Genetic control of development in the parasitic nematode Haemonchus contortus by microRNAs

The parasitic nematode Haemonchus contortus has a major impact on the welfare and economic sustainability of small ruminant farming throughout the world. Increasing drug resistance requires the development of novel therapeutic agents. To further this process, we examined the fundamental biology of development in H. contortus, specifically, the potential role of microRNAs (miRNAs). miRNAs are short, non-coding RNA molecules that negatively regulate gene expression. In the free-living nematode Caenorhabditis elegans, miRNAs regulate a variety of genes including those involved in development. This thesis describes the expression patterns, potential targets and possible functions of miRNAs in H. contortus throughout development.

Studies on Haemonchus contortus infections in Merino sheep

The work described in this thesis was initially directed towards the elucidation of the self-cure phenomenon as a flock phenomenon under natural grazing conditions. As a consequence of these studies it became apparent that several important aspects of the epidemiology and of the clinical syndromes associated with haemonchosis required further investigation. The thesis is therefore essentially a description of haemonchosis of sheep in an endemic area supported, where indicated, by observations obtained by experimental infections. Each of the separate aspects studied is prefaced by a review of the relevant literature and the details of many individual observations which are not included in the text may be found in the various appendices. Some results, for example the individual faecal egg counts and worm burdens at autopsy, were too voluminous to be included and these are available at The Veterinary School of the University of Glasgow.

Caracterização do nematóide de ovinos, Haemonchus contortus, resistente e sensível a anti-helmínticos benzimidazóis, no estado do Ceará, Brasil.

A resistência anti-helmíntica tem maior prevalência entre os nematóides de ovinos e caprinos. No Ceará, estudos demonstraram que esse problema está se disseminando, sendo detectado em aproximadamente 90% das propriedades de ovinos na região do baixo e médio Jaguaribe. H. contortus é o mais prevalente e de maior intensidade em populações parasitárias resistentes a anti-helmínticos em várias regiões do estado do Ceará e do mundo. Este trabalho teve como objetivo caracterizar as unidades produtoras de ovinos quanto ao aparecimento da resistência aos benzimidazóis, isolar uma cepa sensível e resistente aos benzimidazóis e fazer uma caracterização destas cepas. Para cada cepa foi determinado o poder infectante, patogenicidade, a fertilidade das fêmeas, a capacidade do desenvolvimento dos seus ovos até larvas de terceiro estágio sob diversas condições de meio, além da classificação da população quanto à morfologia do processo vulvar das fêmeas e avaliação global da aptidão das larvas de terceiro estágio em desenvolver uma nova infecção. Os fatores que apresentaram valor preditivo para o desenvolvimento da resistência foram tratamentos aplicados na época seca (P=0,03) e rotação de pastagem (P=0,17). Foram isoladas duas cepas, uma considerada sensível (DE50 = 0,20) e uma resistente (DE50 = 1,63). Durante a época seca (32°C), a cepa sensível desenvolveu-se melhor, enquanto na época chuvosa (23°C), a cepa resistente obteve melhor desempenho. Na primeira geração, a cepa resistente apresentou maior patogenicidade, demonstrada pela maior produção de ovos, hematofagismo e melhor estabelecimento das larvas de terceiro estágio. Na segunda geração, produzida com larvas desenvolvidas nas temperaturas que mimetizam as épocas seca e chuvosa, as larvas obtidas com 23°C demonstram uma maior patogenicidade. Acredita-se que nesta geração o genótipo não tenha influenciado as características de vida dos parasitos. A partir destes resultados conclui-se que existem influências do genótipo resistente ou sensível aos benzimidazóis e da temperatura sobre as características de vida do parasito H. contortus isolado no estado do Ceará.

Avaliação da eficácia in vitro de extratos vegetais sobre a inibição da migração de larvas infectante de Haemonchus contortus.

Na ovinocultura, o Haemonchus contortus é o parasita de maior importância em regiões tropicais e subtropicais, causando perdas econômicas aos produtores.

Imunização de ovinos com glicoproteína integral de membrana do intestino de Haemonchus contortus.

2015

Extração de DNA de machos e fêmeas adultos de Haemonchus contortus.

2015

Thymus vulgaris L. essential oil and its main component thymol: anthelmintic effects against Haemonchus contortus from sheep.

2016

Peptídeos ovicidas de Moringa oleifera Lamarck: isolamento, caracterização bioquímica e alvos de ação no modelo biológico Haemonchus contortus.

2016

Indução da resistência ao anti-helmíntico monepantel em Haemonchus contortus (resultados preliminares).

2016

Avaliação da eficácia do óleo essencial de Mentha arvensis, e de seu majoritário mentol, no controle de Haemonchus contortus em ovelhas.

2016

Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections

This trial was carried out in Piracicaba, Sao Paulo State. Brazil. to comparatively evaluate the degree of resistance to naturally acquired gastrointestinal nematode infections in sheep of the following genetic groups purebred Santa Ines (SI), SI crossbred with Dorper (DO x SI), lie de France (IF x SI), Suffolk (SU x SI), and Texel (TE < SI) Fifteen ewes from each group were raised indoors until 12 months of age. At this age, they were moved to pasture that was naturally contaminated by nematode infective larvae and were evaluated from December to May. 2007. Rainfall ranged from 267 mm in January to 37 mm in April Maximum and minimum mean temperatures ranged from 32 5 degrees C to 19 0 degrees C in March and from 25.9 degrees C to 12.8 degrees C in May. There was an increase in the mean number of eggs per gram of feces (EPG) after animals were placed on pasture with significant difference between the SI (80 EPG) and IF x SI (347 EPG) groups in January: and the DO x SI (386 EPG) and TE x SI (258 EPG) groups in May. The highest mean fecal egg count (FEC), 2073 EPG, was recorded for the TE x SI group in February. All groups showed a progressive reduction in body weight throughout the experiment of 12.0% (TE x SI) to 15.9% (SU x SI). In general. the animals with the highest FEC presented the lowest packed cell volumes (PCV): the highest correlation coefficient between FEC x PCV occurred in the SU x SI sheep in January (r = -0.70; P < 0.01). Similarly, there was an inverse relationship between FEC and blood eosinophil Values, with the highest correlation coefficient in the TE x SI sheep in February (r = -0.64; P < 0.05). Immunoglobulin G (IgG) levels against Haemonchus contortus antigens increased in all groups as a result of the exposure to parasites and remained relatively constant until the end of the study, with the exceptions of SU x SI and TE x SI, which showed a rise in IgG levels during the last sampling that coincided with a reduction in mean FEC. In conclusion. crossbreeding Santa Ines sheep with any of the breeds evaluated can result in a production increase and the maintenance of a satisfactory degree of infection resistance, especially against H. contortus and Trichostrongylus colubriformis. the major nematodes detected in this flock. (c) 2009 Elsevier B.V. All rights reserved.

Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-1 2p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC- 1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (1-3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P < 0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P < 0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-1 2p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by TH2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group. (C) 2008 Elsevier B.V. All rights reserved.

mRNA profile of Nellore calves after primary infection with Haemonchus placei

Haemonchus parasites are responsible for many losses in animal production. However, few studies are available, especially of zebu cattle. In this study, we investigated mRNA differences of immune response genes in naive Nellore calves infected with Haemonchus placei, relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000 H. placei 13 larvae and after 7 days lymph node and abomasum tissues were collected. IL-2, IL-4, IL-8, IL-12, IL-13, IFN-gamma, MCP-1, lysozyme, pepsinogen and TNF-alpha genes were evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abomasum histology. In the infected group, IL-4, IL-13 and TNF-alpha were up-regulated in the abomasal lymph node. In the abomasum, IL-13 increased and TNF-alpha was down-regulated (p < 0.05). No differences were detected for mast cells and eosinophil counts in abomasal tissue (p > 0.05). We conclude that for this infection time, there was Th2 polarization but that cellular infiltrate in abomasal tissue takes longer to develop. (C) 2010 Elsevier B.V. All rights reserved.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.

Cleavage of hemoglobin by hookworm cathespin D aspartic proteases and its potential contribution to host specificity

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.