Soil-transmitted helminths are a major cause of morbidity for humans and their livestock. A combination of better sanitation, anthelminthic drugs and vaccines are predicted to reduce the morbidity of these parasites in humans. The drugs currently used to treat these infections, albendazole and mebendazole, are fairly ineffective against Trichuris trichiura (human whipworm), and there are reports of drug resistance arising within parasite populations in Vietnam and Zanzibar. There are also no commercially available vaccines against human STH species, and very few against their veterinary counterparts. The murine whipworm, T. muris, has been used for over 50 years as a model for T. trichiura. These parasites share homology at the genomic and transcriptomic levels, and the immune responses associated with both acute and chronic infection have been well studied using the T. muris mouse model. T. muris excretory/secretory products have been studied in the context of vaccination for over four decades, however relatively little progress has been made towards identifying the molecular components that stimulate protective immunity following vaccination or during acute infection. Here, a stringent selection protocol was developed using chromatography and mass spectrometry methods combined with a measurement of T cell cytokine production. The work presented in this thesis provides a novel framework for identifying potential immunogenic candidates within adult T.muris excretory/secretory products. Exosome-like vesicles isolated from adult T. muris ES were also explored as a source of host protective material. Vaccination with exosome-like vesicles protected male C57BL/6 mice from a subsequent low dose infection, which would ordinarily progress to chronicity, and a number of potential immunogenic candidates were identified. Over the course of this thesis, several important observations were made relating to characteristics of the immune response induced by vaccination with ES. Firstly, proteinaceous material is likely to be responsible for the host protective properties of ES. Secondly, vaccination with ES products stimulates long-lasting immunity. Thirdly, vaccination with ES collected from both larval and adult stages stimulates protective immunity. The number of potential immunogenic candidates has also been narrowed down from over four hundred to just eleven. Given the homology between T. muris and T. trichiura at both the genomic and transcriptomic levels, this work has the potential to advance vaccine design for T. trichiura and other Trichuris parasites.

Düsseldorf, Universiẗat, Diss., 2004.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-12T15:33:27Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_Final_Digital.pdf: 1044188 bytes, checksum: 49fcb98340d75c059b4fc044e603cce8 (MD5) / Made available in DSpace on 2016-04-12T15:33:27Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_Final_Digital.pdf: 1044188 bytes, checksum: 49fcb98340d75c059b4fc044e603cce8 (MD5) Previous issue date: 2015-02-27 / CAPEs / A prevalência de doenças alérgicas, como rinite e asma, é menor em países subdesenvolvidos, onde há uma maior exposição a agentes infecciosos, como os helmintos. A relação entre infecções com T. trichiura e a prevalência das doenças alérgicas e reatividade cutânea ainda não está estabelecida. Os estudos divergem quanto à alteração (potencializar ou reduzir) do quadro clínico e/ou testes cutâneos, bem como, a carga parasitária da população estudada. O estudo teve como objetivo verificar se existe diferença entre a ocorrência de asma alérgica, prick test, níveis séricos das citocinas IL-4, IL-6, IL-10, TNF-α e anticorpos IgE anti-ascaris em crianças com infecção ativa por T. trichiura. Para isto, crianças com ou sem asma foram definidas pelo questionário ISAAC, foram realizados o prick-test, parasitológico (Hoffman/Kato Katz) e coleta de sangue, que foi submetido a cultura (estimulada com PHA) e o sobrenadante coletado para a dosagens das citocinas (CBA). A prevalência de crianças com parasitológico positivo foi de 16,9 % (61/361 crianças), entre essas 27,9 % (17/61) foram positivas para infecção por Trichuris trichiura (12/17) ou co-infectadas por Trichuris trichiura/ Ascaris lumbricoides (5/17). O grupo de pacientes infectados, com ou sem asma, produziram níveis significantemente elevados para todas as citocinas em relação ao grupo controle. Além disso, o grupo dos pacientes infectados sem asma apresentou um tendência maior de produção de IL-6, TNF-alfa e IL-10 que os com asma; os pacientes infectados e asmáticos apresentaram uma menor reatividade no Prick Test quando comprado aos asmáticos não infectados. Então, a infecção por T. trichiura parece modular positivamente os níveis das citocinas TNF-α, IL-10 e IL-6, mas em pacientes asmáticos estes níveis tendem a ser controlados. As reações de hipersensibilidade cutânea imediata parece ser menos frequente em asmáticos quando infectados. Os dados levantam a possibilidade de uma modulação mútua entre asma e tricuríase, favorecendo um estado de maior cronicidade de ambas entidades de doença. / The prevalence of allergic diseases such as rhinitis and asthma is smaller in developing countries, where there is a greater exposure to infectious agents, such as helminths. The relationship between infection with T. trichiura and the prevalence of allergic diseases and skin reactivity is not yet established. Studies differ as to the nature of the change (increase or reduce) in the clinical condition and/or skin tests, as well as the parasite load of the studied population. The study aimed to determine whether there are differences between the occurrence of allergic asthma, prick test, serum levels of IL-4, IL-6, IL-10, TNF-α and anti-Ascaris IgE antibodies in children with active infection by T. trichiura. For this purpose, children with and without asthma were defined by the ISAAC questionnaire, prick-test and parasitological (Hoffman/Kato Katz) were performed and blood samples were collected, which were then subjected to culture (stimulated with PHA) and the collected supernatant for cytokines measurements (CBA). The prevalence of children with positive parasitological was 16.9% (61/361 children), and among these 27.9% (17/61) were positive for Trichuris trichiura infection (12/17) or co-infected with Trichuris trichiura/Ascaris lumbricoides (5/17). The group of infected patients, with or without asthma, produced significantly high levels for all cytokines in the control group. Furthermore, the group of patients infected without asthma showed a greater tendency of IL-6,TNF-α and IL-10 production than those with asthma; infected and asthmatic patients had a lower reactivity in Prick Test when compared to those with asthma who were uninfected. Thus, the infection with T. trichiura positively modulates the levels of TNF-α, IL-10 and IL-6 cytokines, but these levels in asthmatic patients tend to be controlled. The immediate hypersensitivity skin reactions appears to be less common in asthmatics when infected. The data raise the possibility of a mutual modulation between asthma and trichuriasis, favoring a state of chronic course on both disease entities.

The prime aim of the present study was to investigate the welfare of Abyssinian colobus monkeys (Colobus guereza kikuyuensis) held in captivity, by behavioural, endocrinological and parasitological approaches. Five groups of colobus monkeys were studied, in the UK at Port Lympne Zoo, Banham Zoo and Paignton Zoo (separate male and female groups) and in France at La Boissière du Doré Zoo. Overall, the patterns of behavioural activity for captive colobus monkeys agree with observations of wild colobus monkeys, suggesting that there was generally good welfare at all zoos. However, local differences in group composition (i.e. age and gender) or management practices were shown to affect activity budgets. At Banham Zoo, where colobus monkeys had access to a large paddock and food was offered only twice a day, animals spent significantly more time foraging/feeding than other groups which received three meals a day. At Paignton Zoo (adult male only group) animals spent more time resting alone and less time resting socially or being social than other zoos. Generally, across zoos, older, higher ranking animals spent less time moving and more time feeding than younger, more subordinate ones. Social dominance rank and linearity of the hierarchy in the five groups of colobus was determined. Large mixed groups of colobus monkeys including both males and females displayed strong linear dominance hierarchies, but in single sex groups, the strength of the hierarchy was found to depend on local events and group composition. Generally, male colobus monkeys displayed most of the dominance behaviour, however, females were an integral part of the dominance hierarchy with unidirectional dominant behaviour between both genders in large mixed groups. Factors such as life history, which may influence social dominance ranking, were investigated. Index of success (another measurement of dominance rank) was mainly explained by age and gender. Social behaviours such as play behaviour and grooming were also associated with ranking. Older, more dominant animals were more often the recipient of grooming behaviour whereas younger more subordinate animals spent more time playing. Faecal egg counts of the intestinal nematode parasite, Trichuris trichiura, were investigated in relation to individual index of success, age, gender, and husbandry practices at each zoo. No significant differences in egg count were observed between genders. Egg count was explained by the index of success and anti-helmintic practices at zoos. The highest count of eggs were observed at La Boissière du Doré Zoo, probably due to the fact animals were locked in for several months in the winter, therefore favouring re-infection of infective larvae. The lowest count of Trichuris egg was at Paignton Zoo female group, who had the largest enclosure, with very low animal density. Counts of eggs were at their lowest in the winter compared to other season of the year. Differences between zoos seem to result from anti-helmintic practices coupled with the influence of enclosure size and husbandry regimes. Faecal glucocorticoids and their metabolites, in colobus monkeys held in the five groups, were measured by radioimmunoassay and investigated in relation to index of success, levels of aggression, gender, age and reproductive status of females. The overall dominant animals in various groups had higher levels of faecal glucocorticoids. Males had significantly lower faecal glucocorticoid than females, and pregnant females had higher levels than others. The older, higher ranking colobus monkeys initiated significantly more acts of aggression than younger, lower ranking animals. Seasonal variation in faecal cortisol equivalents were observed in the autumn where higher levels were recorded compared to other seasons. Comparison of faecal content of cortisol equivalents of the different groups showed the lowest levels at Port Lympne Zoo and Banham Zoo while the highest levels were at Paignton Zoo (female groups). This suggests that the large multi-male, multi-female groups had the lowest levels of stress and associated high levels of welfare. These studies lead to the recommendation that single sex groups of captive Abyssinian colobus monkeys should be avoided, particularly when this involves breaking down the social structure of established groups.

Intestinal dwelling parasites live in close association with the complex microbiota that inhabit our intestinal tracts. The intestinal helminth, Trichuris muris, depends on these bacteria for egg hatching and successful establishment of infection within the epithelium of the caecum and colon. Infection causes significant alterations to the host intestinal microbiota, including a decrease in bacterial diversity and shifts in proportions of certain bacterial groups. This is accompanied by a decrease in Foxp3+ regulatory T cells and changes to the metabolic potential of the host microbiota, consequently impacting host health. However, the factor(s) driving these changes and the existence and role of its own intestinal microbiota is unknown. Infection of C57BL/6 and immunodeficient SCID mice with a high dose (~ 200 embryonated eggs) and a low dose (~ 20 embryonated eggs) of T. muris was used to determine the impact of worm burden and the adaptive immune system on the host intestinal microbiota, in comparison to naïve controls. Microbiota analysis was performed by 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and Illumina sequencing. This revealed that infection-induced microbiota changes were dose dependent and high level infection caused an increase in the Bacteroidaceae and Enterobacteriaceae families, independently of the host adaptive immune system. Development of a surface sterilisation protocol enabled the internal T. muris microbiota to be analysed by 16S rRNA gene DGGE and fluorescence in situ hybridisation (FISH). The resulting data indicated that T. muris requires its own diverse intestinal microbiota that is derived from, but distinct to, that of its host. A core microbiota is selected and maintained by the parasite regardless of the surrounding host microbiota. The parasite microbiota is important for its fitness, shown in vitro using an antibiotic motility assay and in vivo using germ free (GF) mice. Furthermore, infection with T. muris causes a significant reduction in caecal butyrate concentrations and consequently a decrease in the expression of butyrate transporters in caecal tissue. Interestingly, the T. muris microbiota is able to produce the short-chain fatty acid (SCFA) butyrate, which the parasite is unable to make itself yet secretes into its local environment. Together these strategies promote the long term survival of T. muris within the intestinal niche, adding a new level of complexity to the interaction between the pathogen, the host and their respective microbiotas that underpins successful chronic nematode infection.

The goal of this paper was to determine rate of presence of whipworms of genus Trichuris in bodies of selected ruminants (sheep, roe deer) in certain areas and to morphologically state different species of whipworms using molecular revision and professional literature on samples found during helmitological dissections of selected ruminants. Two hypotheses were stated: H1: species that are found in highest volume in case of roe deer and sheep are whipworms Trichuris discolor and Trichuris ovis H2: these whipworms can not be positively distinguished when using morphometrical methods. Material needed for the study, i.e. the intestines of examined ruminants, was recovered in different areas of Czech Republic. Later were the intestines dissected in a laboratory using standardized procedure and hereby collected samples were analysed. Based on selected methods it was determined that in roe deer the rate of occurence of Trichuris discolor is much higher compared to that of Trichuris ovis. With sheep the difference between rates of presence is smaller. These results confirm the first hypothesis by showing high rate of presence of whipworms in these hosts. Collected females of genus Trichurids were morphometrically differentiated by their sex and in 4 morphotypes. Following this differentiation, the most present were the females of morphotype M2, those with a vulval opening without an everted vagina. The second hypothesis was also confirmed. Multihosting species Trichuris discolor and Trichuris ovis are prevalent in the bodies of roe deer and sheep. Thus we can say the roe deer are a potential source of whipworm contamination to sheep breeding. It can not be excluded that sheep are infected by roe deer and vice versa. Molecular determination is a necessary tool for correct assessment of whipworm species, considering the fact that morphological methods may lead to incorrect results.

Circadian rhythms have been implicated in severity and outcome of infection and disease. Commonly, LPS and bacterial infection have been used to identify the mechanisms behind the difference in immune responses depending on the time of day of the challenge. In this thesis, the colon dwelling nematode parasite Trichuris muris, which elicits a Th2 immune response in resistant mice, was used to identify if circadian rhythms influence infection outcome 3 weeks post infection. C57BL/6 mice infected with 200 eggs of T. muris at ZT0 (7am, lights on) expelled the parasite more efficiently than mice infected at ZT12 (7pm, lights off), which expelled with a delay of several days compared to ZT0 infected mice. Analysis of cell infiltration into the colon during the first days of infection suggested that there was no visible difference in the local immune response. There also were no differences in macrophage and dendritic cell numbers in colon tissue of naïve mice at ZT0 or ZT12. Further experiments examined immunomodulation of the immune response to T. muris by pushing the immune response towards a Th1, by low dose infection, or a Th2 response, by vaccination with excretory/secretory antigen. In both cases any circadian influence was overwritten. Mice infected at ZT0 or ZT12 with only 40 eggs of T. muris were equally susceptible to infection and mice infected at ZT3 10 days after vaccination at ZT0 or ZT12 were equally resistant to infection. Mice food restricted to mid-light phase and infected at ZT0 were not significantly delayed in their worm expulsion or polarised more towards a Th1 immune response compared to ZT0 infected mice fed during the dark phase. Therefore it is unlikely that feeding behaviour during the first days of infection is able to polarise towards a Th1 response and lead to delayed worm expulsion. Transgenic mice were used to dissect the mechanism underlying the delay in worm expulsion in ZT12 infected mice. mPer2::luc mice were used to confirm rhythmic Per2 expression in colon tissue and dendritic cells. Infection of mPer2::luc mice at ZT0 or ZT12 with T. muris showed similar worm expulsion, antibody and cytokine production when infected at ZT0 or ZT12. Bmal1floxLysMcre mice, which lack rhythmic clock gene expression in macrophages and granulocytes, produced a stronger Th2 antibody response in a primary infection at ZT3 than wild-type littermate controls. Newly generated mPer2::lucBmal1floxCD11ccre mice showed the no difference in worm burden and parasite specific antibody production between ZT0 and ZT12 infected mice. Only IL-10 and IL-6 levels were significantly lower in ZT12 infected mPer2::LucBmal1floxCD11ccre mice compared to ZT12 infected wild-type littermates. Confirmation of removal of exon 8 of the Bmal1 gene was not achieved; therefore it is not clear if circadian rhythm in dendritic cells has any impact on the immune response to T. muris or if the mPer2::LucBmal1floxCD11ccre mice and littermate controls both contain circadian rhythm in dendritic cells and therefore cannot be used to identify the role of the dendritic cell clock in the time of day differences in infection outcome. This thesis shows that time of day of the infection impacts on the outcome of infection with Trichuris muris.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-14T20:57:37Z No. of bitstreams: 1 Dissertação_ICS_ Leonardo Nascimento Santos.pdf: 1769409 bytes, checksum: 84530b31d51dfec9bf3c5617b8fb1217 (MD5) / Made available in DSpace on 2013-08-14T20:57:37Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Leonardo Nascimento Santos.pdf: 1769409 bytes, checksum: 84530b31d51dfec9bf3c5617b8fb1217 (MD5) / CAPES; FAPESB / Estudos epidemiológicos e experimentais indicam que helmintos regulam respostas imunes a aeroalérgenos através da estimulação de uma rede de regulatória. Tem sido relatado que infecção por T. trichiura e outros trichuroides levam à proteção contra atopia, doenças alergicas e autoimunes. O objetivo deste estudo foi identificar frações do extrato somático de T. trichiura (TtEFs) com atividade imunomodulatória, avaliando seus efeitos sobre a produção de citocinas por células mononucleares do sangue periférico (CMSP) de doadores saudáveis. Quatorze TtEFs (TtEF1 a TtEF14) foram obtidos pelo fracionamento do extrato somático de T. trichiura por cromatografia líquida de troca iônica. Para avaliar o efeito estimulatório das TtEFs sobre a produção de citocinas (IL-10, IL-12 p40, TNF-α, IL-5 e IL-13) por CMSP, essas células foram obtidas de doadores saudáveis e cultivados na presença das TtEFs (50 ug de proteína/mL). Efeitos inibitórios foram avaliados por incubação das células com as TtEFs e: (a) uma concentração subótima de LPS (4 UE/mL), para IL-10 e TNF-α, (b) LPS e interferon (IFN) γ (100 ηg/mL), para IL-12 p40, e (c) fitohemaglutinina (5 μg/mL), para IL-5 e IL-13. As citocinas foram quantificados por ELISA de captura. Oito das frações (TtEF6, TtEF8 a TtEF14), bem como o extrato somático de T. Trichiura, induziram a produção de IL-10, TNF-α e, em menor quantidade, IL-12 p40. Três das frações (TtEF8, TtEF9 e TtEF10) não induziram significantemente a produção de IL-12 p40. Nenhuma das frações estimulou a produção de IL-13. Estas oito frações que estimularam a produção de IL-10 foram analisados por eletroforese em gel de poliacrilamida na presença de dodecilsulfato de sódio e para a atividade inibitória sobre a produção de citocinas por CMSP estimuladas. Todas as frações inibiram a produção de pelo menos uma citocina, mas nenhuma delas inibiu a produção de IL-10. Apenas uma das frações (TtEF9) inibiu significativamente a produção de todas as outras citocinas analisadas. TtEF10 inibiu a produção de TNF-α, IL-5 e IL-13; TtEF11 reduziu a produção de ambos TNF-α e IL-12 p40; três frações (TtEF6, TtEF12 e TtEF13) inibiram apenas a produção de TNF- α e duas frações (TtEF8 e TtEF14) inibiram significativamente apenas a produção de IL-5. Os resultados obtidos indicam que T. trichiura produz mais de uma molécula com atividade imunomodulatórias. TtEF9, que, além de estimular a produção de IL-10, sub-regula a produção de todas as outras citocinas estudadas, é o melhor candidato para conter uma molécula imunoregulatória que deve ser caracterizada e purificada à homogeneidade, a fim de investigar a sua utilização como um agente terapêutico para doenças imunomediadas. / Salvador

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.

Background: Soil transmitted helminth infections (STHI) are important Neglected Tropical Diseases (NTD). The three main STHI are infections with Ascaris lumbricoides, Trichuris trichiura and hookworms. STHI have a significant effect on the growth and development of children. A national survey for STHI in El Salvador by Pan American Health Organization and Ministry of Health in 2012 in school children aged 8years to 10 years. The survey collected data on age, gender, behavioral habits, and source of drinking water, type of toilet facility used and ecological zone of residence. A) We did an analyses of the data with an aim to determine the prevalence of STHI in El Salvador, assess the risk factors and risk interactions. B) We also aimed to determine the efficacy of urea as a potential additive for inactivation of Ascaris suum in solar toilets. Methods: A) Data from 1310 subjects was analysed for determination of prevalence of STHI in El Salvador. Risk factor assessment was done by chi-square test, unadjusted logistic regression and fully adjusted logistic regression. Risk factor interactions was tested on multiplicative and additive scale. B) Urea was tested for efficacy in inactivation of Ascaris suum ova in 20 solar toilets. Under conditions of controlled pH and moisture, concentration of gas ammonia, peak temperature were measured along with duration of treatment with urea to determine viability of Ascaris suum samples placed in the solar toilets. Results: I) The prevalence of Ascaris lumbricoides in 8-10 year old school children is 2.75%, Trichuris trichiura is 4.1% and hookworm is 1.83%. A) For Ascaris lumbricoides infection: Significant risk in individuals from volcanic chains and central depression compared to those from the mountains. Spring or well water when used as source of drinking water was associated with higher risk of infection when compared with piped water. Higher infection was also associated with open air defecation compared to use of septic tank or flush toilet. Use of sandals or no footwear was associated with a higher risk of infection when compared to use of closed footwear at all times. B) For Trichuris infection: Coastal plains were associated with a higher risk of infection compared to the mountains while rural status was protective against infection. Spring or well water when used as source of drinking water was associated with higher risk of infection when compared with piped water. Use of sandals or no footwear was associated with a higher risk of infection when compared to use of closed footwear at all times. C) For hookworm infection: Risk of infection was higher in individuals from urban regions. Spring or well water when used as source of drinking water was associated with higher risk of infection when compared with piped water. Use of sandals or no footwear was associated with a higher risk of infection when compared to use of closed footwear at all times. Poor handwashing was shown to be protective against infection with hookworm. Significant risk factor interactions were identified for infection with each of the three soil transmitted helminths. II) Urea as an additive at 1%w/w to feces tested in solar toilets showed an inactivation rate of nearly half the Ascaris suum ova samples. Fifty percent or higher inactivation rates were associated with ammonia gas concentrations of 109.5 ppm or higher and duration of treatment of 72 hours or higher. Conclusions: Prevalence of STHI in 8-10 year old school children for 2012 in El Salvador is low. Significant risk factors for STHI in El Salvador are eco-epidemiologic zone, source of drinking water, type of sanitation, use of shoes behavior and urban status of place of residence. Use of urea for inactivation of soil transmitted helminth ova in feces is a possible intervention for environmental control of STHI.