35 cycles ; 72 °C for 7 min. Amplicons (5 µl) were identified on 1.0% agarose gels to verify that they respectly represented about 365 bp、287 bp and 800 bp single bands and sent to the BGI.Write Company (China) for sequencing. For more precision, sequencing of amplicons was done in forward and reverse directions.

Data analysis

Using Seqman biological software pliced and corrected the obtained H. contortus sequences, and subjected to Blast on NCBI to ensure the accuracy of the sequencing results. Through ClustalX1.83 biological software remove the 18 S and 28 S gene fragments at both ends of each sequence. By using the biological software BioEdit (7.2.5.0) analyzed the base content, A + T content and G + C content of ITS2 and Nad4 sequences. The sequences were aligned using the Clustal W program in the MEGA 11 software, and the flanking sequences at both ends were deleted to obtain the ITS and Nad4 nucleotide fragments for subsequent data analysis. Phylogenetic analyses were performed using Neighbor-joining ( NJ ) in MEGA 11 software, set the parameters as the Bootsrap method, replications 1000, and Tamura-Nei model. In order to better observe the intraspecific evolutionary relationship of H. contortus, no setting outgroup, aimed to explore the evolutionary relationship between the IVM-resistant H. contortus in Inner Mongolia Autonomous Region and other H. contortus. To further investigate the genetic relationship among mitochondrial Nad4 gene Haplotype, DnaSP v6 software was used to analyze the variation sites33, simple information sites, nucleotide diversity (pi), haplotype diversity (Hd), Fu and Li’s D, Fu and Li’s F, andTajima’s D and Fu’s Fs. ARLEQUIN 3.5.2.2 software34 calculated the pairwise FST between the IVM-resistant H.contortus samples and other populations, and PopART 1.7 software was used for haplotype Median-Joining network analysis35.

Results

The results of in vitro assays of H.contortus to IVM resistance

The results of fecal egg reduction experiment showed that FECR the highest was 20.35% and the lowest was − 59.09% (Table 3), following the FECR calculation method by Denwood et al. (fecrt.com data analy-sis), judge the results was resistant36. Therefore, the experimental sheep were successfully infected with the IVM-RR of Haemonchus contortus. During the larval development inhibition test, we observed the process of egg development, larval shelling, and development of L1 ~ L3 (Fig. 1). According to the statistical results, established the linear regression equation and LD50 results as follows (Fig. 2) :

Group1: y1 = 1.161x + 2.5251 (R² = 0.9808) LD50 = 189.67ng/mL.

Group2: y2 = 1.1005x + 2.7929 (R² = 0.9639) LD50 = 29.51ng/mL.

Group2: y3 = 1.0392x + 2.7029 (R² = 0.983) LD50 = 162.18ng/mL.

According to Liu (2020) reported the sensitive value of IVM LD50 was < 9ng / mL30, and the resistance value was 15.28ng / mL. Therefore, this test LD50 was greater than the reported resistance value, so it was further determined that IVM-RR were AR. Through the larval locomotor behaviour, it was found that the L3 activity and head swing frequency of the three groups of sheep were maintained at a high level when the IVM concentration was 15.625 µg / mL (Fig. 3). In the second group, IVM could inhibit most of L3 before 15 h, but after 30 h of IVM treatment, L3 recovered at 15.625 µg / mL IVM concentration, indicating that the tolerance concentration of L3 to IVM in this experiment was 15.625 µg / mL, while the resistance degree of the second group was lighter than that of the other two groups, which was consistent with the results of LD50.

Table 3 Results of faecal egg count reduction test.
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Fig. 1
figure 1

Larval development process.

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Fig. 2
figure 2

The results of larval development inhibition test.

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Fig. 3
figure 3

The results of IVM inhibiting L3 locomotor.

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Sequences analyses

30 samples PCR amplified products were consistent with the theoretical size, as shown in Figs. 4 and 5. The complete ITS sequence of 788 bp was obtained by sequencing analysis using universal primers. Homology search was performed between the sequence and the data in GenBank. The ITS1 was 404 bp, the 5.8 S rDNA gene sequence was 153 bp, and the ITS2 was 231 bp. After comparison and analysis by ClustalX 1.83 software, it was found that there were C-T conversion and A-C transversion at some sites of ITS1 sequences. There was no base difference in 5.8 S rDNA, indicating that the 5.8s gene of H.contortus was relatively conserved.

In order to obtain more accurate ITS2 sequences, used universal primers and specific primers to amplify and compare the ITS2 sequences of different periods of H. contortus. The ITS2 lengths of all samples were consistent, which were 231 bp. Of which the A content was 30.74 / 31.17%, the G content was 17.32 / 17.75%, the C content was 15.58 / 15.15%, and the T content was 36.36 / 35.93%. The A + T content of ITS2 sequence was 67.1% / 66.67%, which was significantly higher than the G + C content of 32.9% / 33.33%. The representative ITS2 sequences of H.contortus from all over the world in NCBI were selected for alignment. It was found that the A content in the ITS2 sequence was 29.87% ~ 31.6%, the G content was 15.75% ~ 17.75%, the C content was 14.72% ~ 16.02%, the T content was 35.5% ~ 36.8%, and the A + T content was 65.37% ~ 67.97%, which was significantly higher than the G + C content of 32.03% ~ 33.33%. The base composition of ITS2 sequences in this IVM-RR were in the range of having reported in NCBI H. contortus ITS2, so it shows that ITS2 gene is relatively conservative and stable.

The 30 samples’ nad4 gene (412 bp) were Blasted in NCBI and comparative genetic analysis. The base contents were: A content 31.07 / 31.31 / 31.55%, C content 9.95% / 9.71% / 10.19 / 10.44, G content 11.89 / 10.38 / 12.14%, T content 46.12 / 46.36 / 46.6% / 46.84%, A + T content 77.43% ~ 78.4% was significantly higher than G + C content 21.6% ~ 22.57%. Selected nad4 gene (412 bp) sequences from NCBI, it was found that the A content was 30.58% ~ 32.04%, the C content was 9.47% ~ 10.44%, the G content was 11.65% ~ 12.62%, the T content was 46.12% ~ 47.09%, the A + T content was 77.43% ~ 78.64%, and the G + C content was 21.36% ~ 22.57%. The representative Nad4 sequence (102) selected from NCBI were compared with IVM-RR samples (30) in this experiment, it was found that in the IVM-RR 21 samples’ sequences were all bases G at169 site, accounting for 70%, which was only consistent with the 8 sequence sites selected in NCBI, in NCBI remaining 94 sequences were base A at 169 site (Additional file 1). So the 169th base mutation should continue to be concerned, but whether this site is related to AR needs a large number of samples for verification and analysis. All nad4 (412 bp) sequences were submitted to GenBank ( accession number: PV125660-PV125689 ).

Fig. 4
figure 4

PCR amplification results. (A): specific primers (2-F/R) amplified L3-ITS2; (B): nematode universal primers(NC5/2) amplified L3-ITS; (C): specific primers (A-F/R) amplified L3-ITS2; (D): L3 nad4 genes amplification. M: DNA marker ; 13: negative control. (1–3: group1; 4–6: group2; 7–9: group3; 10–12: Hinggan League)

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Fig. 5
figure 5

PCR amplification results. (A): specific primers (A-F/R) amplified Egg/Adult/Female/male-ITS2; (B): Egg/Adult/Female/male- nad4 genes amplification; (C): negative control; M: DNA marker. (0–3: group1-egg; 4–6:group1-Adult; 7–9:group2-Adult; 10–12:group3-Adult; 13–15:group2-Female; 16–18:group2-male)

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Phylogenetic analysis

In order to compare the genetic relationship between IVM-RR samples and H. contortus in the world, selected ITS2 and nad4 genes to construct phylogenetic tree. ITS2 phylogenetic tree showed that IVM-RR and China, Bangladesh, New Zealand, African, Iran, India, Laos, Italy, Australia, Thailand, USA and other countries all came from the same branch. Branch length was shorter, the evolutionary variation was less than 0.007, and the genetic relationship was closer. The variation rate of ITS2 sequence among different geographical strains was low. The results showed that the H. contortus-ITS2 was relatively conserved within the species, and the variation between different samples were small, variation of ITS2 was no significant correlation with the AR, geographical location and host source of the samples (Fig. 6).The nad4 gene phylogenetic tree showed that the IVM-RR were compared with the representative nad4 gene of 5 countries (China, Greece, Pakistan, Bangladesh, France) (Fig. 7), it was found that the IVM-RR samples were closely related to China, Greece and Pakistan, there was no obvious boundary. But it had a relatively distant relationship with Bangladesh and France, and there were clear groupings and clear boundaries between the two places and other regions.

Fig. 6
figure 6

A phylogenetic tree was constructed using 79 H.contortus ITS-2 gene of IVM-RR, China, Bangladesh, New Zealand, African, Iran, India, Laos, Italy, Australia, Thailand, USA and other countries. Each terminal branch represents a single sequence, according to its geographical origin and host of the worm to be labeled.

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Fig. 7
figure 7

The phylogenetic tree displays the relationship among the 75 nad4 sequences of H. contortus from IVM-RR, China, Greece, Pakistan, Bangladesh and France. Each terminal branch represents a single sequence, according to its geographical origin and host of the worm to be labeled.

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Population genetic structure

From the 30 IVM-RR nad4 amplicons defined 13 different haplotypes, sequence information of the IVM-RR nad4 gene was shown in Table 4, where the Pi was 0.00623, the Hd was 0.926, Tajima’s D value was 1.98026, and Fu’s Fs value was − 5.203. At the same time, Table 4 showed the representative nad4 sequence information of the other five countries, and drawed median joining (MJ) network (Fig. 8) for all haplotypes (n = 104) in Table 4, and calculated the pairwise Fst, Fu and Li ‘s D values and Fu and Li ‘s F values between IVM-RR and five different countries (Table 5). The Fst between populations was 0.10957 ~ 0.98101. It can be found that the genetic differentiation level of IVM-RR was relatively low with China and Pakistan populations, and the Fst values were 0.10957 and 0.17262, respectively. The genetic differentiation level of France population was the highest, and the Fst value was 0.98101, which was consistent with the results of the MJ network.

Table 4 H.contortus nad4 gene sequence analysis. The nucleotide diversity, haplotype diversity and neutrality index of nad4 gene in IVM-RR and five different countries were calculated.
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Table 5 Paired fst, Fu and Li ‘s D and Fu and Li ‘s F values between IVM-RR and five National H.contortus populations were calculated based on the nad4 haplotype sequence.
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Fig. 8
figure 8

The median joining network of 104 nad4 haplotypes representing 132 individuals of H. contortus from the IVM-RR samples of this test and 5 different geographical locations in NCBI. The different coloured dots represent haplotypes from the different populations/locations: RR: IVM-RR; Ch: China; Gr: Greece; Pa: Pakistan; Ba: Bangladesh; Fr: France.

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Discussion

In this study, the genetic diversity and population structure of H.contortus IVM-resistant samples were analyzed. Selected three sheep to cultivate IVM-RR samples which was determined by three methods: Fecal egg count reduction test, larval development inhibition test and larval locomotor behaviour. The results of FECRT showed that the highest FECR was 20.35% and the lowest was-59.09% (Table 3), which was juge as resistance. During the larval development inhibition test, the LD50 values were 189.67ng / ml, 29.51ng / ml, and 162.18ng / ml, respectively, which were greater than the reported sensitivity values30. Through the larval locomotor behaviour test, it was found that the tolerance of IVM remained mostly at IVM = 15.625 µg / mL in this experiment. It is well known that the resistance of H.contortus to IVM has been developed for many years, and there is no clear resistance mechanism and sustainable method to solve the problem. Some studies suggest that the sustainable development of AR has a certain relationship with genetic evolution. As the so-called ' natural selection, survival of the fittest ‘, while chemical drugs are used to deworm, nematodes will gradually adapt or resist corresponding changes in order to cope with and survive. With the continuous development of molecular biology, people have a deeper understanding of the genetic marker molecule ITS rDNA and mitochondrial nad4 gene, and better apply it to the study of nematodes or other parasites. Therefore, in this study, we amplified the ITS2 sequences of IVM-RR at different stages (egg, Adult, L3) by using nematode universal primers and specific primers, respectively. Compared with the H.contortus ITS2 in NCBI, phylogenetic analysis explained that they all came from the same branch, the length of the evolutionary branch was shorter, and there was no significant difference among egg, L3 and adults. The genetic relationship was close, and the variation rate between different geographical strains was low. The results showed that the ITS2 of H.contortus was relatively conservative within the species, and the variation between different samples was small, and there was no significant correlation with AR. In 2017, Kandil et al.determined the genetic diversity and sequence variation of the H.contortus population in Egypt. Nucleotide sequence analysis showed that all nematodes were one genotype without genetic differentiation37. Upon reviewing the relevant literature, we found that some researchers have used ITS markers to analyze the phylogenetic relationships among H. contortus populations, and they all concluded that the sequences were relatively conserved within the populations, and their variation did not show significant correlation with the geographical origin of the strains, the host source, or drug resistance38,39, which was consistent with the results of this study. However, Hafidh et al. (2012) used the ITS2 gene sequence to analyze the evolution of Haemonchus contortus parasitizing in goats, sheep and cattle from the same area of Tunisia, and found that there were obvious differences between these sequences. The author believes that there may be genetic diversity among populations of Haemonchus contortus parasitizing in different ruminants40. Shen DD (2017) found that the genetic differentiation between H. contortus populations in wild blue sheep and domestic ruminants in China was low through phylogenetic analysis, but there was a high degree of gene flow41. Through the analysis of the genetic diversity of ITS2 gene of H.contortus at home and abroad, combined with the results of this study, we found that the genetic variation between H.contortus populations infected by the same host was almost independent of geographical location, the presence or absence of AR and other factors. However, different hosts may affect the genetic changes between H.contortus populations. The genetic phylogenetic tree analysis of this study revealed that the IVM-RR from three sheep hosts and H. contortus from Hinggan League did not differ significantly in genetic differentiation. Of course, if we want to confirm whether the host has an impact on the inheritance of parasitic nematodes, and exclude the individual differences that may exist in itself, need multiple hosts around the world and a large number of samples for genetic differentiation statistics, from the perspective of statistics and probability analysis to get sufficient conclusions.

In addition, we amplified the IVM-RR Nad4 gene in L3, adult, and egg, respectively, and the results from the developmental evolution and haplotype analysis of the nad4 gene showed that 13 haplotypes were obtained out of 30 sequences. The Pi was 0.00623 and the Hd was 0.926. In this study, the IVM-RR nad4 were compared with nad4 of representative H. contortus populations of China, Greece, Pakistan, Bangladesh, France, the Fst the populations was 0.10957 ~ 0.98101, the genetic differentiation level between the IVM-RR population and the Chinese/Pakistan populations is relatively low, and the Fst values are 0.10957 and 0.17262, respectively. The genetic differentiation level with the France population is the highest, and the Fst value is 0.98101, the above results were consistent with the MJ-network results. Meanwhile, in order to grasp the trend of H.contortus genetic information around the world, this study summarized all the nad4 gene sequence information of China, Pakistan, Bangladesh, France, Greece, Yemen, Malaysia, Italy and USA reported in NCBI (Table 6), hoping to provide convenience for subsequent H.contortus genetic research and analysis. Classification of population haplotype frequencies can help to identify common haplotypes for the design of effective vaccines, which plays an important role in controlling and eliminating parasites42. Troell et al. in 200632 analyzed the population genetic structure of H. contortus from 14 countries and 19 populations including 150 H.contortus using mitochondrial nad4 gene as a genetic marker, which revealed the robust population structure of H. contortus globally for the first time. From the results of cluster analysis, found that populations from Asia clustered together, populations from Southeast Asia, Australia and Greece clustered together, and populations coming from the United States and Europe clustered together, suggesting that there is a certain structure of genetic differentiation among populations from different continents, which was consistent with the results of the present study, the resistant strains clustered together with the populations of China and Pakistan, and some parts of Bangladesh clustered together with the populations of Greece is closer, only France has the furthest genetic differentiation, interestingly, this study found that the genetic differentiation map in Fig. 8 was similar to the position on the map between the five countries, which indicates that the genetic differentiation was still related to geographic location and distance, and that the genetic diversity and population structure are more influenced by geographic location. IVM-RR nad4 gene comparison with H. contortus nad4 gene from NCBI revealed that 70% of the IVM-resistant samples in this study were different from other sequences at the 169th base (Additional file 1). Whether it can become a genetic AR marker gene requires a large number of samples for subsequent verification and analysis of this site. Subsequent studies have found low genetic differentiation but high gene flow in H. contortus in different populations of Pakistan, Bangladesh, Lesotho, and China, respectively6,41,43,44. However, the frequency of 1β-tubulin related to benzimidazole resistance at the F200Y locus of H.contortus strains reported in 2014 and 2016 was not significantly higher than that in other regions. Using microsatellite markers for population genetic analysis, it was found that the genetic differentiation between parasitic populations was not obvious. Although it was a high frequency of benzimidazole resistance mutations, the overall genetic diversity of the parasitic population was little or no reduction6,45. The authors believe that the purpose of the high genetic diversity retained by the anthelmintic-resistant parasite population is first to maintain the adaptability of the parasite to the pressure of anthelmintic treatment, and secondly to be related to genetic changes related to anthelmintic selection, such as polymorphism loss and linkage disequilibrium. In addition, global warming and population bottleneck effects will lead to changes in the genetic diversity of originally abundant species. Therefore, paying attention to whether is in a bottleneck period is helpful for the accurate identification of drug resistance targets.

However, from the combination of genetic differentiation and AR, we found that all the studies have certain drawbacks. Firstly, there are relatively few AR samples in this study, If we want to clarify the relationship between AR and genetic diversity, first need multi-regional and multi-group AR samples, and ensure that the sample is single parasite variety that is clearly resistant to a certain anthelmintic, and the same host obtains female, male, egg and L3 respectively, and marks them well. This can not only clarify the changes between different hosts and the same host, but also clarify the changes of the same host from the maternal/paternal line-egg-L1-L2-L3, but the workload of obtaining a single sample is large and requires the cooperation of various regions, of course, it is also necessary to consider whether the degree of AR of IVM resistant strains can cause their genetic genes change; secondly, a single sensitive strain should be cultured in the local area as a control, but the current situation shows that wild single sensitive strains are more difficult to obtain; and finally problem that due to the prevalence of AR and multi-drug resistance, most of the H. contortus related genes that have been reported in the NCBI at the moment are of mixed strains, including the mixture of anthelmintic resistant strains and sensitive strains for the genetic analyses, which will have a certain impact on the analysis of AR in genetic diversity. Although some studies have analyzed the standard sensitive strains and standard resistant strains that have been preserved, a large number of studies have pointed to genetic differentiation related to geographical location. Therefore, the genetic analysis of standard strains does not seem to be applicable to all regions. The best method is to obtain wild pure sensitive strains and wild single resistant strains locally, which seems to be a very heavy task. Of course, in addition to the ITS2 and nad4 marker molecules used in this paper, microsatellite DNA molecular markers, Allozyme markers, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and amplified fragment length polymorphism (AFLP) methods combined with quantitative trait locus (QTL) may be able to find resistance-related genetic loci17,45,46,47,48,49. From the epidemiological reports related to parasitic AR, it can be said that AR has been continuously serious and has not been well controlled. It is hoped that this report can provide a new direction for the study of AR and strive to overcome this problem as soon as possible.

Table 6 Sequence information summary of other nations’ H.contortus nad4 gene.
Full size table

Conclusion

To our knowledge, the present study is the first to report the genetic diversity and population structure about IVM-resistant H.contortus (L3, eggs and adults) in Inner Mongolia, China. The ITS2 of IVM-RR and other H.contortus were from the same branch, and there was no significant difference between different stages of H.contortus development. The intraspecific was relatively conservative, and no significant correlation with anthelmintic resistance. In addition, 70% of the IVM-RR nad4 genes had a mutation at the 169 th base site, and obtained 13 haplotypes from the 30 gene sequences. The nucleotide diversity was 0.00623, and the haplotype diversity was 0.926. The genetic differentiation level of the IVM-RR population was relatively low with the Chinese and Pakistan populations, and the genetic differentiation level with the Bangladesh and France populations was higher, so the H.contortus population structure differentiation seem to be related to geographical location. The finding may provide a reference for the study of anthelmintic resistance.