Medicinal plants attract the attention of many researchers to find natural and safe remedies for various resistant diseases. Leaves of Mitragyna inermis are widely used in traditional veterinary medicine for the treatment of gastrointestinal strongyles of small ruminants. The aim of the current study is to estimate the antioxidant, anthelmintic and the larval toxicity of the aqueous and hydroethanolic extracts of this plant in addition to the hexane, dichloromethane and ethanol fractions of the hydroethanolic extract. Investigation of the most active extract using Ultra Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Electrospray Ionization Mass Spectrometry (UPLC-QToF-ESI-MS). Both plant extracts showed good antioxidant activity by scavenging the 2,2\'-diphényl-1-picrylhydrazyl (DPPH) radical and reducing the ferric ion. Similarly, they were no-toxic to Artemia salina larvae (CL50 > 0.1 μg/mL). Also, they significantly reduced larval migration and motility of Haemonchus contortus adult worms (p < 0.001). The hexane, dichloromethane and ethanolic fractions of the hydroethanolic extract showed low activity compared to crude extracts except for the hexane fraction on H. contortus adult worms (p < 0.001) while it showed a poor result on larvae. It thus appears that the anthelmintic activity of the extract may be linked to the synergistic action of these compounds. The UPLC-QToF-ESI-MS analysis revealed the tentative identification of 15 compounds including 7 alkaloids. The results of the present study confirm the anthelmintic activity of M. inermis in traditional veterinary medicine.

Haemonchus contortus is the most prevalent and pathogenic gastrointestinal nematodes (GINs) in ruminants causing extensive economic losses. It is essential to estimate the efficacy of common commercially available anthelmintics against Haemonchus contortus parasite. Here, we standardized an ex-vivo culture platform for H. contortus and evaluated the efficacy of commonly used anthelmintics namely, albendazole (ABZ), levamisole (LVM), ivermectin (IVM), closantel (CLS) and rafoxanide (RFX). Adult worms were collected from abomasa of slaughtered animals, cultured in MEM, DMEM, M199 or RPMI with or without 20% FBS for up to 72 h. Cultured worms were incubated with ABZ, LVM, IVM, RFX or CLS in DMEM supplemented with 20% FBS at different concentrations (0.5-50 µg/ml) in triplicates and examined at 0, 3, 6, 12, 24, 36 and 48 h post treatment. Of the culture conditions, DMEM supplemented with 20% FBS supported the survival of H. contortus for (P < 0.001) longer period of time which was used in the evaluation of anthelmintics. The efficacy of CLS and RFX were significantly (P < 0.001) higher than other drugs and 100% mortality was observed at 2 µg/ml of CLS and RFX within 12 h post treatment. However, ABZ, LVM, and IVM showed significant effect at the concentration of 50 µg/ml with 48, 36, and 24 h, respectively. Morphological changes included severe cuticle disruption around the buccal cavity, posterior region and vulva as well as loss of cuticle structure integrity coupled with expulsion and fragmentation of digestive components of parasites when treated with 50 µg/ml of ABZ, LVM, and IVM and 2 µg/ml of RFX and CLS. Collectively, DMEM supplemented with 20% FBS can be used as ex-vivo culture platform for maintenance of H. contortus, and RFX and CLS can be used as the promising drugs for the prevention, control and treatment of H. contortus infections.

Bridelia ferruginea (Euphorbiaceae) and Mitragyna inermis (Rubiaceae) are two plants of the beninese pharmacopeia used in vivo for the control of gastrointestinal nematodes (GINs) in small ruminants. The objective of the present study is to explore the mechanism of bioactive compounds involved in the action of these two plants on the third-stage infective larvae (L3s) of Haemonchus contortus. Thus, sheathed L3s of H. contortus were incubated with acetone extracts of B. ferruginea and M. inermis at concentrations of 0, 150, 300, 600 and 1200 µg/mL for 3 h at 25°C. The L3s were then washed and artificially submitted to exsheathment in the presence of sodium hypochlorite solution. The role of tannins was verified by adding a tannin inhibitor, polyvinyl polypyrrolidone (PVPP), to the acetone extracts of these two plants for 2 h at 25°C. Acetone extracts from B. ferruginea and M. inermis inhibited the exsheathment of H. contortus larvae (p < 0.001) and this inhibitory effect was dose-dependent for M. inermis at the concentrations tested. Treatment of B. ferruginea and M. inermis extracts with PVPP was associated with a partial restoration of the exsheathment kinetics of H. contortus larvae (p < 0.001), confirming the predominant role of tannins but also the residual role of other secondary metabolites. These in vitro results suggest that these plants are endowed with anthelmintic (AHs) properties and therefore likely to be used as alternatives to synthetic molecules.

Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. Haemonchus contortus is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against H. contortus is investigated in this study. Out of which, three isolates-Comamonas testosteroni, Comamonas jiangduensis, Pseudomonas weihenstephanesis-show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of C. testosteroni and P. weihenstephanesis show 46% adult mortality, whereas C. jiangduensis shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of H. contortus egg. It is concluded that the effect of bacterial culture filtrates against H. contortus is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.

The prevention, treatment and control of Haemonchus contortus have been increasingly problematic due to its widespread occurrence and anthelmintic resistance. There are very few descriptions of recombinant antigens being protective for H. contortus, despite the success of various native antigen preparations, including Barbervax. We recently identified an H. contortus excretory–secretory antigen, H. contortus adhesion-regulating molecule 1 (HcADRM1), that served as an immunomodulator to impair host T-cell functions. Given the prophylactic potential of HcADRM1 protein as a vaccine candidate, we hereby assessed the efficacies of HcADRM1 preparations against H. contortus infection. Parasitological and immunological parameters were evaluated throughout all time points of the trials, including fecal egg counts (FEC), abomasal worm burdens, complete blood counts, cytokine production profiles and antibody responses. Active vaccination with recombinant HcADRM1 (rHcADRM1) protein induced protective immunity in inoculated goats, resulting in reductions of 48.9 and 58.6% in cumulative FEC and worm burdens. Simultaneously, passive administration of anti-HcADRM1 antibodies generated encouraging levels of protection with 46.7 and 56.2% reductions in cumulative FEC and worm burdens in challenged goats. In addition, HcADRM1 preparations-immunized goats showed significant differences in mucosal and serum antigen-specific immunoglobulin G (IgG) levels, total mucosal IgA levels, haemoglobin values and circulating interferon-γ, interleukin (IL)-4 and IL-17A production compared to control goats in both trials. The preliminary data of these laboratory trials validated the immunoprophylactic effects of rHcADRM1 protein. It can be pursued as a potential vaccine antigen to develop an effective recombinant subunit vaccine against H. contortus under field conditions.

Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/β-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory-secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

BACKGROUND: Gastrointestinal nematodes could release excretory-secretory (ES) proteins into the host environment to ensure their survival. These ES proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions, especially in chronic infections. In our preliminary study, Haemonchus contortus adhesion-regulating molecule 1 (HcADRM1) was identified from H. contortus ES proteins (HcESPs) that interacted with host T cells via liquid chromatography-tandem mass spectrometry analysis. However, little is known about HcADRM1 as an ES protein which may play a pivotal role at the parasite-host interface.
METHODS: Based on bioinformatics approaches, multiple amino acid sequence alignment was conducted and the evolutionary relationship of HcADRM1 with ADRM1 orthologues was extrapolated. Employing RT-qPCR and immunohistochemistry assays, temporal transcriptional and spatial expression profiles of HcADRM1 were investigated. Using immunostaining approaches integrated with immunological bioassays, the immunomodulatory potentials of HcADRM1 on goat T cells were assessed.
RESULTS: We hereby demonstrated that HcADRM1 with immunodiagnostic utility was a mammalian ADRM1 orthologue abundantly expressed at all developmental stages of H. contortus. Given the implications of ADRM1 proteins in cell growth, survival and development, we further investigated the immunomodulatory property of HcADRM1 as an individual ES protein acting at the parasite-host interface. The rHcADRM1 stimuli notably suppressed T cell viability, promoted intrinsic and extrinsic T cell apoptosis, inhibited T cell proliferation and induced cell cycle arrest at G1 phase. Simultaneously, rHcADRM1 stimuli exerted critical controls on T cell cytokine secretion profiles, predominantly by restraining the secretions of interleukin (IL)-4, IL-10 and interferon-gamma.
CONCLUSIONS: Importantly, HcADRM1 protein may have prophylactic potential for anti-H. contortus vaccine development. Together, these findings may contribute to the clarification of molecular and immunomodulatory traits of ES proteins, as well as improvement of our understanding of parasite immune evasion mechanism in H. contortus-host biology.

The α/β-hydrolase domain (ABHD) proteins belonging to α/β-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.

The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and β-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.

UNASSIGNED: This study aimed to explore the phytochemical constituents and anthelmintic activities of four Cassia spp. leaves against Haemonchus contortus.
UNASSIGNED: The extracts were prepared from four species of Cassia spp. (C. siamea, C. fistula, C. surattensis, and C. spectabilis). Phytochemical screening of the extract was done based on the Harborne method. Evaluation of the anthelmintic activities against H. contortus was done in vitro using infective larvae (L3) migration inhibition assay (LMIA). Measurement of larvae migrating was conducted through a nylon filter with a pore size of 20 μm. The doses of Cassia spp. extract implemented were 25, 50, 100, and 200 mg/ml.
UNASSIGNED: Tannins, alkaloids, phenol hydroquinone, flavonoids, steroids, triterpenoids, and saponins were present in all the extracts, whereas alkaloids were absent in C. fistula. No triterpenoids were found in C. surattensis and C. spectabilis. Movement of H. contortus larvae was significantly inhibited after exposure to Cassia extracts at various dosage levels (p < 0.05). The test results using LMIA on L3 H. contortus showed the lowest inhibition in the negative control. Among the species of Cassia, the C. surattensis (at 200 mg/ml) showed the highest (p < 0.05) inhibition level on the larvae. The latter result corresponded to the effect of albendazole.
UNASSIGNED: Compared to other Cassia spp., C. surattensis exhibited the highest inhibition against L3 H. contortus. However, the inhibition effect of C. surattensis was still lower as compared to albendazole.