In vitro evaluation of the potential of mites of the family Macrochelidae (Acari: Mesostigmata) as macrobiological agents against the nematode Haemonchus contortus (Strongylida: Trichostrongylidae).

Small ruminants (sheep and goats) constantly suffer from endoparasitoses caused by gastrointestinal nematodes. Among these, the species Haemonchus contortus (Rudolphi, 1803) is considered to be the one of greatest importance within sheep farming. This nematode is difficult to control due to its resistance to most commercial anthelmintics. The aim of the present study was to assess the potential of macrochelid mites as macrobiological agents for controlling endoparasitoses of sheep caused by the nematode, H. contortus. For this, novel in vitro methodology was used, in which assessments were made not only of the predatory ability but also the population growth of mite species (Macrocheles merdarius, Macrocheles robustulus and Holostaspella bifoliata) when offered larvae of the nematode, H. contortus. The predatory ability of the mites, M. merdarius and H. bifoliata were efficient regarding their predatory ability against H. contortus nematode larvae. The mite, M. merdarius exhibited the highest predation rate with mean distribution values for the treated group of 18656 ± 10091 and for the control group of 1178 ± 712 (P < 0.0001). The species, H. bifoliata presented the highest population growth rate, with a percentage acarid recovery rate of 263% in relation to the number added initially. The data from this in vitro predation experiment suggest that, M. merdarius and H. bifoliata showed promise as macrobiological agents for controlling gastrointestinal endoparasitoses of sheep caused by the nematode, H. contortus given that both species reduced the population of this helminth by more 70% and the number of mites recovered was three times greater than the number added.

Influence of probiotic supplementation on parasitological parameters in lambs.

The control of parasitosis is based on the use of anthelmintics. However, its long-term and indiscriminate use can select populations of resistant nematodes. New alternatives such as probiotics are being studied to solve this problem. This study aimed to investigate the effects of an oral probiotic containing six different bacterial strains and the yeast Saccharomyces cerevisiae on the blood biochemistry, parasitological, and histological parameters of naturally infected lambs. Forty-two weaned Texel or Ile de France crossbred lambs aged 86.9 ± 8.0 days and weighing 27.4 ± 3.7 kg were randomly allocated into three groups (n = 14 lambs). The control group (CG) was fed a basal diet without probiotic supplementation. The treatment group 1 g (T1G) was fed a basal diet with commercial probiotic supplementation at a dose of 1 g/lamb/day. The treatment group 5 g (T5G) was fed a basal diet with commercial probiotic supplementation at a dose of 5 g/lamb/day. The experimental period was 84 days, where the groups undergo mild natural infection. Every two weeks the hematocrit, total protein, albumin, globulin, fibrinogen, plasma protein, fecal egg count (FEC), and fecal consistency score were evaluated. Twenty lambs were slaughtered for histological evaluation of the rumen and abomasal wall and for counting abomasal nematodes. The area, length, and number of eggs from the recovered Haemonchus contortus female uteri were measured. Data were analyzed using analysis of variance (ANOVA) and Tukey's test (p ≤ 0.05). The area and length of Haemonchus contortus females from T1G were smaller (p < 0.01) than those of CG and T5G. The number of eggs from the H. contortus female uterus was lower (p < 0.01) in T1G and T5G. Evaluating the incidence of diarrhea, the T1G and T5G groups had a higher prevalence with a 35% and 39% score of zero, respectively (p < 0.05), while the CG group had 22%. All other evaluated parameters showed no significant differences (p > 0.05) between the groups. The probiotic had a beneficial effect on the gastrointestinal health of the weaned lambs through a decrease of the area, length, and the number of eggs of female H. contortus and an improvement in fecal consistency.

Immunization with thiol-binding proteins from Haemonchus contortus adult worms partially protects goats against infection during prepatency.

To contribute of the knowledge of the immune mechanisms underlying the response to the immunization of goats with thiol-binding proteins fractions (PBS-TSBP) from Haemonchus contortus (H. contortus) adult worms, this study analyzed the degree of protection and the immune responses developed against the parasite after vaccination with this antigenic complex during the time-elapsing between challenge with L3 of the parasite and the development of adult worms, evidenced by the appearance of first faecal eggs (prepatent period or prepatency). Goat kids immunized with PBS-TBSP generated an immune response during the prepatency which translates into a reduction in the number of worms, as well as a lower reduction on packed cell volume and plasma protein levels in relation to the non-vaccinated animals. As previously described in other studies carried out after the prepatent period, this protection was associated with a systemic humoral response. At the local level, a specific humoral response was also observed, together with an immune-inflammatory infiltrate in the gastric mucosa of MCH-II + cells and CD4+ lymphocytes, whose number was associated with a reduction in the number of worms and an increase in plasma proteins. A high peripheral eosinophilia was detected, but no corresponding increased infiltration of the gastric mucosa by eosinophils or globular leukocytes was observed. In agreement with previous data on the immunolocalization of the antigens used here, the results obtained contribute to the idea that these may be excretion/secretion (E/S) products necessary for parasite survival, whose inactivation during the larval and/or pre-adult stages may have contributed to immunoprotection.

H11-induced immunoprotection is predominantly linked to N-glycan moieties during Haemonchus contortus infection.

Nematodes are one of the largest groups of animals on the planet. Many of them are major pathogens of humans, animals and plants, and cause destructive diseases and socioeconomic losses worldwide. Despite their adverse impacts on human health and agriculture, nematodes can be challenging to control, because anthelmintic treatments do not prevent re-infection, and excessive treatment has led to widespread drug resistance in nematode populations. Indeed, many nematode species of livestock animals have become resistant to almost all classes of anthelmintics used. Most efforts to develop commercial anti-nematode vaccines (native or recombinant) for use in animals and humans have not succeeded, although one effective (dead) vaccine (Barbervax) has been developed to protect animals against one of the most pathogenic parasites of livestock animals - Haemonchus contortus (the barber's pole worm). This vaccine contains native molecules, called H11 and H-Gal-GP, derived from the intestine of this blood-feeding worm. In its native form, H11 alone consistently induces high levels (75-95%) of immunoprotection in animals against disease (haemonchosis), but recombinant forms thereof do not. Here, to test the hypothesis that post-translational modification (glycosylation) of H11 plays a crucial role in achieving such high immunoprotection, we explored the N-glycoproteome and N-glycome of H11 using the high-resolution mass spectrometry and assessed the roles of N-glycosylation in protective immunity against H. contortus. Our results showed conclusively that N-glycan moieties on H11 are the dominant immunogens, which induce high IgG serum antibody levels in immunised animals, and that anti-H11 IgG antibodies can confer specific, passive immunity in naïve animals. This work provides the first detailed account of the relevance and role of protein glycosylation in protective immunity against a parasitic nematode, with important implications for the design of vaccines against metazoan parasites.

Sheep immune-stimulated with Saccharomyces boulardii show reduced prolificacy of Haemonchus contortus.

Haemonchus contortus is the most pathogenic parasite for sheep. The objective was to evaluate immunomodulation of the probiotic Saccharomyces boulardii in sheep experimentally infected with H. contortus. Twenty-four sheep were divided into three groups: one infected with 500 H. contortus larvae/day for 26 days and supplemented with S. boulardii (40 ml with 1 × 108 CFU/ml/day); a control group only infected with H. contortus but not supplemented; and a naïve group that never came into contact with either parasites or S. boulardii. To assess the humoral immune response, production of specific serum IgG anti-somatic H. contortus antigen was evaluated through indirect ELISA. To assess the cellular immune response, cell populations and cytokine (IL-4, IL-5 and IL-10) production were evaluated through flow cytometry. For parasitological analyses, the counts of eggs per gram of faeces (EPG) and larvae per faecal culture were assessed. At all the study points, the concentration of IgG anti-H. contortus was higher (p < .05) in the S. boulardii group than in the other groups. The cell analysis revealed that there were significantly higher numbers (p < .05) of cells expressing MHC-II and significantly higher numbers (p < .05) of eosinophils in the mucosa in the S. boulardii group. Significant expression of IL-10 was observed only in the control infected group. There were significant reductions (p < .05) in EPG and larval counts in the S. boulardii supplemented group. These results show that S. boulardii supplementation modulated the immune response against H. contortus, thereby reducing its infection.

Hepatocellular carcinoma-associated antigen 59 and ADP-ribosylation factor 1 with poly (lactic-co-glycolic acid): A promising candidate as nanovaccine against haemonchosis.

Haemonchus contortus (H. contortus) ADP-ribosylation factor 1 (Hc-ARF1) and Hepatocellular carcinoma-associated antigen 59 (Hc-HCA59) are recognized to largely regulate the immune responses of host cells. However, studies about the protective efficacy of the two molecules are poorly unknown. In this research, combinations of recombinant Hc-HCA59 (rHc-HCA59) and Hc-ARF1 (rHc-ARF1) proteins were amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticles adjuvant in order to investigate their protection potential against H. contortus in goats. The results demonstrated that the levels of IgG, IgA, IgE, and IL-4 were noticeably enhanced in the rHc-HCA59 and rHc-ARF1 (rHc-HCA59+rHc-ARF1) group before H. contortus third-stage larvae (L3) challenge. After the L3 challenge, the levels of IL-17, IL-9, and TGF-ß were considerably upregulated in the rHc-HCA59+rHc-ARF1 group. In the meantime, the abomasal worm burdens and the fecal eggs were reduced by 63.2% and 69.4% respectively in the rHc-HCA59+rHc-ARF1 group. According to the studies, PLGA nanoparticles immobilized with rHc-HCA59 and rHc-ARF1 proteins conferred partial protection and were expected to be a potential candidate for developing nano vaccines to combat goat haemonchosis.

Investigation of the combined efficacy of two Haemonchus contortus vaccines in weaner Merino sheep.

This study was conducted to investigate whether co-administration of Barbervax® (Bvax) with Haemonchus contortus surface larval antigen (HcsL3) would increase the protective efficacy and duration of protection against H. contortus infection in weaner Merino sheep. A total of 132 10-month-old weaned Merino ewe lambs were randomly allocated into six treatment groups (n = 22). Sheep were vaccinated four times with either Barbervax® (Bvax), H. contortus L3 surface larval antigen (HcsL3), combined vaccination (Bvax + HcsL3), Bvax + AlOH, HcsL3 + Saponin or remained as unvaccinated controls. Aluminium hydroxide (AlOH) and saponin adjuvants were included in HcsL3 and Bvax vaccines respectively. The first three vaccinations were given at 4 week intervals and the fourth vaccination provided as booster, 9 weeks later. All animals were treated with Zolvix™ (monepantel 25 mg/mL, Elanco) at the third vaccination and commencing two weeks later, artificially trickle infected with H. contortus L3. Worm egg count (WEC), packed cell volume (PCV), antibody titre and bodyweight were measured throughout the study as was specific antibody directed against each antigen using ELISA. The administration of Bvax and HcsL3, alone or in combination, induced an antibody response against HcsL3 but only the Bvax and the combined treatment elicited an antibody response to the Bvax antigen. The targeting of HcsL3 by each vaccine was confirmed by immunofluorescence staining of H. contortus L3. However, only the booster vaccination in the Bvax treatments reduced WEC to levels below untreated controls. The HcsL3 vaccine did not reduce WEC in this experiment and co-administration with Bvax did not improve the efficacy and duration of protection against H. contortus infection.

The excretory-secretory antigen HcADRM1 to generate protective immunity against Haemonchus contortus.

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 excretory­secretory 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.

Taenia hydatigena larvae vesicular concentrate reduces the egg shedding of Haemonchus contortus associated with the overexpression of abomasal cytokines in lambs.

The expression patterns of some cytokines were compared by RT-qPCR between lambs with and without Taenia hydatigena larvae vesicular concentrate (ThLVC) administration and subsequent infection with Haemonchus contortus. Lambs that received ThLVC prior to infection with H. contortus showed lower (p < 0.03) cumulative FEC (AUC = 18450 ± 3384) than infected lambs who did not receive ThLVC (AUC = 31081 ± 3277). Lambs infected with H. contortus, in general, overexpressed Th1 and Th2 cytokines in abomasal mucosa and abomasal lymph nodes, which seems to indicate a generalized and nonpolarized activation of the immune response by H. contortus. The main immunomodulatory effects of ThLVC were observed in the abomasal fundic region. The lambs that were given ThLVC prior to infection strongly overexpressed most of the studied cytokines representing the Th1 (IFNγ and IL2) and Th2 profiles (IL4, IL5, IL6 and IL10), proinflammatory cytokines (SOD1 and PRDX6) and IgE receptor; in contrast, lambs that were infected but did not receive ThLVC only moderately overexpressed IFNγ, IL4 and IL6. The absence of the significant overexpression of cytokines in lambs that only received ThLVC suggests that this derived from T. hydatigena does not have a stimulating effect per se; however, the presence of H. contortus did produce the highest expression (p < 0.01) cytokine profile among lambs that received ThLVC prior to infection compared to those who did not receive it, so its effect seems to be immunomodulatory and not only immunostimulatory.

Protective efficacy of Barbervax® in Merino weaner sheep trickle infected with five doses of Haemonchus contortus infective larvae.

Barbervax® protects sheep against H. contortus infection; however, the level of protection afforded by the vaccine at different levels of larval challenge under field conditions has not been reported. Therefore, this study was designed to investigate the protective efficacy of Barbervax® at increasing doses of trickle infection with H. contortus third stage larvae (L3). Merino lambs (220) were randomly allocated to challenge treatment groups and received Barbervax® vaccinations at approximately 8, 11, 15 and 22 weeks of age or not vaccinated (control group). Animals within each treatment group were subjected to one of five levels of H. contortus L3 trickle infection (0, 300, 600, 1200 and 2400 L3/week in two split doses). Trickle infections started two weeks after the third vaccination (week 0). Worm egg count (WEC), packed cell volume (PCV), antibody titre and bodyweight were measured at week 2 (start of trickle infection), week 5 and then every two weeks for 11 weeks. Vaccinated weaners had a significantly (P < 0.0001) lower faecal WEC than unvaccinated control animals. Vaccination induced significant specific antibody responses that were not influenced by level of trickle infection. Vaccination significantly reduced the number of weaners requiring salvage anthelmintic treatment (9.4% vaccinated weaners versus 33.6% unvaccinated). Vaccine protective index based on WEC was similar at all challenge levels (overall mean> 60%) and at the higher challenge levels did not reduce H. contortus infection to levels that would not require anthelmintic treatment. Therefore, it is suggested that under conditions of high larval challenge the use of Barbervax® should be integrated with other control methods.

Haemonchus contortus hepatocellular carcinoma-associated antigen 59 with poly (lactic-co-glycolic acid): A promising nanovaccine candidate against Haemonchus contortus infection.

Hepatocellular carcinoma-associated antigen 59 (HCA59), one of significant excretory/secretory products of Haemonchus contortus (HcESPs), is identified to have immunomodulatory eff ;ects on host cells. However, protection potential of the molecule in H. contortus remains poorly understood. In this study, H. contortus recombinant HCA59 protein amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticle adjuvant was tested for its protection against H. contortus infection in goats. Fifteen goats were allocated into three groups. On days 0 and 14, rHCA59 group was immunized with PLGA nanoparticles encapsulated with recombinant protein HCA59 (rHCA59-PLGA) respectively. Positive control group was unvaccinated, but challenged with H. contortus third stage larvae (L3). Negative control group was unvaccinated and unchallenged with L3. Goats in rHCA59 group and positive control group were challenged with 8000 H. contortus L3 after 14 days of the second immunization. Following immunization, high level of sera IgG, IgA, and IgE, as well as significantly high production of IL-4 and IL-9 was produced in rHCA59 group. After L3 challenge, the level of IL-17 and TGF-ß in rHCA59 group increased obviously. Meanwhile, the fecal eggs and the abomasal worm burdens in rHCA59 group was reduced by 44.1 % and 54.6 %, respectively. The studies suggested that rHCA59-PLGA nanoparticles conferred partial protection and could be a good candidate for the development of nanovaccines against H. contortus infection in goats.

Evaluation of protection in grazing lambs immunised with different doses of Haemonchus contortus gut membrane glycoproteins in Southern Brazil.

A vaccine containing doses ranging from 2 µg to 50 µg of integral membrane proteins from Haemonchus contortus intestinal cells (H11 and H-gal-GP complex) has been shown to be effective for lambs. A vaccine for H. contortus was tested in two-month old grazing Corriedale lambs during an eight-month trial on the outskirts of Bagé, Rio Grande do Sul, Brazil. The animals were kept in a single paddock and allocated to five similar groups according to weight, sex and faecal egg counts (FEC). Vaccinated lambs received 0, 2, 5, 10 or 50 µg of the same antigen diluted in QuilA adjuvant. Vaccine injections were given at days 0, 21, 49, 91, 133, and 175. Lambs were sampled weekly for FEC, packed cell volume (PCV), and plasma ELISA antibody titre. Lambs with PCVs ≤15 % were drenched with levamisole (7.5 mg/kg body weight) as a salvage treatment. During days 77-98 an artificial challenge was administered to increase parasite transmission. Post-artificial challenge (from day 98-217), the FEC of the vaccinated lambs were 59.3 % lower than those of the control lambs. Antigen dose correlated with changes in PCV (r = 0.387 p-value < 0.001). Vaccinated lambs also had higher PCV than controls (p < 0.001; contrast analysis). Salvage treatment was needed in 16.7 % of vaccinated lambs and 88 % of control lambs.

Protection studies of an excretory-secretory protein HcABHD against Haemonchus contortus infection.

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/ß-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.

Artificial Haemonchus contortus infection as a strategy to induce protective immune response to natural infection in Pelibuey lambs.

The objective of this study was to evaluate the reduction in nematode faecal egg count (FEC) in Pelibuey lambs segregated as resistant (RES), susceptible (SUS) and intermediate (INT) to gastrointestinal nematodes. Twenty-nine weaned Pelibuey lambs, aged five months old, free of nematode infection, were used. Nine lambs were RES, six were SUS and 14 were INT lambs. The study consisted of two phases: in Phase 1 the lambs were infected experimentally with Haemonchus contortus. In Phase 2, the lambs were naturally infected by grazing. Faecal and blood samples were taken every week. The packed cell volume and total protein were quantified. The FEC value (FECmax) per lamb was recorded together with a natural reduction in FEC in the two phases. The data were analysed with a model of measures repeated over time. During Phase 1, the RES lambs showed the lowest FEC (1061 ± 1053) compared to the other groups (INT: 2385 ± 1794 eggs per gram of faeces (EPG); and SUS: 3958 ± 3037 EPG). However, in Phase 2 no significant differences (p > 0.05) were observed between the groups of lambs (RES: 275 ± 498 EPG; SUS: 504 ± 1036 EPG; and INT: 603 ± 1061 EPG). At the end of Phase 1, the FEC of RES lambs was naturally reduced by 75.5% in respect to FECmax (p < 0.05), and at the end of Phase 2 the reduction in FEC was 90% in respect to FECmax (p > 0.05); the same behaviour was observed in RES and SUS lambs. It is concluded that the artificial infection in the lambs induced a more rapid immune response in RES than SUS lambs, and all lambs developed high acquired resistance by continuous infection.

Evaluation of Barbervax® vaccination for lambing Merino ewes.

The Barbervax® vaccine, directed against Haemonchus contortus, has been commercially available in Australia since 2014. However, to date the optimal timing for booster vaccination of lambing ewes has not been identified. Therefore the aim of this experiment was to compare the efficacy of Barbervax® when administered to ewes one week prior to the commencement of lambing versus at lamb marking 7 weeks later. A total of 400 single bearing Merino ewes, run on a commercial sheep property in the New England region of New South Wales, previously primed with Barbervax® were enrolled in the project. The experiment incorporated a cross-over design with two treatments imposed pre-lambing; Barbervax® booster vaccination with anthelmintic (n = 200) or anthelmintic alone (n = 200). At lamb marking, there were four treatment groups (n = 100) and anthelmintic was administered to all treatment groups. The four treatment combinations were Barbervax® booster vaccination at pre-lambing and lamb marking (V-V), booster vaccination only at pre-lambing (V-A), booster vaccination only at lamb marking (A-V) or anthelmintic only at both at pre-lambing and lamb marking (A-A). Pre-lambing Barbervax® booster vaccination of ewes induced an antibody response and, when used in conjunction with anthelmintic, significantly reduced worm egg counts (WEC) assessed at 7 weeks (lamb marking) and 13 weeks later, relative to treatment with anthelmintic alone. Booster vaccination plus anthelmintic treatment at lamb marking was not effective at reducing WEC compared to anthelmintic treatment alone. Body weight and packed cell volume did not differ between the treatment groups throughout the trial. In conclusion Barbervax® booster vaccination at pre-lambing provides additional protection for reproducing ewes given an effective short acting anthelmintic at this time.

Anthelmintic resistance and common worm control practices in sheep farms in Flanders, Belgium.

In contrast to many other European countries, no data were available on the presence of anthelmintic resistance in gastrointestinal nematodes in sheep in Belgium. A faecal egg count reduction test was performed in 26 sheep flocks in Flanders, Northern Belgium. Results indicated widespread resistance against benzimidazoles (albendazole, fenbendazole and mebendazole), with treatment failure on all 8 farms investigated. Haemonchus contortus and Teladorsagia circumcincta were the predominant species after treatment failure. Amino acid substitutions associated with benzimidazole resistance were detected at the codon positions 167 (8%) and 200 (92%) of the isotype-1 beta tubulin gene in H. contortus, codon positions 198 (47%) and 200 (43%) in T. circumcincta and position 200 (100%) in T. colubriformis. Resistance against macrocyclic lactones (ivermectin, doramectin and moxidectin) was recorded on 7 out of 20 flocks, mainly in H. contortus and T. circumcincta. Treatment failure was also observed for closantel (in combination with mebendazole) and for monepantel, on one farm each. Trichostrongylus spp. were implicated with resistance against monepantel. A questionnaire survey on farm management and worm control measures indicated that worm control was often not sustainable. Ewes and lambs were treated frequently (on average 2.6 and 3.2 times per year), mostly without weighing. Only few sheep farmers (9%) regularly used faecal egg counts to monitor worm infections. Despite the FECRT showing otherwise, most of the farmers perceived the efficacy of anthelmintics as very good (30%) or good (54%).

Long spelling periods are required for pasture to become free of contamination by infective larvae of Haemonchus contortus in a humid subtropical climate of São Paulo state, Brazil.

The objective of this trial was to evaluate the period of spelling necessary for a pasture to become free of contamination by infective larvae of gastrointestinal nematodes (GIN) of sheep, in different seasons of the year, as well as to determine when the greatest pasture contamination occurs and how long it lasts. An area was divided into four paddocks, one for each season (spring, summer, autumn, and winter). In order to contaminate the paddocks with free living stages of GIN, eight ewes, naturally infected, grazed on each paddock for 14 consecutive days, starting on the following dates: autumn, on April 4, 2017; winter, on July 4, 2017; spring, on September 26, 2017; summer, on January 2, 2019. At the beginning and end of the grazing period, faecal samples were taken directly from the rectums of the ewes to count eggs per gram of faeces (EPG) and for faecal cultures. Every 14 days pasture samples were collected to assess the number of infective larvae (L3) per kilogram of dry matter. At the end of the 14 day ewe grazing period, 21 stakes were placed where there were faeces on the paddock. Subsequently, every 14 days, the faeces located at three of the stakes were collected and the L3 were recovered. After the exit of the ewes, monthly, two tracer lambs, free of helminth infection, were allocated into the paddock for 14 days. At the end of this period they were housed in covered stalls for 28 days. Faeces from the lambs were collected for individual EPG counting and faecal culture at 21 and 28 days after grazing. Infective larvae recuperation was observed from faeces and pasture in all seasons. In the autumn, spring, and summer, high EPG counts were observed in the first tracer lambs (8521, 4800, and 8064 EPG, respectively), while in winter, high infection (14132 EPG) of the animals was observed only from the second pair of tracer lambs. For a pasture to become "clean", 322 days, 350 days, 294 days, and 182 days following contamination were necessary, respectively, in the autumn, winter, spring, and summer. In autumn, spring, and summer, massive contamination of the pasture with L3 occurred soon after an area had been grazed by infected sheep, while in winter this took a little longer. The contamination persisted, approximately, from a minimum of six months post contamination in summer to up to almost one year post contamination in winter.

Haemonchus contortus transthyretin domain - containing protein (HcTTR): A promising vaccine candidate against Haemonchus contortus infection.

Haemonchus contortus transthyretin domain-containing protein (HcTTR) with 136 amino acids belongs to a transthyretin-like (TTL) family member. In our previous study, it was reported that HcTTR was a novel antagonist of the goat cytokine Interleukin 4 (IL-4), and was involved in the regulation of host immune responses, implying that it might be applied for therapeutic strategies or vaccine development against the infection of H. contortus. Thus, the protective capacities of HcTTR against H. contortus infections via active and passive immunization trials were examined. For the passive protection trials, H. contortus-infected goats were intravenously immunized twice with 5 mg of total IgG containing anti-rHcTTR goat polyclonal antibodies. The results showed that the significant rates of reduction in egg shedding and worm burden was 58.12% and 64.61%, respectively, as compared with the positive control group. For the active protection trials, local goats were vaccinated twice with 500 µg of recombinant HcTTR to generate antigen-specific circulating antibodies, resulting in 63.7% reduction in eggs shedding and 66.4% reduction in worm burdens after H. contortus challenge. In both passive and active trials, the immunized goats displayed higher mucosal IgA levels and less anaemic compared to the challenged positive controls. Pen trials indicated that HcTTR generated partial immune protective effects against H. contortus challenge and it could be a promising vaccine candidate for development of effective strategy to control H. contortus.

A novel technique for retrospective genetic analysis of the response to vaccination or infection using cell-free DNA from archived sheep serum and plasma.

Genetic variation is associated with differences in disease resistance and susceptibility among individuals within a population. To date, molecular genetic analyses of host responses have relied on extraction of genomic DNA from whole blood or tissue samples. However, such samples are not routinely collected during large-scale field studies. We demonstrate that cell-free genomic DNA (cfDNA) may be extracted and amplified from archived plasma samples, allowing retrospective analysis of host genetic diversity. This technique was also applicable to archived serum samples up to 35 years old and to different ruminant species. As proof of concept, we used this cfDNA approach to genotype the major histocompatibility complex (MHC) class II DRB1 locus of 224 Merino sheep which had participated in field trials of a commercial Haemonchus contortus vaccine, Barbervax®, in Australia. This identified a total of 51 different DRB1 alleles and their relative frequencies. This is the first study to examine host MHC diversity using DNA extracted from archived plasma samples, an approach that may be applied to retrospective analyses of genetic diversity and responses to vaccination or infection across different species and populations.

Immunization with recombinant rHc23 partially protects lambs against trickle infections by Haemonchus contortus.

BACKGROUND: Haemonchosis is one of the most economically important parasitic diseases affecting small ruminants all over the world. Chemotherapeutic control has several shortcomings (limited anthelmintic arsenal, frequent resistance) and is hardly affordable by many farm economies. A recombinant antigen (rHc23) was shown to induce significant protection in vaccination trials with single dose challenges and different adjuvants. RESULTS: Lambs were vaccinated with 100 µg rHc23/dose + bacterial immunostimulant (BI) (LPS from Escherichia coli + Propionibacterium acnes extract) (days - 2, 0, 7 and 14) and subjected to a trickle infection with two dosages [6x, 1000 infective larvae (L3) or 6x, 2000 L3]. Vaccinated lambs showed a significant antibody response against rHc23 and Haemonchus contortus soluble extract as assessed by ELISA and Western blot (WB). Fecal egg counts (epg) along the experiment of vaccinated and BI treated lambs were significantly reduced. All vaccinated animals showed total egg output and abomasal helminth burdens (median, average) lower than those from unvaccinated or BI-treated animals lambs although differences were not statistically significant. CONCLUSIONS: Vaccination with 100 µg rHc23/dose + BI against H.contortus trickle infections apparently induced lower epg values and helminth burdens at the end of the experiment. Intragroup individual variations did not allow to obtain conclusive results and more research is needed including adjuvants and larger groups of animals to validate the potential value of rHc23 as candidate to develop a recombinant vaccine for lambs haemonchosis.