Haemonchus contortus is one of the most economically important parasites infecting small ruminants worldwide. It is a blood-sucking nematode that feeds on blood from capillaries in the abomasum of ruminants, especially for cattle, sheep, and goats (Hoberg and Zarlenga, 2016). Infections with the nematode can cause anemia, weight loss, or even deaths in severely affected animals. In China, H. contortus is ubiquitously distributed in the whole country with variable prevalence among different provinces (Wang et al., 2006; Liu et al., 2009; Ma et al., 2014; Yang et al., 2016).

Current control strategies against H. contortus primarily rely on repeated anthelmintic treatments. However, the widespread use of anthelmintic drugs has resulted in serious drug resistance problems worldwide in domestic animals (Kaplan, 2004; Kaplan and Vidyashankar, 2012; Kotze et al., 2014). For example, benzimidazole and ivermectin have been used heavily to control nematode infections in China, resulting in the development of drug resistance (Cai et al., 2007; Zhao et al., 2010). Therefore, the emergence of anthelmintic resistance in H. contortus necessitates development of new intervention strategies. One of the possibilities is the rational strategy of discovering new anti-parasite drugs and vaccines, built on the deep understanding of the key molecules in the processes of development and reproduction. For H. contortus, clear insights into the biological processes at the molecular level might identify key molecules as new drug targets (Britton et al., 2016). In addition, development of immunological control against livestock nematode infections is urgently needed. Significant protection against H. contortus has been achieved following vaccination with native protein extracts, demonstrating that vaccination is feasible (Newton and Meeusen, 2003; Mohandas et al., 2016b; Nisbet et al., 2016). In China, the promising vaccine candidates from native protein extracts have been studied, providing some insights in the development of commercial vaccines against H. contortus (Yan and Li, 2006; Zhao et al., 2012; Zhou et al., 2014).

This paper reviews research progress in China on H. contortus. Particular areas of importance include epidemiological investigation, population genetics, and detection of anthelmintic resistance by applying conventional approaches and molecular methods, structural and functional studies on key molecules in signaling pathways regulating development, interaction between parasites and host cells and vaccine development. The proposed formulation of these views is conducive to identify areas for future research and expedite possibilities for new or updated control measures.

Haemonchus contortus is a blood-feeding nematode infecting small ruminants (Taylor et al., 2016). Animals infected with H. contortus can show a series of symptoms, including anemia, emaciation, diarrhea or even death under heavy burden. Damages caused by H. contortus can lead to billions of economic losses to the breeding industry (He et al., 2011; Roeber et al., 2013a; Emery et al., 2016), especially for young animals. What’s more, arrested larvae of H. contortus are related to the spring rise and can lead to a mass of deaths (Blitz and Gibbs, 1971). China is a large and civilized country with a long traditional history of 5000 years and is also a traditional agricultural country. It locates in Asia, east of the world. Rich vegetation and water resources make it suitable for the development of livestock breeding industry, including cattle, sheep, and goats. In recent 20 years, with the improvement of people’s living standard and increasing supports from the government (e.g., Luo et al., 2005; Ni et al., 2007), more and more farmers turn to breed cattle, sheep, and goats, so the population of cattle, sheep, and goats has been over 300 million since 2008¹. A large number of cattle, sheep, and goats also provide a suitable environment for the development and spread of bacteria, virus, and parasites, including H. contortus which is an important parasitic nematode that can lead to considerable economic losses to the breeding industry of cattle, sheep, and goats. Understanding the epidemiology of H. contortus is essential for preventing and controlling of this species.

Until now, there are about 170 Chinese publications (Data based on literature search at CNKI) and five English publications (Data based on literature search at Google Scholar) reported the investigation of H. contortus infection of cattle, sheep, and goats in most provinces in China, except for Hainan, Hong kong, Macao, and Taiwan. To understand H. contortus infection in ruminant in China, post-mortem diagnosis was the most common method used in the investigations (e.g., Chen et al., 2000; Wang et al., 2005; Yang, 2005), and DNA-based methods were also applied in recent years (Yang et al., 2016, 2017). From the reported data, H. contortus is a dominant or key species in most goat farms (e.g., Wei et al., 2013; Zhao et al., 2016; Jiang et al., 2017), and the infection rate ranges from 28 to 100% in the investigated goat farms (e.g., Cao et al., 1995; Chen et al., 2000; Wang et al., 2005, 2006; Yang, 2005; Ma et al., 2014; Yang et al., 2016), and from 0 to 92% in the investigated sheep farms (e.g., Han et al., 1984; Wang et al., 2006, 2014c; Xin, 2010), and from 0 to 61.8% in the investigated cattle farms (e.g., Li et al., 2002; Liu et al., 2009). Surveys on the H. contortus infection of both cattle and goats in areas of Chongqing (Li et al., 2001), Guizhou (Yang et al., 2014) and Yunnan (Li et al., 2002) indicated the prevalence of H. contortus infection in goats is higher than that in cattle in these investigated areas. However, surveys in Hebei (Yang and Wang, 1984) indicated the prevalence of H. contortus infection in sheep was lower than that in cattle. Most of the investigated animals were 0.5- to 4-years-old, but no comparison on the effect of age on the infection of H. contortus was made in all the investigations. What’s more, the convenience of trans-regional transport of cattle, sheep, and goats accelerated the spread as well as anthelmintic resistance of H. contortus (Li et al., 2007).

To further understand the occurrence and infection patterns of H. contortus, Qin et al. (2003) reported the seasonal incidence of predominant gastrointestinal nematodes of sheep in Bashang Altiplano Area, Hebei province. Bashang Altiplano refers to the Altiplano area at 400–1300 m altitude laying in the most north of Hebei province where winter is long and cold and January is the coldest month with the average temperature ranging from -11 to -19°C whereas July is the hottest month with the average temperature ranging from 18 to 20°C, and the annual precipitation is 350–400 mm. The results indicated there were two peak infection periods (March to June; September to January) for H. contortus within a year, and the seasonal dynamics of H. contortus had obvious spring rise phenomenon. Then, Song et al. (2007) reported the seasonal incidence of egg output and worm burden of H. contortus adult of sheep in Songhuajiang area, Heilongjiang province, China, and found egg output and worm burden of H. contortus adults reached two peaks within a year. Egg output and worm-burden increased from late April to late May and reached a peak at late May, and then they decreased immediately until late July and reached the second peak at early August and decreased again, and then kept at a low level till to next April. Winter in Songhuajiang area is long and cold, and summer is hot and rainy when July is the hottest month with the average temperature ranging from 20 to 25°C whereas January is the coldest month with the average temperature below -20°C, and the annual precipitation is about 500 mm. Obviously, warm weather in summer would promote the development of H. contortus in these two areas, and cold weather in winter would inhibit the development, thus leading to the seasonal dynamics. No data reflecting the peak of transmission in these two areas were related with calving season. Sharing similar climatic characteristics, the seasonal dynamics of H. contortus in both areas are alike, however, the infection peak of H. contortus in Songhuajiang area was 1 month later in winter than that in Bashang Altiplano area due to colder weather in January, but 1 month earlier in summer than that in Bashang Altiplano area due to warmer weather in July. Knowledge of the infection and seasonal dynamics for H. contortus can help us choose the optimum time for effective deworming and decrease the cost.

Knowledge of genetic variation of H. contortus can provide a foundation for understanding the spread of anthelmintic resistance alleles and making the strategy of control of haemonchosis. Until now, three studies have been conducted to explore population genetics of H. contortus in China using mitochondrial nicotine amide dehydrogenase subunit 4 (nad4) gene, microsatellite markers and the isotype-1 β-tubulin gene (Yin et al., 2013, 2016; Zhang et al., 2016), respectively.

Yin et al. (2013) amplified nad4 gene from 152 H. contortus from sheep and goats from seven different regions in China and analyzed population genetic diversities. As a result, 142 haplotypes of nad4 gene were identified. The nucleotide diversities and haplotype diversities were 0.0178–0.0369 and 0.993–1.000, respectively, similar with previous reports from different countries. Population genetics analysis revealed that high nucleotide variation (92.4%) was partitioned within population. They concluded that high variations within population, low genetic differentiation and high gene flow among different populations were present in H. contortus in China. Yin et al. (2016) then used eight different microsatellite makers to study the genetic variation within and among H. contortus populations including 184 adult male worms from seven distinct populations from sheep and goats in China. They found that all eight microsatellite markers were highly polymorphic with high heterozygosity and inbreeding coefficient (F_IS). Moreover, various analysis including AMOVA, F_ST, phylogenetic, structure, mantel test and population dynamics revealed high within-population variation, low population genetic differentiation and high gene flow for H. contortus in China. In addition, Zhang et al. (2016) used the isotype-1 β-tubulin gene to explore the population genetics of H. contortus in China. In this study, 132 of H. contortus sequences of isotype-1 β-tubulin gene from eight different populations were analyzed. Finally, high haplotype diversities (0.455–0.939) and nucleotide diversities (0.018–0.039) were calculated within each population. The pairwise F_ST and AMOVA analysis revealed that high gene flow and low genetic differentiation were present among populations. From the above studies, although three different genetic markers were used, a fairly similar picture of population genetic structure of H. contortus in China was identified.

The main method for controlling parasitic nematodes including Haemonchus contortus is based on the application of anthelmintics, however, long-term and unreasonable usage of anthelmintics has led to the emergency and development of anthelmintic resistance. Anthelmintic resistance in H. contortus has been studied and reported in many parts of the world. A brief review was published by Kaplan and Vidyashankar (2012) reporting the existence of anthelmintic resistance in many countries, including United States, Brazil, South Africa, Australia, New Zealand, and European countries. Nevertheless, up to now, there is no review about anthelmintic resistance in H. contortus in China. Over the last two decades, 15 studies have been conducted to detect anthelmintic resistance in parasitic nematodes in the fields using fecal egg count reduction test (FECRT) and (or) egg hatch assay (EHA). However, only five reports focused on or referred to H. contortus. In addition to conventional in vivo and in vitro methods used in those five reports, molecular methods were also utilized to detect benzimidazole resistance in four studies and one study also explored candidate genes for ivermectin resistance in H. contortus using a new genome-wide SNP analysis. Hence, we reviewed the research results on anthelmintic resistance in H. contortus in China using in vivo test, in vitro test and molecular test to detect benzimidazole (BZ) resistance and single nucleotide polymorphism (SNP) analysis in ivermectin (IVM) resistant H. contortus.

Haemonchus contortus can enter into the arrested development (diapause) during the infective L3s (iL3s) of free-living life stage or early L4 of the parasite stage to protect them against hostile environment. Understanding this developmental change might identify key molecules as new drug targets and provide new insight into prevention and control of H. contortus. Here, we reviewed the studies on 10 genes which are related to diapause of H. contortus and their structures and functions have been characterized at the molecular level (Table 1).

It was found that infective L3 (iL3s) of parasitic nematodes have significant similarities in anatomy, behavior and biology with the dauer stage of free-living nematode Caenorhabditis elegans. Thus, the “dauer hypothesis” deems that the iL3 of parasitic nematodes is developmentally and functionally analogous to the dauer stage of C. elegans and is regulated by similar molecular mechanism (Hotez et al., 1993; Blaxter, 1998; Bürglin et al., 1998; Hu, 2007). Insulin-like signaling pathway governs dauer development in C. elegans and is conserved in several species of parasitic nematodes including H. contortus. Five genes were found to belong to the insulin-like signaling pathway in H. contortus: fork head transcription factor encoding gene Hc-daf-16 (Hu et al., 2010), insulin receptor encoding gene Hc-daf-2 (Li et al., 2014b), phosphoinositide 3-kinases (PI3Ks) component catalytic subunit encoding gene Hc-age-1 and regulatory subunit encoding gene Hc-aap-1 (Li et al., 2014a) and phosphoinositide dependent protein kinase-1 gene Hc-pdk-1 (Li F.C. et al., 2016). These genes all had expression patterns in the intestine that were consistent with those of the homologous genes of C. elegans correspondingly, indicating a similar location where they regulate the growth and development. It was found that Hc-daf-2 can partially rescue the C. elegans daf-2 mutant (CB1379) worm (Li et al., 2014b), suggesting to some extent, Hc-daf-2 has functional similarity to Ce-daf-2. Downstream of Hc-DAF-2 is the PI3K kinase with two subunits encoded by Hc-age-1 and Hc-aap-1, respectively. These two molecules can interact strongly with each other demonstrated by yeast two-hybrid method. However, Hc-age-1 can’t rescue age-1-deficient strain of C. elegans (CY246) like Ce-age-1. The possible interpretation may be because that the exogenous Hc-age-1 subunit can’t bind to the endogenous Ce-aap-1 subunit in vivo to constitute the functional PI3K (Li et al., 2014a). Interestingly, Hc-pdk-1 displayed conserved functional domains which are crucial for the phosphorylation of downstream signaling. Its function may be similar to Ce-pdk-1, promoting arrested development (Li F.C. et al., 2016). At the transcriptional level, RNAseq analysis showed that these four genes have highest transcriptional expression levels in iL3, which indicated that these genes could play an important role in regulating iL3 against external unfavorable environment. In summary, the findings from these studies provide evidences of the functional conservation of insulin-like signaling between H. contortus and C. elegans. Additionally, the reconstructed insulin-like signaling pathway of H. contortus from transcriptomic and genomic data sets for this nematode by a bioinformatic approach further confirmed its existence in H. contortus (Mohandas et al., 2016a). However, although the basic skeleton of insulin-like signaling pathway in the H. contortus is built, the mechanism about how these genes and this pathway function in the development of H. contortus need to be further studied in the future.

In China, in addition to the discovery of insulin signaling pathways, researchers are currently working on other signaling pathways such as TGF-beta signaling pathway which is also vital for normal development of C. elegans and it is important to understand their roles in H. contortus diapause in the iL3. What’s more, as H. contortus iL3s are able to protect themselves from desiccation, genes involved in this biological process were also identified and their functions were studied. The mRNA differential display RT-PCR was used to screen differentially expressed genes in L3s upon desiccation, among the 58 differentially expressed gene transcripts, two transcripts with highest transcription were named Hc-ubq and Hc-gst based on their homologs ubiquitin in C. elegans and glutathione-S-transferase in H. contortus, respectively. Silencing Hc-ubq or Hc-gst by RNAi in L3s of H. contortus reduced the survival rate, suggesting that they may contribute to the nematode desiccation tolerance (Yang et al., 2015). In addition, silencing Hc38, which was first discovered by Northern blot and highly expressed in the intestinal microvilli by in situ localization (Hartman et al., 2001), by soaking iL3 of H. contortus in dsRNA which were then used to infect sheep reduced the amount of egg and worm burden by 50 and 48.6%, respectively, suggesting Hc38 is involved in the development of H. contortus (He et al., 2013).

In addition to diapause in iL3, H. contortus can enter into diapause in early L4 in the parasitic stage. The larvae with arrested development have shorter-length, reduced body metabolic rate and rod-shaped crystals appeared in the intestinal tract. Staying as early L4 in the host and not spawning can protect them against the cold weather. For diapause in this stage, three genes were found highly expressed. They are Hc-daf-22 (encoding 3-ketoacyl-CoA thiolase), Hc-maoc-1 (encoding enoyl-CoA hydratase) and Hc-hsp-70 (encoding heat shock protein 70). Hc-daf-22 and Hc-maoc-1 shared similar characteristics and functions with their homologs in C. elegans, Ce-daf-22 and Ce-maoc-1, respectively, and may play important roles in peroxisomal β-oxidation and the development of H. contortus (Guo et al., 2016; Ding et al., 2017). Hc-hsp-70 is highly conserved in other nematodes, especially in Caenorhabditis. Over-expression of Hc-hsp-70 induced down-regulation of hsp-1 of C. elegans, which suggested that Hc-hsp-70 might have similar function to Ce-hsp-1 of involvement in the parasite invasion and survival (Zhang et al., 2013). Hc-fau, a homolog of human fau and C. elegans Ce-rps30 (encoding ribosomal protein S30), have a conserved ribosome protein S30 domain and a diverged ubiquitin-like (UBiL) protein domain. The S30 is mainly expressed in the nucleus while the UBiL is strongly expressed in the cytoplasm. Both of them have effect on egg-laying and life span of C. elegans, suggesting that they may have potential functions in regulating L4 diapause in H. contortus (Yan B. et al., 2014).

Excessive use of anthelmintics for prevention and control of parasitic nematode diseases threatening human and animals have caused serious issues regarding anthelmintic resistance and drug residues worldwide (Saddiqi et al., 2011; Papadopoulos et al., 2012). As an alternative strategy, vaccine is a possible option for controlling parasitic nematodes including H. contortus (Bassetto and Amarante, 2015). Substantial progresses have been made during the past two decades in identifying several potential antigens from H. contortus as they can stimulate prominent levels of protective immunity in the immunized hosts (Knox et al., 2003; Tak et al., 2015). Here, we summarized the molecular properties and the protective efficacy of principal candidate antigens by using recombinant subunit vaccine and DNA vaccination. We hope that these insights will contribute to the study of molecular explorations of antigens and promote the development of H. contortus vaccines research in the future.

To discover useful vaccine antigens, it is crucial to understand the mechanisms of the immune regulation. Some studies have been carried out in China to explore the mechanism of immuno-regulation during parasite invasion by investigating the interaction between parasite molecules and host cells. To do so, the host peripheral blood mononuclear cells (PBMC) from goat blood were isolated by venipuncture. PBMC is actually a mixture of subpopulations of functional cells, which mainly includes lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells (Wang et al., 2014a). All of the subpopulations are critical to reflect interaction mechanism between the host cells and many important parasite molecules. For example, H. contortus galectin peptides recombinant Hco-gal-m/f (rHco-gal-m/f) were cultivated with PBMC of goats, investigated the effect of rHco-gal-m/f to induce apoptosis in the PBMC (Sun et al., 2007a). A combined proteomic and transcriptomic analysis had also been performed to understand the mechanisms underlying the immunomodulation induced by rHco-gal-m/f of H. contortus on goat PBMC. The findings demonstrated that rHco-gal-m/f could bind to the surface of goat PBMC and behaved as the suppressors of inflammatory response to facilitate the immune evasion of H. contortus (Wang et al., 2014b).

On the basis of the above study, further work by yeast two-hybrid screening found two Hco-gal-m and -f binding partners, transmembrane protein 147 (TMEM147) and transmembrane protein 63A (TMEM63A). The interaction of galectin with TMEM147 mainly mediated cell proliferation, cell apoptosis, and cytokine transcription in goat PBMC. This membrane protein, together with TMEM63A, was also involved in the regulation of galectin on phagocytosis and nitric oxide production of goat PBMC. However, TMEM63A might play a greater role than TMEM147 in the regulation of galectin in the migration and IFN-γ transcription of goat PBMC. These findings provided new perspective to the elucidation of the mechanisms involved in immune evasion by nematodes and in parasite–host interactions (Yuan et al., 2015; Li Y. et al., 2016).

Excretory and secretory products as vaccine candidate antigens of H. contortus contain various proteins, which can stimulate or depress the host immune response and are involved in the pathogenesis of the worms. Research showed that the H. contortus excretory and secretory products (HcESPs) displayed suppressive potential on the goat PBMC in vitro. HcESPs inhibited the productions of IL-4, IFN-γ, increased the suppressive cytokine IL-10, enhanced the inflammatory modulator IL-17, suppressed the production of chemical factor nitric oxide, decreased the cell proliferation and activated the cell migration (Gadahi et al., 2016d). Meanwhile, Gadahi et al. (2016c) also reported the interaction of proteins from HcESPs at different developmental stages to goat PBMC in vivo using liquid chromatography-tandem mass spectrometry. A total of 407 HcESPs that interacted with goat PBMC at different time points were identified. This study identified the secreted H. contortus 14-3-3 protein as a goat PBMC-interacting protein in all parasitic stages. In a follow-up study, recombinant protein of H. contortus 14-3-3 isoform 2 (rHcftt-2) decreased the production of IL-4 and suppressed the proliferation of goat PBMC in vitro (Gadahi et al., 2016a). In addition, a 24 kDa H. contortus excretory/secretory protein (HcES-24) also showed to have important antigenic function. The immune interactions between recombinant protein of HcES-24 and goat PBMC demonstrated that IL-4, IL-10, IL-17 and cell migration were increased. Nevertheless, the interaction significantly suppressed the PBMC proliferation and NO production. The findings showed that the rHcES-24 played important regulatory effects on the goat PBMC (Gadahi et al., 2016b).

Haemonchus contortus has the capacity to cause huge economic losses in China. Over the past two decades, studies in China on H. contortus were carried out covering a range of research areas including epidemiological survey, population genetic studies, detection of anthelmintic resistance, diapause genes related with development, vaccine development and immuno-regulation. Until 2017, most provinces in China have reported H. contortus infection with high prevalence in goats, sheep, and cattle. It was found that high genetic variation existed in Chinese H. contortus and most genetic variations were distributed within population, and there was high gene flow among populations, indicating drug resistance could be rapidly spread. Resistance against BZ was detected in several H. contortus populations, emphasizing the importance of detection and monitoring drug resistance for optimizing control strategy. Encouraging is the emergence of studies on screening the SNPs in the genome of H. contortus resistant against ivermectin as ivermectin resistance has been found in many places in China discovered by a recent nation-wide research project (funded by Special Fund for Agro-scientific Research in the Public Interest). Fundamental studies into structure and function of diapause related genes and the mechanism of immuno-regulation will provide insights into the molecular mechanism on developmental change and immune invasion of the parasite. In addition, vaccine development has also been attempted. These achievements are visible and provide valuable bases for future research exploration in these areas.

In addition to the above mentioned research areas, more research work should be carried out to assess the relationship between the infection rate/intensity and the severity of haemonchosis in order to implement more cost-effective control strategy. The usefulness of molecular methods in detecting drug resistance in the field should be evaluated in order to detect the emergency of drug resistance as early as possible and monitor its development to decrease the adverse influence of drug resistance. The extent of drug resistance should be investigated in a nationwide scale to understand the seriousness of drug resistance in China for which is still almost unknown. More sensitive and specific methods should be developed for use in the field and for detecting mixed infections. Finally, more fundamental studies relating to the biology of this parasite and its interaction with hosts of domestic animals are still needed to better understand the biology of H. contortus and the disease it causes in order to establish more useful diagnostic methods, develop effective drugs and vaccines, which will contribute to the preventing and controlling H. contortus in China.

MH conceived and designed the project. CW, FL, ZZ, XY, AA, and MH contributed to the writing of the manuscript with the input from XL and AD. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.