Cyathostomins are potentially life threatening parasitic nematodes of adult horses and are highly prevalent worldwide. Infected animals may be asymptomatic or show clinical signs of weight loss, diarrhoea and colic. Third and fourth stage larvae spend a large proportion of their lifecycle encysted in the large intestinal wall where they cannot currently be detected ante mortem. Mast cells are commonly found at interfaces to the external environment, such as the rectum, and these cells and the proteinases they produce have been implicated in protective host immune responses against nematode infection in animals. Previous studies have demonstrated an increase in caecal mast cell proteinase expression during cyathostomin infection. Prior to this study, there were two known equine mast cell proteinases, which had been purified and characterised from a mastocytoma (equine tryptase [eqTRYP] and equine mast cell proteinase-1 [eqMCP-1]). However, as many mammalian species express multiple closely-related chymases it was hypothesised that other equine mast cell proteinases exist that have not yet been characterised and which may be more closely associated with the level of worm burden. The primary objective of this study was to investigate the recruitment of mast cells to the large intestine in cyathostomin infected horses and the expression of mast cell proteinases in response to infection. A further aim was to evaluate the potential of associated mast cell proteinase assays or rectal biopsy mast cell enumeration for utility in diagnostic tests to estimate cyathostomin mucosal burden. A secondary objective was to explore the existence of further mast cell proteinases and the relationship of these enzymes to cyathostomin mucosal burden. Optimised sampling protocols, parasitological, histological and immunohistochemistry techniques were performed to enumerate cyathostomin mucosal burden and to characterise the mast cell populations in the caecum, right ventral colon (RVC) and rectum of naturally infected horses (n=28). Mast cell populations correlated throughout the intestine, providing further evidence of the common mucosal system. EqMCP-1 and eqTRYP labelled mast cells were identified throughout the large intestine. Significant positive linear relationship existed between rectal proteinase-labelled mucosal mast cell populations and both the combined total cyathostomin mucosal burden (CTMB; eqMCP-1, p=0.018; eqTRYP, p=0.048) and the combined total luminal burden (CTLB; eqMCP-1, p=0.009; eqTRYP, p=0.007). Concentrations of eqMCP-1 and eqTRYP in (i) serum, (ii) local serum from venous blood draining the large intestine, and (iii) large intestinal tissue homogenates were assessed using ELISA. There was no significant correlation identified between local and peripheral serum proteinase concentrations suggesting that peripheral serum proteinase levels are not representative of the local proteinase response. There was however a significant negative relationship between peripheral serum eqMCP-1 concentrations and the CTMB, which could relate to the activation and sequestering of proteinases within the gut lumen. Concentrations of eqMCP-1 and eqTRYP measured in local serum did not significantly positively correlate with cyathostomin mucosal burden. There was a significant association observed between intestinal tissue levels of eqMCP-1 and eqTRYP and the CTMB in the RVC (p < 0.023), providing support for their role in the immune response. Four proteinase sequences, equine tryptase (TLP1), Granzyme B-like (GZMBL), putative equine Mast Cell Proteinase-1 (CLP1) and Granzyme(BGH)-like (GZM(BGH)L), were sequenced and the local transcription levels of each of these enzymes assessed using quantitative reverse-transcription PCR. The expression of TLP1 was closely correlated with GZMBL expression, and there was a significant positive relationship observed between TLP1 and GZMBL transcript levels and combined total mucosal burden in the RVC. Both GZM(BGH)L and CLP1 transcript levels were also positively correlated with each other, but the levels of these transcripts were not statistically correlated to any of the cyathostomin parasitological measures assessed here. This work has provided the basis for further rectal biopsy studies to examine the important dynamics of the mast cell response to cyathostomin infection. The results from this thesis, with the demonstration of novel proteinases, are encouraging for further investigation into equine mast cell proteinases and their role in cyathostomin infections.

Made available in DSpace on 2016-04-28T20:15:31Z (GMT). No. of bitstreams: 1 2008 - Melissa Carvalho Machado do Couto Chambarelli.pdf: 767679 bytes, checksum: 28c75b5091c80e9da0651ba953952d51 (MD5) Previous issue date: 2008-02-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The knowledge of cyathostomin infective larvae population level presents onto pasture is important to epidemiological purposes, estimating parasitic risk infection to horses and helping on setting up integrated control programs. The present study was elaborated in three parts: first is about the migratory dynamic and survival of cyathostomin infective larvae on Bermuda grass pasture for 12 months. Feces and grass samples were collected weekly at 8 a.m., 1 p.m. and 5 p.m. The samples were weighted and processed by Baermann technique. Higher survival of L3 was found at dry season, 15 and 12 weeks on feces and sward respectively, at rainy season the survival was smaller. The infective larvae were recovered during three times and the Kruskal-Wallis test did not present significance among them. At the second part, the ecology of cyathostomin infective larvae was studied for 24 months. During this period, samples of feces and grass (apex and base) were collected weekly. Samples were processed the same way as in the first part of the study. In the feces, cyathostomin L3 survived for up to 15 weeks, with higher recovery during the rainy period (46,228 kg-1.dh) and on the grass for up to 12 weeks. The recovery of L3 was greater during the dry period in the grass base (1,868 kg-1.dh) rather than in the apex (809 kg-1.dh). The L3 migration from feces to grass varied during the period. The last part of the study is about the effect of irrigation on cyathostomin infective larvae migration in Bermuda grass pasture during the four seasons of the year. Fecal masses of naturally infected horses were placed on Bermuda grass pasture at the beginning of each season. Samples of feces and grass were collected every two weeks at 8 a.m. and 5 p.m. until the end of each season from September/2007 to September/2008. Samples were processed the same way as in the first part of the study. The Kruskal-Wallis non parametric test showed a significant difference for L3 recovery in each season. The test did not present significance between the two times sampling. These results suggest that in conditions of Baixada Fluminense tropical climate horses are in permanent risk of infection. / O conhecimento do grau de contamina??o das pastagens pelas larvas infectantes de ciatostom?neos ? importante para os prop?sitos epidemiol?gicos, determinando o risco de infec??o para os eq?inos e podendo tamb?m auxiliar no estabelecimento de estrat?gias de controle integrado. O estudo foi elaborado em tr?s partes: a primeira relata o estudo da din?mica migrat?ria e a sobreviv?ncia de larvas infectantes de ciatostom?neos em pastagem coast cross durante 12 meses. Para este estudo foram coletados amostras de fezes e gram?nea com intervalos regulares de sete dias em tr?s hor?rios diferentes (8, 13 e 17 horas). As amostras foram pesadas e processadas segundo a t?cnica de Baermann. A sobreviv?ncia das L3 foi de at? 15 semanas nas fezes e 12 semanas na gram?nea no per?odo seco e de nove e oito semanas respectivamente para o per?odo chuvoso. No per?odo chuvoso, maior n?mero de L3 foi recuperado nas fezes e no per?odo seco na gram?nea. N?o foi observada diferen?a significativa entre os hor?rios de coleta pela an?lise n?o param?trica de Kruskal-Wallis. Na segunda parte experimental foi estudada a ecologia das larvas infectantes de ciatostom?neos por 24 meses. Durante este per?odo, foram realizados coletas semanais de fezes e gram?nea (?pice e base). O processamento das amostras seguiu o mesmo protocolo realizado na primeira parte do experimento. Nas fezes as L3 sobreviveram por at? 15 semanas, ocorrendo uma maior recupera??o das larvas durante o per?odo chuvoso (46.228 kg-1.ms). Na gram?nea, a sobreviv?ncia foi de at? 12 semanas. A recupera??o das L3 foi mais intensa durante o per?odo seco na base (1.868 kg-1.ms) e no ?pice (809 kg-1.ms) da gram?nea. A migra??o das L3 das fezes para a gram?nea variou durante todo o per?odo. A ?ltima parte do estudo observou a migra??o de larvas infectantes de ciatostom?neos em pastagem coast cross irrigada e n?o irrigada durante as quatro esta??es do ano. Massas fecais, de eq?inos naturalmente infectados foram depositadas nos canteiros de coast cross no in?cio de cada esta??o.A amostragem de fezes e gram?nea foi realizada quinzenalmente em dois hor?rios distintos (8 e 17 horas) at? o final de cada esta??o, no per?odo de setembro/2007 a setembro/2008. O processamento das amostras foi o mesmo descrito na primeira parte experimental. O teste n?o param?trico de Kruskal Wallis evidenciou uma diferen?a significativa na recupera??o de larvas infectantes entre as esta??es do ano. N?o foi observada uma varia??o significativa na recupera??o de L3 nos diferentes hor?rios de coleta. Os resultados sugerem que em condi??es tropicais de Baixada Fluminense, RJ os animais est?o em permanente risco de infec??o.

Made available in DSpace on 2016-04-28T20:15:31Z (GMT). No. of bitstreams: 1 2008 - Simone Quinelato final.pdf: 1288089 bytes, checksum: 806c125a6ebd7474f17e8efb57adbc3f (MD5) Previous issue date: 2008-02-29 / The knowledge of biology and epidemiology of cyathostomin free living stages have been helping the development of control programs that limitis anthelmintic use. However little is known about environmental larvae dynamics, mainly in tropical climate. Studies about pasture contamination can help to estimate parasitic risks for animals and to set up integrate control programs. The present study was elaborated in three complemented parts: first, it was evaluated the distribution, recovery and survival of cyatostomin infective larvae of equines in feces and pasture during 15 months. Fecal samples were monthly placed on Tifton 85 (Cynodon spp. cv. Tifton 85) pasture, from september of 2003 to september of 2004. Grass and feces were collected weekly, at 8 a.m., 1 and 5 p.m. and processed by Baermann technique. The results indicate that the environmental conditions were favorable for L3 development and survival. In feces, more L3 was recovered during the rainy period and on the grass in dry period. L3 survival was higher in the dry period for feces and grass samples. L3 were recovered during the three times of collection and no significant difference was observed among three times. The second part was more wide-ranging and approaches the distribution, recovery and survival of cyathostomin infective larvae in feces and pasture for february 2005 to march 2007. Seven days after the deposit, sample of feces and grass were collected weekly at 8 a.m., 1 p.m. and 5p.m., from three different field sites. Grass sample was divided into base (0-20 cm) and apex (20-40 cm). The samples were processed by the Baermann technique for L3 recovery. In the rainy period, more infective larvae were recovered on the feces and grass apex. In the dry period, the recovery was higher only on the grass base, as well as the L3 survival on feces and grass. More larvae were recovered at 8 a.m., except from the grass apex, where the highest recovery was at 1 p.m. The third part refers to the seasonal distribution and recovery of cyathostomin infective larvae in feces, pasture and soil. In the beginning of the seasons feces samples were placed on experimental Tifton 85 plot. The collections began one week after deposit, and later every 15 days. L3 recovered were higher in the autumn and winter and smaller in the spring and mainly in the summer. More L3 were recovered in the morning, although no statistic difference has been observed between the collection times in each season. The soil didn't demonstrate to be potential L3 reservoir, seen the low larvae recovery in this study. / O conhecimento da biologia e epidemiologia das fases pr?-paras?ticas de ciatostom?neos de eq?inos t?m contribu?do para o desenvolvimento de programas de controle que limitem a utiliza??o de anti-helm?nticos. No entanto, pouco se conhece sobre a din?mica das larvas no ambiente, principalmente nas regi?es de clima tropical. Estudos sobre o grau de contamina??o das pastagens por larvas infectantes de ciatostom?neos podem auxiliar na determina??o do risco de infec??o dos animais e fornecer dados para o estabelecimento de programas de controle integrado. O presente estudo foi dividido em tr?s etapas complementares: na primeira etapa, avaliou-se a distribui??o, recupera??o e sobreviv?ncia de larvas infectantes (L3) de ciatostom?neos de eq?inos nas fezes e na pastagem no per?odo de 15 meses. Massas fecais foram depositadas mensalmente na gram?nea Tifton 85 (Cynodon spp. cv. Tifton 85) no per?odo de setembro de 2003 a setembro de 2004. Gram?nea e fezes foram coletadas a cada sete dias, ?s 8, 12 e 17h e processadas pela t?cnica de Baermann. De acordo com os resultados, as condi??es ambientais foram favor?veis para o desenvolvimento e sobreviv?ncia das L3. Nas fezes, a recupera??o das L3 foi superior durante o per?odo chuvoso e na gram?nea no per?odo seco. A sobreviv?ncia das larvas foi superior no per?odo seco, tanto para as amostras de fezes quanto para as de gram?nea. As L3 foram recuperadas durante os tr?s hor?rios de coleta e n?o se observou diferen?a significativa entre os hor?rios. A segunda etapa foi mais abrangente e aborda o estudo da distribui??o, recupera??o e sobreviv?ncia de larvas infectantes de ciatostom?neos de eq?inos nas fezes e na pastagem no per?odo de fevereiro de 2005 a mar?o de 2007. Sete dias ap?s cada dep?sito de massa fecal foram realizadas coletas de amostras de fezes e gram?nea ?s 8, 13 e 17 h, de tr?s pontos distintos em sentido hor?rio. As amostras de gram?nea coletadas foram divididas em base (0-20 cm) e ?pice (20-40 cm), tendo sido processadas pela t?cnica de Baermann para recupera??o das L3. No per?odo chuvoso, as larvas infectantes foram recuperadas em maior n?mero das fezes e do ?pice da gram?nea. No per?odo seco, a recupera??o foi superior na base da gram?nea, assim como a sobreviv?ncia das L3 nas fezes e na gram?nea. Maior n?mero de larvas foi recuperado ?s 8 h, exceto na base da gram?nea, onde a maior recupera??o de L3 ocorreu ?s 13 h. A terceira parte refere-se ? distribui??o sazonal e recupera??o de larvas infectantes de ciatostom?neos nas fezes, na pastagem e no solo. Ao in?cio de cada esta??o do ano foram depositadas amostras de fezes no canteiro experimental de Tifton 85. As coletas iniciaram-se uma semana ap?s o dep?sito e posteriormente a cada 15 dias. As L3 foram recuperadas em maior quantidade no outono e inverno e em menor quantidade na primavera e principalmente no ver?o. No hor?rio da manh? as larvas foram recuperadas em maior n?mero, embora n?o tenha sido observada diferen?a estat?stica entre os hor?rios de coleta em cada esta??o do ano. O solo n?o demonstrou ser potencial reservat?rio de L3, visto a baixa recupera??o de larvas neste estudo.