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Control strategies against the parasitic nematode Teladorsagia circumcincta are problematic under current sheep management systems. Infection with the parasite, particularly in young lambs, results in significant production losses therefore sustainable worm control is being sought. It has been established that variation in resistance to T. circumcincta is under genetic control and the development of resistance is an acquired characteristic and has an immunological basis. This project investigated the immunological response to infection, of lambs with predicted resistance or susceptibility to T. circumcincta. Specifically, the study aimed to identify immune response-associated genes that were differentially-expressed in resistant and susceptible lambs and attempted to identify mutations in these genes. This study was part of a long term project that aims to identify genetic marker/s to aid in marker-assisted selection (MAS) for resistance to T. circumcincta. Real time reverse transcription-quantitative polymerase chain reaction (real time RTqPCR) was performed on abomasal mucosa and lymph nodes from 55 lambs used in a previous experiment. The lambs had been either trickle-infected with 2,300 infective larvae every two days over three months (infected resistant/susceptible, n=45) or sham-dosed (non-infected control, n=10). Lambs were ranked in relation to faecal egg count (FEC) and adult worm count (AWC) at post mortem; zero or low FEC (resistant) to high FEC (susceptible). Histopathology showed only mild pathological changes in the abomasal mucosa of resistant lambs but heavy lymphocytic inflammatory infiltration in the mucosa and submucosa of infected susceptible animals. Measurements of a range of cytokine transcripts and cell markers associated with the four major CD4+ T cell subsets identified IL6, IL21, and IL23A as significantly increased by at least two-fold in abomasal lymph nodes and abomasal mucosa of susceptible lambs in comparison to resistant animals. Highly significant (P<0.02) positive correlations were found between IL6 (ρ=0.35), IL21 (ρ=0.54) and IL23A (ρ=0.38) transcript levels and AWC. Similarly, there were highly significant (P<0.01) positive correlations between FEC and IL6 (ρ=0.41), IL21 (ρ=0.65) and IL23A (ρ=0.31). In contrast, significant negative correlation (P<0.04) between IL23A with IgA antibody levels (ρ=-0.31) was found. There was also a significant positive correlation (P<0.03) of TGFB1 levels with AWC (ρ=0.42) and FEC (ρ=0.32) in the abomasal mucosa. These data suggests that susceptibility to T. circumcincta is linked to the activation of the inflammatory TH17 T cell subset and that this chronic inflammatory response was inappropriate to clear worm infection. High resolution melt analysis failed to identify single nucleotide polymorphisms in the coding regions of IL21 and IL21R. This is the first report of the involvement of TH17 response in GI worm infection in sheep. Similar gene expression studies involving the known upstream and downstream players of the TH17 response could be done.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

In an experiment investigating the influence of M. graminicola on rice yield, M. graminicola caused as significant yield reduction in Azucena but not in Bala indicating that this variety is tolerant. The influence of abiotic (nitrogen and water) and biotic (vesicular arbuscular mycorrhiza - VAM) factors on the nematode/rice interaction were also studied. Nitrogen stress affected Azucena plants but not Bala. Drought did not influence susceptibility of either variety but caused a significant reduction in root weight which was greater when nematodes were present in both rice varieties.

Com o objetivo de avaliar o efeito de isolados de Trichoderma em cultivares de alface foram conduzidos dois experimentos divididos em dois capítulos. O primeiro com a finalidade de avaliar o efeito do Trichoderma na qualidade de mudas de alface e o segundo a sua potencialidade no controle de Meloidogyne enterolobii em plantas adultas de alface. O experimento I foi conduzido em casa de vegetação na Universidade Federal do Tocantins, em delineamento inteiramente casualizado em esquema fatorial, com 5 repetições. Foram utilizados três isolados de Trichoderma (UFT201, UFT202 e UFT205) e duas cultivares comerciais de alface (Elba e Solaris). Para este experimento foram avaliadas as seguintes características: Altura de plantas, diâmetro do caule, comprimento da raiz, número de folhas, massa seca da parte aérea e raiz, massa seca total, índice de qualidade de Dickson e eficiência relativa. Para o capítulo II foram conduzidos dois experimentos com a utilização das cultivares Elba e Solaris, dois isolados de Trichoderma UFT201 e UFT205, um isolado de nematoide M. enterolobii e a cultivar de tomate Santa Clara, usada como testemunha hospedeira padrão dos nematoides. As variáveis avaliadas foram: Comprimento da raiz, diâmetro da cabeça, massa fresca da raiz, massa fresca total, massa seca da parte aérea, eficiência relativa, número de galhas, tamanho médio de galhas, posicionamento de galhas, índice de massas de ovos e índice de reprodução. No experimento I, em geral a presença de Trichoderma não resultou em mudas de melhor qualidade, quando comparados à testemunha. O fator tempo pode ter influência, visto que o trabalho foi realizado em condição de mudas. Para o experimento II, houve redução no número de galhas e massas de ovos de nematoides nos tratamentos inoculados com Trichoderma. Através dos parâmetros que determinam a presença do nematoide na planta, foi possível constatar para as duas cultivares o potencial antagônico do Trichoderma no controle de M. enterolobii. / In order to evaluate the effect of Trichoderma isolates in lettuce cultivars two experiments were conducted, divided into two chapters. The first, in order to evaluate the effect of Trichoderma in lettuce seedlings, and the second, its potential in control of Meloidogyne enterolobii in lettuce adult plants. The first experiment was conducted in a greenhouse at the Federal University of Tocantins, in a factorial randomized design, with five repetitions. Three Trichoderma strains were used (UFT201, UFT202, and UFT205), and two commercial cultivars of lettuce (Elba and Solaris). For this experiment the following characteristics were evaluated: plant height, stem diameter, root length, number of leaves, dry weight of shoot and root, total dry weight, quality index Dickson and relative efficiency. For Chapter II two experiments were conducted with the use of Elba cultivars and Solaris, two isolates of Trichoderma UFT201 and UFT205, an isolate of nematode M. enterolobii and tomato cultivar Santa Clara, used as the default host a witness of the nematodes. The variables evaluated were: root length, diameter of the head, fresh root mass, fresh mass total shoot dry mass, relative efficiency, number of galls, average size of galls, positioning of galls, egg mass index and reproduction index. In the first experiment, in general the presence of Trichoderma did not result in better quality seedlings compared to the control. The time factor may play a role, since the work was done on seedlings. For the second experiment, there was a reduction in the number of galls and egg masses of nematodes in treatments inoculated with Trichoderma. By parameters that determine the presence of nematodes in the plant, it was established for both cultivars the antagonistic potential of Trichoderma to control M. enterolobii.

Resumo: Os objetivos do trabalho foram isolar, identificar, comparar meios de cultura para crescimento, esporulação de fungos nematófagos e testá-los no controle de Meloidogyne exigua e M. paranaensis em laboratório, casa de vegetação e em cafezal infestado. Foram testados dois isolados de Arthrobotrys oligospora e um de Arthrobotrys sp., A. musiformis, Monacrosporium robustum e Paecilomyces liIacinus. Os dados obtidos revelaram que esses fungos ocorrem em diferentes agroecossistemas, têm exigências nutricionais especiais e apresentam diferentes níveis de predação para os nematóides estudados. Os meios, farelo de arroz, extrato de levedura e ágar, e o de fubá e ágar propiciaram crescimento e esporulação adequados para a maioria dos isolados estudados. A mistura de palha de café com farelo de arroz foi um substrato adequado para formulação desses fungos. Uma aplicação de 1 ou 2 L da mistura de partes iguais desse substrato colonizado pelos fungos, proporcionou a redução da população dos nematóides. No período estudado a cultura tratada não esboçou sinais de recuperação, indicando que cafezais depauperados, em solos degradados, não se recuperam com uma aplicação. Os dados também possibilitaram inferir que o controle biológico dos nematóides do cafeeiro será tanto mais efetivo quanto mais cedo forem iniciados os tratamentos, e a aplicação sistemática dos fungos, com isolados mais agressivos contra os nematóides presentes, deve ser efetuada. / Abstract: The objective of the research was to isolate, identify and compare culture media for growth and sporulation of nematode antagonistic fungi, and to evaluate the efficiency of these agents for biological control of Meloidogyne exigua and M. paranaensis under laboratory, greenhouse and field environmental conditions. Two isolates of Arthrobotrys oligospora, one of Arthrobotrys sp. and one of each species of A. musiformis, Monacrosporium robustum and Paecilomyces liIacinus, were also evaluated. It was observed that those fungi were very common under distinct agroecosystems, having special nutrition requirements, and also different pathogenicity levels for the nematodes above related. The culture media prepared with rice meal, malt extract and agar as well as the com meal agar favoured adequate growth and sporulation of most the isolates evaluated. The mixture of coffee straw with rice meal showed to be an adequate substrate for these fungi formulation under experimental trials. Just one application of one or two liters of a mixture of equal parts of the substrate colonized by the fungi, reduced the population of nematodes. However, the treated coffee trees did not show any sign of recovery during the period of study, indicating that weak coffee trees under poor soil conditions could not recover with just one treatment. The data also show that biological control of coffee nematodes can be more effective as early as the treatment with the fungi is applied, and, considering that it is a perennial crop, the fungi systematic application along with the selection of more aggressive isolates against the nematodes, must be done. / Orientador: Jaime Maia dos Santos / Coorientador: Rita de Cássia Panizzi / Banca: Marineide Mendonça Aguillera / Banca: Carlos Eduardo de Mendonça Otoboni / Banca: David Ariovaldo Banzatto / Banca: João Carlos de Oliveira / Doutor