Genomic Regions Associated with Resistance to Gastrointestinal Parasites in Australian Merino Sheep.

The objective of this study was to identify genomic regions and genes associated with resistance to gastrointestinal nematodes in Australian Merino sheep in Uruguay, using the single-step GWAS methodology (ssGWAS), which is based on genomic estimated breeding values (GEBVs) obtained from a combination of pedigree, genomic, and phenotypic data. This methodology converts GEBVs into SNP effects. The analysis included 26,638 animals with fecal egg count (FEC) records obtained in two independent parasitic cycles (FEC1 and FEC2) and 1700 50K SNP genotypes. The comparison of genomic regions was based on genetic variances (gVar(%)) explained by non-overlapping regions of 20 SNPs. For FEC1 and FEC2, 18 and 22 genomic windows exceeded the significance threshold (gVar(%) ≥ 0.22%), respectively. The genomic regions with strong associations with FEC1 were located on chromosomes OAR 2, 6, 11, 21, and 25, and for FEC2 on OAR 5, 6, and 11. The proportion of genetic variance attributed to the top windows was 0.83% and 1.9% for FEC1 and FEC2, respectively. The 33 candidate genes shared between the two traits were subjected to enrichment analysis, revealing a marked enrichment in biological processes related to immune system functions. These results contribute to the understanding of the genetics underlying gastrointestinal parasite resistance and its implications for other productive and welfare traits in animal breeding programs.

In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus.

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

1H-NMR Metabolomic Study of the Mushroom Pleurotus djamor for the Identification of Nematocidal Compounds.

Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (1H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC50 290.8 µg/mL) and basidiomes (EC50 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the 1H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the 1H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC50 of 237.7 µg/mL. The results obtained showed that chemometric analyses of the 1H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.

Revisiting anthelmintic resistance in sheep flocks from São Paulo State, Brazil.

Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.

Crude Extracts of Pleurotus Spp. and the Presence of Their Proteins in the Ovicidal and Larvicidal Activity of Haemonchus contortus.

Mushrooms of the genus Pleurotus have shown nematophagous activity as it produces many chemical compounds and enzymes affecting parasitic nematodes. This study aimed to extract the inhibitory activity of the five strains of the fungus Pleurotus spp. It was evaluated against eggs and larvae of Haemonchus contortus. The extract of P. ostreatus obtained the highest level of inhibition of eggs at 97.6% (1341 µg/mL) followed by P. pulmonarius (EPP) at 81.2% (774 µg/mL). The extract selected for evaluation against larvae was P. pulmonarius, showing no effect for L3 larvae, but for L4 larvae an immobility effect of 56.93% was observed at 900 µg/mL. The protein profile showed the presence of 23 protein bands in the extract. The crude extract of P. pulmonarius showed degradation of tissues both inside the eggs and larvae L1. Metabolites produced by Pleurotus mushrooms can consider using in agriculture sustainable by utilizing in producing of ovicidal and larvicidal against H. contortus instead of chemical compounds.

In vitro and in vivo anthelmintic response of the seeds of Amomum subulatum roxb and Vitex negundo / Atividade anti-helmíntica in vitro e in vivo das sementes de Amomum subulatum roxb e Vitex negundo

The current study was designed to check the anthelmintic activities of some local plants. Seeds of Amomum (A.) subulatum and Vitex (V.) negundo in different solvents were subjected to in vitro (adult motility assay; AMA and egg hatch assay; EHA) and in vivo (faecal egg count reduction test; FECRT) anthelmintic activity testing protocols using Haemonchus (H.) contortus as an experimental model. The results of AMA, EHA, and FECRT were statistically analysed through linear regression and Duncan multiple range test. In AMA test, at 50 mg mL-1 concentration, the percent mortality of H. contortus was higher in A. subulatum than V. negundo, whereas, in EHA test, A. subulatum was proven better ovicidal (LC50=14.2 µg mL-1) than V. negundo (LC50= 65.7405 µg mL-1). The FECRT also indicated the better efficacy of A. subulatum than V. negundo against natural infection of gastrointestinal (GI) parasites. The crude powder of plants used in this study showed 29.6% to 57.7% anthelmintic. The reduction rate was found higher for A. subulatum (3 g kg-1) as compared to V. negundo (7 g kg-1). Reagrding efficacy analysis of solvents used for plants extract, ethyl acetate and chloroform were found better in increasing ovicidal activity in adult worms (in vitro testing), whereas, the crude aqueous methanol was found better than the crude powders in in vivo testing. It will be beneficial to document the indigenous knowledge to standard scientific procedures for their validation. This study will help to motivate the farmers to make a better choice of cultivation of the indigenous plants because of their varying efficacies as an alternative preventive approach against the GI parasitic infections.

O presente estudo foi desenhado para verificar as propriedades anti-helmínticas de algumas plantas locais. Sementes de Amomum (A.) subulatum e Vitex (V.) negundo em diferentes solventes foram submetidas à análise de atividade anti-helmíntica in vitro (ensaio de motilidade de adultos; AMA e teste de eclosão de ovos; EHA) e in vivo (teste de redução da contagem de ovos nas fezes; TRCOF), usando o Haemonchus (H.) contortus como modelo experimental no protocolo de teste. Os resultados dos testes AMA, EHA e TRCOF foram analisados ​​estatisticamente por meio de regressão linear e teste de Duncan. No teste AMA, na concentração de 50 mg mL-1, o percentual de mortalidade de H. contortus foi maior com o uso de A. subulatum do que com V. negundo, enquanto, no teste EHA, A. subulatum apresentou maior ação ovicida (LC50=14,2 µg mL- 1) do que V. negundo (LC50= 65,7405 µg mL-1). O TRCOF também indicou a melhor eficácia do uso de A. subulatum do que de V. negundo contra a infecção natural de parasitas gastrointestinais (GI). O extrato bruto seco das plantas utilizadas neste estudo apresentou 29,6% a 57,7% de atividade anti-helmíntica. A taxa de redução observada com o uso de A. subulatum (3 g kg-1) foi maior que com o uso de V. negundo (7 g kg-1). Em relação à análise da eficácia dos solventes utilizados para o extrato de plantas, o acetato de etila e o clorofórmio apresentaram maior ação ovicida em vermes adultos (testes in vitro), enquanto o extrato bruto metanólico aquoso apresentou maior eficácia do que os extratos brutos secos em testes in vivo. Consideramos vantajoso documentar o conhecimento indígena relativos aos procedimentos científicos padronizados, para sua validação. Este estudo irá servir de motivação para que os agricultores façam escolhas melhores referentes ao cultivo das plantas indígenas devido às suas diferentes eficácias comprovadas, servindo como alternativa para a abordagem preventiva contra as infecções parasitárias GI.

Validation of targeted selective treatment (TST) methodology for gastrointestinal parasites of adult sheep in different physiological states.

We aimed to validate a targeted selective treatment (TST) methodology for treating parasitic gastrointestinal infections in ewes in different physiological states using parasitological and hematological parameters. Forty ewes were monitored from December 2021 to June 2022 and evaluated during various physiological stages in their life cycle. Before starting the experiment, a fecal egg count (FEC) reduction test was performed to evaluate the efficacy of the anthelmintic (AH) treatment. Weekly assessments were performed based on the Famacha© (F) system and body condition score (BCS), and ewes were subjected to AH treatment when necessary, with their physiological states recorded. Ewes were treated when they presented F ≥ 3, BCS ≤ 2.0 (when F = 2), or submandibular edema. Parasitological, i.e., FEC, and hematological, i.e., hematocrit (Ht), parameters were evaluated monthly to determine the efficiency of the TST methodology. Comparisons between the mean Ht and FEC values in ewes subjected to AH treatment and untreated ewes were performed using analysis of variance, followed by Tukey's test. Spearman's correlation was performed to determine the correlation between the variables, i.e., F scores, BCS, Ht, and FEC. All tests were performed at a significance level of 5 %. During the experimental period, 1138 evaluations were performed. The main reason for AH treatment was F ≥ 3. Ewes in early pregnancy, lactation and late pregnancy received comparatively more AH treatments than the other physiological states. Ewes in late pregnancy and lactation exhibited lower mean Ht values (23.5 % and 22.9 %) and higher mean FEC values (3269 and 1426) compared with those in early pregnancy (30.2 % and 727 EPG). In addition, a statistically significant difference was observed in the Ht and FEC values of ewes that presented submandibular edema (P < 0.001) compared with those that did not exhibit submandibular edema. The genus Haemonchus sp. showed a 96.4 % prevalence in coprocultures. A positive correlation existed between F scores and FEC (r = 0.3819) and a negative correlation between F scores and Ht (r = -0.4728). Ewes that needed AH treatment had lower mean Ht values than ewes that did not need the treatment (19.2 % × 29.3 %; P < 0.001) and higher mean FEC values (8747 × 1163; P < 0.001), confirming that these ewes needed AH treatment. The TST methodology based on F scores, BCS, and submandibular edema could effectively identify individuals in the herd needing AH treatment, identifying 13 % additional cases requiring treatment than using only the F score criterion.

Molecular interaction analysis of the lignans from Piper cubeba in complex with Haemonchus contortus phosphomethyltransferase.

In vitro larvicidal assays carried out previously by our research group with cubebin, dihydrocubebin and hinokinin, lignans extracted from the fruits of Piper cubeba, against Haemonchus contortus larvae showed strong action larvicidal these compounds. Hinokinin was the most active (EC50 = 0.34 µg/mL) with strong action on the cuticle of the larvae as observed by scanning electron microscopy of the L3 stage. Therefore, to understand the mechanism of action of these compounds in silico studies were carried out using the enzyme phosphomethyltransferase of Haemonchus contortus that contain PMT-1 and PMT-2 di-domains responsible for phosphocholine synthesis, which is one of the main lipids in nematodes. This pathway is not found in mammals, so this enzyme is an important biological target for the development of new anthelmintics. Results of molecular docking, molecular dynamic and a density functional theory calculations studies with the three lignans show few interactions with PMT-1. However, hinokinin has important interactions with PMT-2, that can deactivate the enzyme and interrupt the phosphocholine synthesis, which is an essential compound for the development and maintenance of the nematode cuticle and its survive. Therefore, the previous results of the in vitro assay allied with in silico results, now realized; suggest that hinokinin may be a possible selective target for the development of new anthelmintics against Haemonchus contortus since the PMT-2 domain is present in this nematode.

Enhanced mucosal response in sheep harbouring ßA beta-globin against haemonchosis.

Association between ovine ß-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity ßA ➔ ßC switch during anaemia, but there are no studies regarding the involved local host responses. Phenotypic parameters and local responses were evaluated in sheep from two ß-globin haplotypes naturally infected with Haemonchus contortus. Morada Nova lambs were monitored at 63, 84 and 105 days of age for faecal egg counts and packed cell volume (PCV) under natural infection with H. contortus. At 210 days of age, lambs of Hb-AA and Hb-BB ß-globin haplotypes were euthanised, and the fundic region of abomasum was sampled for evaluation of microscopic lesions and relative expression of genes related to immune, mucin and lectin activities. Lambs harbouring the ßA allele presented an improved resistance/resilience against clinical haemonchosis, showing higher PCV during infection. Hb-AA animals presented increased eosinophilia in the abomasum compared to Hb-BB animals, accompanied by higher Th2 profile, mucin and lectin activity transcripts, while the inflammatory response was increased in Hb-BB animals. This is the first report to demonstrate an enhanced local response in the primary site of H. contortus infection related to ßA allele of ß-globin haplotype.

Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. THE AIMS OF OUR STUDY: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. MATERIALS AND METHODS: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). RESULTS: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 µg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 µg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. CONCLUSIONS: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.

Assembly and Analysis of Haemonchus contortus Transcriptome as a Tool for the Knowledge of Ivermectin Resistance Mechanisms.

Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.

Effect of commercial tannins on parasitic infection and immunity of lambs naturally infected with Haemonchus contortus.

This study aimed to evaluate the effects of different doses of quebracho extract tannins (Schinopsis lorenzii) (EQ) included in the basal diet (DB) on the count of eggs per gram of feces (EPG), count of parasites in the abomasum, hematological, and immunological parameters in lambs naturally infected with Haemonchus contortus. Forty-eight lambs were evaluated for 42 days and divided into four groups (n = 12): CONT- lambs that received DB without EQ supplementation; G1%- DB with EQ at 1% of dry matter intake (DMI) level; G3%- DB with EQ at the 3% of DMI level; G6%- DB with EQ at the 6% DMI level. For EPG counts, feces were collected weekly. For counting abomasal parasites, at the end of the experiment (D42) half of the lambs in each group were slaughtered. For hematological and immunological analysis, venous blood samples were collected at D0, D21, and D42 to perform the erythrocyte count, mean cell volume (MCV), hematocrit, differential leukocyte count, total plasma protein dosage, and serum IgA class anti-H. contortus using an indirect enzyme immunoassay (ELISA). In the EPG count, there was no significant difference (p > 0.05) between the groups compared with the control. Analysis of the abomasal parasite count showed Haemonchus contortus in its entirety; however, there was no significant difference (p > 0.05) in the number of males, females, and total parasites. No differences (p > 0.05) were observed in erythrocyte count, MCV, hematocrit, and total plasma protein. The average total leukocyte, monocyte, eosinophil, and basophil counts showed no difference (p > 0.05). However, the averages of segmented neutrophils in the G1% and G3% groups presented a reduction in the percentage of cells (p < 0.05) between D21 and D42. Regarding the lymphocyte count, a difference was observed in the CONT and G3% groups at D21 and D42 (p < 0.05), when compared to D0. The analyses of the average absorbance of anti-Haemonchus contortus serum IgA showed no difference (p > 0.05), when the groups were analyzed over time or when compared between them. It can be concluded that the inclusion of different doses of tannins from EQ did not alter the parasitological, hematological, and immunological parameters of lambs infected with H. contortus.

3'-Demethoxy-6-O-Demethylisoguaiacin and Norisoguaiacin Nematocidal Lignans from Artemisia cina against Haemonchus contortus Infective Larvae.

Artemisia cina is a plant used in traditional Chinese medicine as a remedy for parasitic diseases. This study describes the isolation and chemical characterization of anthelmintic compounds of A. cina against Haemonchus contortus infective larvae (L3) through lethal testing. Previously, three extracts-n-hexane (HexAc), ethyl acetate (EtOAc) and methanol (MeOAc)-were evaluated at concentrations of 4 to 0.5 mg/mL, resulting in the HexAc extract with the greatest effect of 76.6% mortality of the larvae at 4 mg/mL. Then, this was chemically fractioned by polarity, obtaining seven fractions (C1F1-C1F7), and, when evaluated at concentrations from 2 to 0.25 mg/mL, the 2 mg/mL C1F5 fraction produced an effect against the nematode H. contortus of 100% mortality of the larvae. Thus, this fraction was fractionated again by column chromatography, obtaining twelve subfractions (C2F1-C2F12) which were evaluated from 1 to 0.125 mg/mL, with the C2F5 subfraction causing a nematicidal effect of 100% mortality. NMR analysis of one (1H, 13C and DEPT) and two dimensions (COSY, HSQC and HMBC) and mass spectrometry of this fraction allowed us to identify the mixture of 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin. Therefore, it can be assumed that the mixture of these compounds is responsible for the anthelmintic effect. These results indicate that A. cina containing anthelmintic compounds and might be used as an antiparasitic drug against H. contortus.

EVALUATION OF PELIBUEY LAMBS BORN TO MOTHERS PHENOTYPICALLY SEGREGATED ACCORDING TO RESISTANCE TO GASTROINTESTINAL NEMATODES IN THE HUMID TROPICS OF MEXICO.

The objective was to evaluate the effect of the resistance of ewes to gastrointestinal nematodes (GIN) on the parasitologic and productive performance of their lambs during preweaning and postweaning periods. Offspring from 44 Pelibuey ewes classified as resistant or susceptible to GIN were evaluated. During the preweaning period, birth weight (BW), live weight (LW) at 35 and 70 days of age, and live weight changes (LWC) were determined in 73 lambs. During the postweaning period, 20 lambs to susceptible or resistant ewes were evaluated. The lambs were challenged under natural GIN infections and artificial infection with L3 of Haemonchus contortus. The fecal egg count (FEC), hematocrit (HT), peripheral eosinophil count, plasma protein, LW, and LWC were determined. There were no differences among parameters evaluated during preweaning (P > 0.05). The type of lambing (single or multiple) affected the BW, LW, and LWC (P < 0.01). The sex (male) of the lamb had a positive effect on BW at 35 and 70 days of age (P < 0.05). After weaning, lambs of susceptible ewes showed higher FEC than resistant ewes (P < 0.05). The HT of lambs to resistant ewes was higher than those the susceptible group (P < 0.01). In conclusion, offspring of resistant ewes show low FEC compared with those susceptible during the postweaning stage.

Biological activity of cinnamaldehyde, citronellal, geraniol and anacardic acid on Haemonchus contortus isolates susceptible and resistant to synthetic anthelmintics / Atividade biológica dos compostos cinamaldeído, citronelal, geraniol e ácido anacárdico em isolados de Haemonchus contortus susceptíveis e resistentes a anti-helmínticos sintéticos

Parasitism by gastrointestinal nematodes is a challenge for small ruminant farming worldwide. It causes productive and economic losses, especially due to parasite resistance to conventional anthelmintics. Natural compounds with antiparasitic activity are a potential alternative for controlling these parasites especially when considering the widespread occurrence of anthelmintic resistance. Our objective was to evaluate the activity of anacardic acid, geraniol, cinnamaldehyde and citronellal on Haemonchus contortus isolates with different levels of anthelmintic resistance profiles. These compounds were tested using egg hatch assays (EHAs), larval development tests (LDTs) as well as LDTs on mini-fecal cultures, on the Haemonchus contortus isolates Kokstad (KOK-resistant to all anthelmintics), Inbred-Strain-Edinburgh (ISE-susceptible to all anthelmintics) and Echevarria (ECH-susceptible to all anthelmintics). Effective concentrations to inhibit 50% (EC50) and 95% (EC95) of egg hatching and larval development were calculated. Results for EHA and LDT for all tested compounds, considering EC50 and EC95 values, showed low variation among the studied isolates with most RF values below 2x. All studied compounds showed efficacy against egg hatching and larval development of H. contortus isolates regardless of anthelmintic resistance profiles. The compounds with the smallest EC50 and EC95 values were cinnamaldehyde and anacardic acid making them promising candidates for future in vivo studies.(AU)

A infecção por nematoides gastrintestinais é um dos principais desafios na produção de pequenos ruminantes e ocasiona perdas produtivas, principalmente, devido à resistência anti-helmíntica. Bioativos com atividade anti-helmíntica são potencial alternativa para o controle desses parasitos em especial, considerando-se a ampla incidência de resistência anti-helmíntica nos rebanhos. O objetivo deste estudo foi avaliar a atividade biológica do ácido anacárdico, geraniol, cinamaldeído e citronelal em isolados de Haemonchus contortus com diferentes perfis de resistência anti-helmíntica. Foram realizados testes de eclosão de ovos (TEO), testes de desenvolvimento larvar (TDL) e TDLs em minicoproculturas, utilizando-se o isolado Kokstad (resistente a todos os anti-helmínticos), o isolado Inbred-Strain-Edinburgh (suscetível) e o isolado Echevarria (suscetível). Foram calculadas as concentrações efetivas para inibir 50% (CE50) e 95% (CE95) da eclodibilidade dos ovos e do desenvolvimento larvar. Resultados de TEO e TDL apresentaram baixa variação entre os diferentes isolados para um mesmo composto testado com fatores de resistência geralmente abaixo de 2x. Todos os compostos estudados mostraram eficácia contra a eclosão de ovos e desenvolvimento larvar de isolados de H. contortus independente do perfil de resistência anti-helmíntica dos mesmos. Os compostos que apresentaram atividade nas menores concentrações foram cinamaldeído e ácido anacárdico, sendo estes os componentes mais promissores para futuros estudos in vivo.(AU)

Castor bean cake for the control of parasites in pasture-finished sheep.

The purpose of this study was to evaluate castor bean cake as alternative input in the control of gastrointestinal parasites in sheep raised on irrigated pasture under continuous stocking. The treatments consisted of sheep supplemented with standard diet and pasture fertilized with urea; sheep supplemented with alternative diet and pasture fertilized with urea; sheep supplemented with standard diet and pasture fertilized with in natura castor bean cake; and sheep supplemented with alternative diet and pasture fertilized with in natura castor bean cake. A randomized complete block design (CBD) was used, with 16 replications (sheep), with repeated measures over time, the plots being the treatments, and the subplots the collection times. Infective nematode larvae in the pasture (L3.g DM-1), number of eggs per gram of feces (EPG), globular volume (GV), and total plasma protein (TPP) were evaluated. For adult gastrointestinal parasite counts, CBD was also used with six replications. Except for FAMACHA© grade, all variables had effect (P < 0.01) of the time factor. The average number of L3.g DM-1 and EPG were 126 and 841, respectively, with no effect (P > 0.05) of the treatment factor. The values observed for GV and TPP were higher than 25.9% and 6.4 g·dL-1, respectively, which were considered normal. As organic fertilizer, the fractionated application of in natura castor bean cake does not reduce the contamination of pastures by nematode larvae. The evaluated feeds improve the resilience of the sheep to infection by gastrointestinal parasites. The treatments using castor bean cake reduced the adult parasites present in the abomasum of sheep.

Bio-directed Chemical Study of Pleurotus ostreatus Spent Substrate and Its Nematicidal Activity.

PURPOSE: In the present study, the nematicidal activity of the chemical fractionation of the spent substrate of the edible mushroom Pleurotus ostreatus against eggs and L3 larvae of Haemonchus contortus was evaluated. METHODS: The hydroalcoholic extract of the spent substrate was subjected to a bipartition with ethyl acetate giving two fractions: one aqueous (F. Ac) and one organic (F. AcOET). Both fractions were evaluated against eggs and L3 larvae at different concentrations (5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and 2% methanol, PBS and thiabendazole (5 mg/mL) as controls. Chemical fractionation of F. AcOET was performed in open column chromatography where 76 fractions were obtained and when analyzed by thin layer chromatography (TLC) were grouped into 11 mixtures (R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11). These mixtures were evaluated at 10 mg/mL against eggs and L3 larvae. The data obtained were analyzed by ANOVA and a Tukey test in the SAS V9 program. RESULTS: The results showed that, in the evaluation of F. AcOET and F. Ac, for the inhibition of egg hatching, the highest percentages were 78.80 and 76.89% at 5 mg/mL, respectively. As for the percentage of larval mortality, F. AcOET obtained 60.91 and F. Ac 29.77% at 5 mg/mL. The results of the evaluations of the mixtures showed that mixtures R4, R5 obtained 100 and 95.41% larval mortality and mixtures R6 and R7 presented 100% inhibitory activity of egg hatching, so these were analyzed by gas chromatography mass spectrometry finding compounds such as vanillin, ß-sitosterol, ρ-methyl ρ-hydroxycinnamate and ρ-hydroxybenzaldehyde. CONCLUSION: The results of the present study demonstrate that the spent substrate of P. ostreatus has potential anthelmintic activity against H. contortus. Moreover, by reusing and taking advantage of this substrate, its environmental pollution effects can be reduced.

Improvement of cell suspension cultures of transformed and untransformed Carica papaya cell lines, towards the development of an antiparasitic product against the gastrointestinal nematode Haemonchus contortus.

Parasitic diseases have a major impact on human and animal health worldwide. Despite the availability of effective anti-parasitic drugs, their excessive and uncontrolled use has promoted the emergence of drug resistance, severely affecting ecosystems and human health. Thus, developing environmentally friendly antiparasitic treatments is urgently needed. Carica papaya has shown promising effects against infectious diseases. C. papaya embryogenic calluses were genetically modified by our research team to insert immunogenic peptides with the goal of developing an oral anti-cysticercosis vaccine. Among these callus cell lines, one labeled as CF-23, which expresses the KETc7 immunogenic peptide, induced the highest protection levels against experimental cysticercosis. In the process of designing a natural antiparasitic product based on C. papaya that simultaneously induced immunity against cysticercosis, both transformed (SF-23) and untransformed (SF-WT) suspension cultures were produced and optimized. Our results showed a better duplication time (td) for SF-23 (6.9 days) than SF-WT (13.02 days); thus, the SF-23 line was selected for scale-up in a 2-L airlift bioreactor, reaching a td of 4.4 days. This is the first time that a transgenic line of C. papaya has been grown in an airlift bioreactor, highlighting its potential for scale-up cultivation in this type of reactor. Considering the previously reported nematocidal activity of C. papaya tissues, their activity against the nematode Haemonchus contortus of aqueous extracts of SF-WT and SF-23 was explored in this study, with promising results. The information herein reported will allow us to continue the cultivation of the transgenic cell suspension line of C. papaya under reproducible conditions, to develop a new anti-parasitic product.

Population replacement of benzimidazole-resistant Haemonchus contortus with susceptible strains: evidence of changes in the resistance status.

The spread of anthelmintic resistance (AR) in nematode populations threatens the viability of sheep production systems worldwide, and warrants the adoption of sensitive, practical, and standardized tests to detect AR. The aim of this study was to characterize the replacement of an Haemonchus contortus population resistant to benzimidazoles (BZDs) by a susceptible one, by means of both phenotypic and genotypic techniques. Phenotypic methods to assess BZD resistance included in vivo tests, such as the fecal egg count reduction test (FECRT), and in vitro tests, such as the egg hatch assay (EHA). Additionally, genotypification of polymorphisms associated with BZD resistance by sequencing a fragment of the isotype 1 ß-tubulin gene was carried out. The initial, BZD-resistant population (initial Balcarce population) exhibited an egg count reduction (ECR) of 59.3%. Following refugium replacement, the final population (final Balcarce population) exhibited an ECR of 95.2%. For the initial Balcarce population, the median effective dose (ED50) for the EHA was 0.607 µg thiabendazole (TBZ)/mL, with a rate of eclosion at a discriminating dose (EDD) of 0.1 µg TBZ/mL of 76.73%. For the final Balcarce population, ED50 was 0.02 µg TBZ/mL, and EDD was 1.97%. In the initial population, 93% of the analyzed individuals exhibited genotypic combinations associated with BZD resistance (53% Phe/Phe167-Tyr/Tyr200, 37% Phe/Tyr167-Phe/Tyr200, and 3% Phe/Tyr167-Glu/Leu198). Conversely, no combination associated with resistance was found in individuals from the final population. All of the tests were useful for detecting AR to BZDs. The results from the genetic and phenotypical studies were consistent, and the resulting information greatly aided in interpreting the outcomes of the population replacement and the potential impact of this strategy on management of AR.

Investigating the benefits of targeted selective treatment according to average daily weight gain against gastrointestinal nematodes in Morada Nova lambs.

The objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, based on the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: control (CT), routine (RT, treated every 42 days), and targeted selective (TST, treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. The anthelmintic resistance of parasites was assessed by the EPG count reduction test (FECRT), RESISTA-Test©, and molecular test. For CT, RT, and TST groups, the following results were obtained: mean EPG values were 4665.1, 3063.5, and 3462.1 in the rainy season and 4475.1, 1341.7, and 2863.4 in the dry season, respectively; mean PCV values were 32.1, 33.4, and 32.3% in the rainy season and 33.9, 36.0, and 35.1% in the dry season; mean DWG readings were 0.087, 0.101, and 0.094 kg in the rainy season and 0.102, 0.113, and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1, and 76.4% in the rainy and 90.7, 12.4, and 64.8% in the dry season, respectively; in the RESISTA-Test©, the LC50 values were 0.482, 1.926, and 0.117 µg.mL-1 in the rainy and 0.437, 0.851, and 0.045 µg.mL-1 in the dry season, respectively; the frequencies of the homozygous-resistant genotype were 57.1, 71.4, and 40.0% in the rainy and 47.8, 55.9, and 41.9% in the dry season. In conclusion, TST reduced the development of resistance through refugia maintenance, without productive losses, contributing to the sustainability of sheep breeding.