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1.
Exp Parasitol ; 218: 107980, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32877640

RESUMO

Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one 'Katahdin' crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3-68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.


Assuntos
Acacia/química , Anti-Helmínticos/uso terapêutico , Hemoncose/veterinária , Extratos Vegetais/uso terapêutico , Doenças dos Ovinos/tratamento farmacológico , Animais , Anti-Helmínticos/isolamento & purificação , Anti-Helmínticos/farmacologia , Fezes/parasitologia , Feminino , Flavonoides/química , Ácido Gálico/química , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Haemonchus/efeitos dos fármacos , Haemonchus/isolamento & purificação , Hematócrito/veterinária , Contagem de Ovos de Parasitas/veterinária , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Distribuição Aleatória , Ovinos , Doenças dos Ovinos/parasitologia
2.
Exp Parasitol ; 217: 107957, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32687847

RESUMO

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.


Assuntos
Anti-Helmínticos/farmacologia , Haemonchus/genética , Taxa de Mutação , Tiabendazol/farmacologia , Tubulina (Proteína)/genética , Animais , DNA de Helmintos/isolamento & purificação , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Frequência do Gene , Marcadores Genéticos , Genótipo , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/classificação , Haemonchus/efeitos dos fármacos , Dose Letal Mediana , Óvulo/efeitos dos fármacos , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/parasitologia
3.
PLoS One ; 15(6): e0235072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574178

RESUMO

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.


Assuntos
Abomaso/efeitos dos fármacos , Sistema Digestório/efeitos dos fármacos , Hemoncose/tratamento farmacológico , Haemonchus/efeitos dos fármacos , Preparações de Plantas/farmacologia , Plantas Medicinais/química , Doenças dos Ovinos/tratamento farmacológico , Abomaso/microbiologia , Abomaso/parasitologia , Animais , Anti-Helmínticos/farmacologia , Sistema Digestório/microbiologia , Sistema Digestório/parasitologia , Quimioterapia Combinada , Fezes/microbiologia , Fezes/parasitologia , Feminino , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/fisiologia , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Fitoterapia/métodos , Plantas Medicinais/classificação , Ovinos , Doenças dos Ovinos/parasitologia , Especificidade da Espécie
4.
Parasit Vectors ; 13(1): 154, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228657

RESUMO

BACKGROUND: Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet. RESULTS: An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-ß1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. CONCLUSIONS: Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.


Assuntos
Cabras/parasitologia , Haemonchus/enzimologia , Haemonchus/genética , Leucócitos Mononucleares/imunologia , Metiltransferases/genética , Metiltransferases/imunologia , Metiltransferases/isolamento & purificação , Animais , Anticorpos Anti-Helmínticos/sangue , Proliferação de Células , Clonagem Molecular , Citocinas/metabolismo , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Regulação da Expressão Gênica , Hemoncose/parasitologia , Hemoncose/veterinária , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/imunologia , Masculino , Metiltransferases/metabolismo , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência de Proteína
5.
Parasit Vectors ; 13(1): 151, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32204731

RESUMO

BACKGROUND: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. METHODS: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. RESULTS: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. CONCLUSIONS: Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.


Assuntos
Hemoncose/parasitologia , Haemonchus/enzimologia , Haemonchus/genética , Metaloproteinase 12 da Matriz/genética , RNA Interferente Pequeno , Animais , Proliferação de Células , China , Feminino , Técnicas de Silenciamento de Genes , Cabras/parasitologia , Haemonchus/patogenicidade , Proteínas de Helminto/genética , Leucócitos Mononucleares , Masculino , Óxido Nítrico/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Análise de Sequência
6.
Parasite Immunol ; 42(6): e12707, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32118305

RESUMO

AIMS: Most breeds of goat are more susceptible to nematode infection than sheep, and this appears to be a consequence of less effective immune responses. Several papers have considered the effectiveness of eosinophils and immunoglobulin A (IgA) in goats but differences in the induction of responses have not been studied in the same detail. The aim of this study was to look at the induction of eosinophil and IgA responses in Boer goats reared indoors under intensive conditions. METHODS AND RESULTS: The goats were experimentally infected with a low dose of 2400 Haemonchus contortus, Trichostrongylus spp. and Oesophagostomum spp. at a 6:1:1 ratio. Faecal egg counts (FEC), packed cell volume (PCV), IgA activity against third-stage larvae and peripheral eosinophilia were measured twice a week for eight weeks. The infection generated an IgA response but did not significantly increase peripheral eosinophilia in the 25 infected kids compared with the 4 control animals. FEC was not associated with IgA activity or eosinophilia. CONCLUSION: A detailed analysis of IgA and eosinophil responses to deliberate nematode infection in Boer goats showed that there was an increase in nematode-specific IgA activity but no detectable eosinophil response. In addition, there was no association between increased IgA activity or eosinophilia with egg counts and worm burdens. These suggest that IgA and eosinophils do not act to control nematode infection in goats.


Assuntos
Eosinofilia/imunologia , Doenças das Cabras/parasitologia , Cabras/imunologia , Hemoncose/veterinária , Haemonchus/imunologia , Imunoglobulina A/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Eosinofilia/parasitologia , Eosinófilos/imunologia , Fezes/parasitologia , Feminino , Doenças das Cabras/imunologia , Cabras/parasitologia , Hemoncose/imunologia , Hemoncose/parasitologia , Hematócrito/veterinária , Contagem de Leucócitos , Masculino , Oesophagostomum/imunologia , Contagem de Ovos de Parasitas/veterinária , Trichostrongylus/imunologia
7.
Vet Parasitol ; 279: 109060, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32143013

RESUMO

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.


Assuntos
Criação de Animais Domésticos , Hemoncose/veterinária , Haemonchus/fisiologia , Doenças dos Ovinos/prevenção & controle , Animais , Brasil , Fazendas , Fezes , Pradaria , Hemoncose/parasitologia , Hemoncose/prevenção & controle , Haemonchus/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/fisiologia , Estações do Ano , Ovinos , Doenças dos Ovinos/parasitologia , Fatores de Tempo
8.
Vet Parasitol ; 278: 109040, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32007679

RESUMO

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants around the world, seriously hampering the healthy development of the sheep industry. The control of this parasite mainly depends on anthelmintics, however, drug resistance of H. contortus has become a serious problems worldwide. Previous studies demonstrated that the E198A (GAA to GCA), a single nucleotide polymorphism (SNP) in the isotype-1 ß-tubulin gene is associated with benzimidazole resistance in H. contortus. However, only PCR-RFLP and ARMS-PCR methods have been previously used for the detection of the E198A mutation. In the present study, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of the E198A SNP in H. contortus. The results showed that optimization of LAMP reaction reagents and conditions could achieve this. The resulting amplicons were visualized by adding hydroxynaphthol blue dye (HNB) prior to amplification. The color of LAMP products amplified without DNA or from DNA from worms with the E198A homozygous susceptible genotype was still violet, but the products with DNA from worms with the E198A heterozygous genotype or the E198A resistant homozygous genotype changed to sky blue. The specificity of this method was further verified by sequencing, which confirmed the successful LAMP detection of the E198A mutation with high specificity. In conclusion, the developed LAMP method has high specificity and good reproducibility for screening the E198A SNP of isotype-1 ß-tubulin gene of H. contortus of field samples without using sophisticated equipment, providing useful technique for the rapid detection and thus prevention and control of benzimidazole resistant H. contortus infections.


Assuntos
Hemoncose/veterinária , Haemonchus/isolamento & purificação , Proteínas de Helminto/análise , Técnicas de Amplificação de Ácido Nucleico/veterinária , Polimorfismo de Nucleotídeo Único , Doenças dos Ovinos/parasitologia , Tubulina (Proteína)/análise , Animais , China , Genes de Helmintos , Hemoncose/parasitologia , Masculino , Técnicas de Amplificação de Ácido Nucleico/métodos , Ovinos
9.
Vet Res ; 51(1): 9, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024546

RESUMO

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.


Assuntos
Variação Genética/imunologia , Hemoncose/veterinária , Doenças dos Ovinos/prevenção & controle , Vacinação/veterinária , Vacinas/imunologia , Animais , Austrália , Hemoncose/parasitologia , Hemoncose/prevenção & controle , Haemonchus/imunologia , Plasma/imunologia , Estudos Retrospectivos , Soro/imunologia , Ovinos , Doenças dos Ovinos/parasitologia , Vacinas/administração & dosagem
10.
Vet Parasitol ; 279: 109045, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32045836

RESUMO

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.


Assuntos
Doenças das Cabras/prevenção & controle , Hemoncose/veterinária , Haemonchus/imunologia , Proteínas de Helminto/uso terapêutico , Imunização Passiva/veterinária , Vacinação/veterinária , Vacinas/imunologia , Animais , Feminino , Doenças das Cabras/parasitologia , Cabras , Hemoncose/parasitologia , Hemoncose/prevenção & controle , Masculino , Proteínas Recombinantes/imunologia , Vacinas Sintéticas
11.
Vet Parasitol ; 279: 109054, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32065932

RESUMO

This study aimed to evaluate the dynamics of Haemonchus contortus and Haemonchus placei infections and hybridization between these species in grazing sheep without contact with cattle. On January 14, 2014, sixteen young sheep were infected with 4000 infective H. placei third-stage larvae L3; 11 days later, another group n = 16 was infected with 4000 H. contortus L3. The establishment rates of H. contortus and H. placei L3 were, on average, 61.6 % and 56.8 %, respectively, in the permanent sheep. After the establishment of patent infections, all permanent sheep were allocated together in the same clean pasture where they grazed for the next 12 months. Euthanasia of a sample of the permanent sheep was performed every three months: in May, August, November and February. Two weeks before the sheep were removed for euthanasia, 2 worm-free tracer sheep were introduced to the pasture to evaluate the larval population in the field. The tracer sheep grazed alongside the permanent sheep for 2 weeks. Then, they were housed indoors for 20 days; at the end of this period, they were euthanized. Parasites were recovered from the permanent and tracer sheep and identified using morphological and molecular techniques. A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using species-specific primer pairs. Of these specimens, only two (0.46 %) male worms were identified as hybrids: one was recovered from a permanent animal euthanized in August and the other from a tracer sheep that grazed in May. The last detection of adult H. placei worms occurred in sheep euthanized in May (approximately 3.5 months after the beginning of the grazing period). The morphological evaluation of the L3 produced in fecal cultures showed that H. placei were progressively replaced by H. contortus populations starting in March. The last trace of H. placei L3 was found in August, when a small percentage (0.5 %) of infective larvae with H. placei morphology was identified in a fecal culture. In conclusion, hybridization between H. contortus and H. placei can occur in the field during coinfection. It was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, with concomitant natural reinfection with H. contortus, the H. placei population showed a rapid decrease and was eliminated within a few months in an environment without cattle.


Assuntos
Coinfecção/veterinária , Hemoncose/veterinária , Haemonchus/fisiologia , Doenças dos Ovinos/epidemiologia , Animais , Coinfecção/epidemiologia , Coinfecção/parasitologia , Fazendas , Feminino , Hemoncose/epidemiologia , Hemoncose/parasitologia , Haemonchus/genética , Haemonchus/crescimento & desenvolvimento , Herbivoria , Hibridização Genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Ovinos , Doenças dos Ovinos/parasitologia , Especificidade da Espécie
12.
Vet Parasitol ; 279: 109053, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32109653

RESUMO

Gastrointestinal nematodes are a major constraint in sheep production. Breeding for resistance has proven to be an effective and feasible approach to address this problem. The use and investigation of genetic markers for resistance traits could accelerate genetic progress and lead to a better understanding of underlying molecular mechanisms. Thus, the aim of this study was to evaluate if five single nucleotide polymorphisms SNPs OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 and OAR15_59871543 are associated with resistance and resilience traits in a flock of the Morada Nova sheep breed. Lambs were submitted to two consecutive parasite challenges by oral infection with 4000 infective larvae L3) of Haemonchus contortus. Fecal egg counts (FEC), packed cell volume (PVC) and body weight were measured every one or two weeks for 42 days in each trial. DNA samples from 287 lambs, 131 ewes and 4 rams were amplified by ARMS-PCR or PCR-RFLP and genotypes were determined. Analysis of variance (ANOVA) was used for association analyses between genotypes and phenotypes. In case of significant association, the allele substitution effect was calculated based on a linear model. OAR2_14765360 and OAR12_69606944 were associated with FEC, and OAR12_69606944 also had significant effects on PCV and weight gain, showing favourable associations of the CC genotype with all evaluated traits. Both OAR6_81718546 and OAR11_62887032 were associated with weight gain, and OAR6_81718546 had an additional effect on PCV. OAR15_59871543 was not polymorphic in the population. OAR6_81718546 and OAR12_69606944 presented significant allele substitution effects of -1.06 ±â€¯0.52 kg for the T allele on final body weight and 0.74 ±â€¯0.32 for the C allele in PCV of the same sampling date, respectively. This is the first report of SNPs associated with gastrointestinal nematode resistance in this sheep breed. Our findings support the existence of quantitatice trait loci (QTL) for resistance and resilience in linkage disequilibrium with the polymorphic SNPs and suggest their future use for explorations of these traits in Morada Nova sheep.


Assuntos
Resistência à Doença/genética , Hemoncose/veterinária , Haemonchus/genética , Polimorfismo de Nucleotídeo Único , Doenças dos Ovinos/genética , Animais , Brasil , Hemoncose/genética , Hemoncose/parasitologia , Ovinos , Doenças dos Ovinos/parasitologia
13.
Parasite Immunol ; 42(5): e12703, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32043596

RESUMO

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF-1α) was cloned and expressed. The influences of recombinant HcEF-1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF-1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF-1α was bound on surface of PBMCs. Moreover, the productions of IL-4, TGF-ß1, IFN-γ and IL-17 of cells were significantly modulated by the incubation with rHcEF-1α. The production of interleukin IL-10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF-1α was increased significantly, whereas MHC-I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF-1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host-parasite relationship.


Assuntos
Doenças das Cabras/parasitologia , Hemoncose/veterinária , Haemonchus/imunologia , Proteínas de Helminto/imunologia , Leucócitos Mononucleares/imunologia , Fator 1 de Elongação de Peptídeos/imunologia , Animais , Apoptose , Movimento Celular , Proliferação de Células , Doenças das Cabras/genética , Doenças das Cabras/imunologia , Doenças das Cabras/fisiopatologia , Cabras , Hemoncose/imunologia , Hemoncose/parasitologia , Hemoncose/fisiopatologia , Haemonchus/genética , Proteínas de Helminto/genética , Interleucina-17/genética , Interleucina-17/imunologia , Óxido Nítrico/imunologia , Fator 1 de Elongação de Peptídeos/genética
14.
Vet Parasitol ; 277: 109020, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31896019

RESUMO

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per µl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.


Assuntos
Criação de Animais Domésticos/métodos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/parasitologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Hemoncose/veterinária , Haemonchus/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Bovinos , Hemoncose/diagnóstico , Hemoncose/parasitologia , Compostos Orgânicos/análise , Especificidade da Espécie , Turquia
15.
Anim Genet ; 51(2): 330-335, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31900974

RESUMO

Resistance to gastrointestinal nematodes has previously been shown to be a moderately heritable trait in some breeds of sheep, but the mechanisms of resistance are not well understood. Selection for resistance currently relies upon faecal egg counts (FEC), blood packed cell volumes and FAMACHA visual indicator scores of anaemia. Identifying genomic markers associated with disease resistance would potentially improve the selection process and provide a more reliable means of classifying and understanding the biology behind resistant and susceptible sheep. A GWAS was conducted to identify possible genetic loci associated with resistance to Haemonchus contortus in Katahdin sheep. Forty animals were selected from the top and bottom 10% of estimated breeding values for FEC from a total pool of 641 sires and ram lambs. Samples were genotyped using Applied Biosystems™ Axiom™ Ovine Genotyping Array (50K) consisting of 51 572 SNPs. Following quality control, 46 268 SNPs were included in subsequent analyses. Analyses were conducted using a linear regression model in plink v1.90 and a single-locus mixed model in snp and variation suite. Genome-wide significance was determined by a Bonferroni correction for multiple testing. Using linear regression, loci on chromosomes 2, 3, 16, 23 and 24 were significantly associated at the genome level with FEC estimated breeding values, and we identified a region on chromosome 2 that was significant using both statistical analyses. We suggest a potential role for the gene DIS3L2 for gastrointestinal nematode resistance in Katahdin sheep, although further research is needed to validate these findings.


Assuntos
Resistência à Doença/genética , Gastroenteropatias/veterinária , Loci Gênicos , Estudo de Associação Genômica Ampla/veterinária , Hemoncose/veterinária , Doenças dos Ovinos/genética , Animais , Gastroenteropatias/genética , Gastroenteropatias/parasitologia , Hemoncose/genética , Hemoncose/parasitologia , Ovinos , Doenças dos Ovinos/parasitologia
16.
Parasite Immunol ; 42(1): e12679, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31630404

RESUMO

Host protective immunity to Haemonchus contortus (Hc) infection in parasite-resistant St. Croix (STC) sheep is initiated early and characterized by an influx of innate cells and robust interleukin-4 (IL-4) production, resulting in T-helper type 2 immune (Th2) responses. The purpose of these studies was to elucidate the source of early IL-4 production. Neutrophils were isolated from whole blood, and populations >98% purity were cultured with larval or adult antigen to access cytokine production. Interleukin-4 and IL-13 were measured in sample supernatant using an ovine-specific enzyme-linked immunosorbent assay (ELISA). Neutrophils exposed to HcLA peaked in IL-4 production at 30 minutes (STC, 3153.65 pg/mL and SUF, 4665.22 pg/mL). A similar trend was observed in IL-13 production by 6 hours (STC, 391.02 pg/mL and SUF, 419.6 pg/mL). Adult antigen stimulation resulted in low cytokine production when compared to HcLA stimulation (STC IL-4, 6.04 pg/mL and SUF, 8.05 pg/mL, respectively; STC IL-13, 10 pg/mL and 12.5 pg/mL; P < .001), and no breed differences were observed. Mixed immune cell assays revealed an ability of neutrophils to induce IL-4 production in peripheral blood mononuclear cell (PBMC). Taken together, these data implicate neutrophils as a potential effector cell responsible for Th2 initiation.


Assuntos
Antígenos de Helmintos/imunologia , Hemoncose/veterinária , Interleucina-13/imunologia , Interleucina-4/imunologia , Neutrófilos/imunologia , Doenças dos Ovinos/imunologia , Animais , Cruzamento , Ensaio de Imunoadsorção Enzimática , Hemoncose/imunologia , Hemoncose/parasitologia , Haemonchus/crescimento & desenvolvimento , Haemonchus/imunologia , Interleucina-13/biossíntese , Interleucina-4/biossíntese , Larva/imunologia , Leucócitos Mononucleares/imunologia , Ovinos , Doenças dos Ovinos/parasitologia , Carneiro Doméstico , Células Th2/imunologia
17.
J Immunol Res ; 2019: 3562672, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815153

RESUMO

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H. contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L3. 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L3, and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1ß expression levels (P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed (P < 0.05). Higher levels (P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels (P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H. contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response.


Assuntos
Aminopeptidases/genética , Resistência à Doença/genética , Hemoncose/imunologia , Imunidade Inata , Doenças dos Ovinos/imunologia , Receptor 2 Toll-Like/genética , Aminopeptidases/imunologia , Animais , Fezes/parasitologia , Feminino , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Expressão Gênica , Hemoncose/genética , Hemoncose/parasitologia , Hemoncose/patologia , Haemonchus/imunologia , Haemonchus/patogenicidade , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-33/genética , Interleucina-33/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Ovos de Parasitas , Fenótipo , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/patologia , Células Th2/imunologia , Células Th2/parasitologia , Células Th2/patologia , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
18.
Vet Res ; 50(1): 104, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783921

RESUMO

Understanding the immunological basis of resistance to gastrointestinal nematode infections in livestock is important in order to develop novel methods of parasite control such as vaccination or genetic selection for parasite resistance. The present study aimed to investigate differences in immune response between parasite resistant Santa Ines and susceptible Ile de France sheep breeds to natural Haemonchus contortus infection. Parasitological parameters, humoral immunity, local and circulating cellular immune responses were evaluated in 19 Santa Ines and 19 Ile de France lambs undergoing different anthelmintic treatments regimens: suppressive treatments (SUP) or targeted selective treatments (TST) over a 5-month grazing period. Santa Ines lambs had significantly lower Haemonchus faecal egg count and worm burden compared to Ile de France regardless of treatment regime. In addition, circulating blood eosinophils count and parasite-specific IgG levels were significantly higher and more rapidly induced in Santa Ines lambs. Abomasal immune responses were generally greater in the resistant breed, which had significantly higher levels of parasite-specific IgA in mucus, and elevated number of globule leukocytes and CD3+ T cells within the abomasal mucosal. Furthermore, numbers of POU2F3+ epithelial cells, a tuft-cell specific transcription factor, were also elevated in the Santa Ines breed, suggesting that this breed is better able to initiate T-helper type 2 immune responses within the abomasum. In conclusion, the differential immunological responses detailed here are relevant to understanding resistance to gastrointestinal nematodes in other host breeds, as well as to resistance breeding as a sustainable control approach for parasitic infections.


Assuntos
Imunidade Adaptativa , Suscetibilidade a Doenças/veterinária , Gastroenteropatias/veterinária , Hemoncose/veterinária , Haemonchus/fisiologia , Imunidade Inata , Doenças dos Ovinos/imunologia , Animais , Brasil , Gastroenteropatias/imunologia , Gastroenteropatias/parasitologia , Hemoncose/imunologia , Hemoncose/parasitologia , Imunidade Celular , Imunidade Humoral , Ovinos , Doenças dos Ovinos/parasitologia , Carneiro Doméstico
19.
Vet Parasitol ; 276: 108973, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785456

RESUMO

The objective of this study was to measure the effect of the nutritional status of Creole goat kids on the host responses and the nematode population established after an experimental H. contortus infection. Fifty six kids were fed with 4 diets corresponding to 2 nutritional statuses: the low nutritional status (HAY, hay ad libitum and HB, Hay ad libitum + banana) and the high nutritional status (HS, hay ad libitum + soya meal and HSB, hay ad libitum + banana + soya meal). For each diet, 8 kids were experimentally infected with 10,000 H. contortus infective larvae (L3) and 6 kids were kept as non-infected controls. From the day of infection until 6 weeks post-infection, samples were collected to measure individual intake, total tract digestibility, parasitological and hematological parameters. The dry matter intake (DMI), the average daily gain (ADG), the crude protein (CP) and the digestible CP intake were higher in goats fed the HS and HSB diets, but no statistically significant interaction between the nutritional status and the infection was observed. The packed cell volume (PCV), the red blood cell counts (RBC) and the mean corpuscular volume (MCV) were higher with the HS and the HSB diets. In kids with the high nutritional status the nematode burden and pathophysiological impact of the infection were significantly lower but not the FEC. In conclusion, this reduced establishment rate was associated with an increased production of eggs by the female parasites and suggested a phenomenon of density-dependent prolificacy of H. contortus probably inherent to the fitness of the parasite population.


Assuntos
Doenças das Cabras/parasitologia , Hemoncose/veterinária , Haemonchus/crescimento & desenvolvimento , Estado Nutricional/fisiologia , Anemia/prevenção & controle , Anemia/veterinária , Ração Animal/análise , Ração Animal/normas , Animais , Dieta/veterinária , Digestão , Ingestão de Alimentos , Eosinófilos/citologia , Fezes/química , Fezes/parasitologia , Feminino , Doenças das Cabras/fisiopatologia , Doenças das Cabras/prevenção & controle , Cabras , Hemoncose/parasitologia , Hemoncose/fisiopatologia , Contagem de Leucócitos/veterinária , Masculino , Contagem de Ovos de Parasitas/veterinária , Pepsinogênio A/sangue
20.
Rev Bras Parasitol Vet ; 28(4): 644-651, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800886

RESUMO

This study aimed to evaluate the anthelmintic activity of Eucalyptus citriodora essential oil and citronellal on sheep gastrointestinal nematodes. Essential oil composition was determined by gas chromatography mass spectrometry. The substances were evaluated in vitro using adult worm motility test (AWMT) and transmission electron microscopy (TEM). The acute toxicity test in mice and the fecal egg count reduction test (FECRT) in sheep were performed. Citronellal was confirmed as the essential oil major constituent (63.9%). According to the AWMT, 2 mg/mL of essential oil and citronellal completely inhibited Haemonchus contortus motility at 6 h post exposure. H. contortus exposed to essential oil and citronellal exhibited internal ultrastructural modifications. The lethal dose 50 values in mice were 5,000 and 2,609 mg/kg for essential oil and citronellal, respectively. E. citriodora essential oil reduced sheep epg at 14 days post treatment by 69.5% (P<0.05). No significant differences were observed in epg between the citronellal and negative control groups (P>0.05). The interaction between citronellal and other constituents in the essential oil may be relevant for its in vivo anthelmintic activity. Thus, E. citriodora essential oil and citronellal pharmacokinetic studies may help elucidate the anthelmintic activity of these compounds.


Assuntos
Anti-Helmínticos/farmacologia , Eucalyptus/química , Hemoncose/veterinária , Haemonchus/efeitos dos fármacos , Óleos Voláteis/farmacologia , Doenças dos Ovinos/parasitologia , Ovinos/parasitologia , Animais , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hemoncose/parasitologia , Haemonchus/isolamento & purificação , Haemonchus/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Óleos Voláteis/isolamento & purificação , Contagem de Ovos de Parasitas , Testes de Sensibilidade Parasitária
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