The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.

Evasion of an immune response mounted by a host is fundamental to the survival of a parasite. Immune evasion can be mediated in many ways from the production of molecules by the parasite which mimic cytokines produced by the human immune system to hiding from the immune system by locating within host cells. The production of immune cell mediating molecules in excretory secretory products is another means by which the parasite can tailor its surroundings to facilitate prolonged survival. The hypothesis of immunosuppression by parasite products, in particular members of the Venom Allergen Like (VAL) family, is key to this thesis. VAL proteins are members of the much larger SCP/TAPS family, which covers proteins from parasitic helminths such as Heligmosomoides polygyrus (H.polygyrus)to the free-living nematode Caenorhabditis elegans. These nematodes may have one or more genes encoding proteins that contain the SCP/TAPS domains often choosing to express these proteins at critical points within the helminths lifecycle. Phylogenetic analysis of a selection of these proteins revealed that their classification could be determined based upon the number of SCP/TAPS domains. Alternatively the presence or absence of the signal sequence combined with conserved cysteine residue data could be used. Further investigations into possible functions of the VAL proteins from H.polygyrus were carried out using recombinant protein produced in an insect cell expression system. To further examine the function of VAL genes a system that allows the heterologous expression of a gene in the well-documented Leishmania infection setting was employed. In vitro and in vivo studies were carried out which examined various infection parameters. Parasite infectivity in bone marrow derived macrophages in vitro along with cytokine production was observed. In vivo the development of lesions and subsequent parasite recovery from infected mice gave indications of changes in virulence that could be attributed to the presence and expression of the HpVAL genes. The ability of parasites to ameliorate symptoms of allergic and autoimmune diseases is now well documented with the most extreme use of this knowledge resulting in administration of an active parasitic infection as a treatment regime. We hope to identify individual molecules from a parasite that is known to reduce allergic symptoms in the allergic airway inflammation (AAI) model and produce these in a more structured and regulated fashion. It is plausible that VAL proteins from H.polygyrus may possess these regulatory properties, as has been shown for the excretory secretory products (HES) of the parasite; to that end HpVAL-1 and HpVAL-4 were tested in the allergic airway inflammation model and were shown to reduce both cell numbers in the bronchioalveolar lavage fluid and eosinophilia. Finally, the position of the parasite and products secreted by the parasite was examined. Directly labelled HES and recombinant VAL proteins were used to identify binding sites inside the parasite and within the parasites’ locality in the host i.e. the gut. Confocal microscopy revealed binding of HES to the parasites surface and internal structures and of both HES and HpVAL-4 to goblet cells and Paneth cells inside the gut. Paneth cells may affect parasite survival by influencing the gut microbiota and goblet cells have been shown to influence parasite persistence by production of mucus. Thus HES and more specifically HpVAL proteins may, through their interactions with these cells, interfere with mechanisms employed by the host to expel the parasite.

Urinary schistosomiasis, which is caused by the blood fluke Schistosoma haematobium, is a tropical disease infecting over 100 million people in sub-Saharan Africa. Infection typically involves repeated re-infection with long-lived parasites, and field studies have demonstrated that protective immunity takes many years to develop in humans. In communities with endemic schistosomiasis, distinctive patterns of infection and schistosome-specific antibody responses are seen, including a peaked age-infection curve, a highly aggregated distribution of infection intensities among individuals, and an age-related switch in the subclasses of antibody produced. The antibody switch, which occurs naturally in older children, is also seen in younger children following treatment with the antihelminthic drug praziquantel, which kills adult worms. This study aimed to identify the important mechanisms underlying the slow development of protective immunity, using a mathematical modelling approach. Deterministic population-level and stochastic individual-based models were developed that describe how levels of infection and antibody change with age for individuals living in endemic communities. These models were used to explore different hypotheses for the slow development of protective immunity: (1) that schistosome parasites actively suppress protective immune responses; (2) that dying worms provide the main antigenic stimulus for protective immunity and (3) that a threshold level of antigen must be experienced before a protective immune response is initiated. Models were assessed for their ability to simultaneously reproduce different robust patterns of infection and antibody responses identified in cross-sectional and post-treatment field data from Zimbabwe. It was found that significant immunosuppression by schistosomes was not consistent with population-level patterns of infection intensity, including the peaked age-infection curve. In order to explain both age-related and post-treatment changes in infection intensity and antibody responses, including the antibody switch, protective antibody responses had to be stimulated by antigens from dying worms. Additionally, it was shown that these protective responses reduced worm fecundity rather than reducing rates of re-infection. An antigen threshold was found to be consistent with observed field patterns, but was not necessary to explain them. From a large number of possible models that were considered, a single model structure and a subset of parameter combinations were identified that were consistent with field data. This model was used to predict the longer-term impact of mass-treatment programmes upon the development of protective immunity, and the consequent effects on infection levels.

The mammalian intestinal tract is highly colonised with a diverse bacterial microbiota. The importance of this bacterial presence is now recognised; these bacteria contribute both to the nutritional status of their hosts and are required for the development of a competent immune system. In addition, the composition of the microbiota is likely important in influencing how the immune system reacts to antigens, as the presence of specific bacterial species can promote differentiation of T cells towards specific effector or regulatory fates. Though the ability of the microbiota to influence infections with bacterial and viral agents has been reported, whether the microbiota can affect a parasitic infection has not yet been described. It is likely, due to millions of years of co-evolution within mammalian hosts, that helminths have co-opted mechanisms of the microbiota to manipulate the host’s immune system, in order to promote their own survival. In this thesis, the immune parameters required for expulsion of a primary infection with the murine gastrointestinal helminth parasite Heligmosomoides polygyrus are examined, and whether the microflora influence these parameters in order to modulate susceptibility is explored. Firstly, a multiparameter analysis of H. polygyrus infection was performed in two mouse strains which differ in susceptibility to a primary infection, to identify both immune factors and microbial populations which correlate with susceptibility to infection. BALB/c mice exhibited a stronger T helper (Th)2-type response to H. polygyrus excretory-secretory antigen (HES), produced high numbers of intestinal granulomas following infection and were better able to expel H. polygyrus, whereas the more susceptible C57BL/6 strain produced higher levels of inflammatory Th1 cytokines in response to HES. High levels of duodenal Lactobacillus/Lactococcus species positively correlated with H. polygyrus persistence within the BALB/c host, as did high levels of Enterobacteriaceae in the C57BL/6 host. Furthermore, the abundance of both of these bacterial groups was elevated in H. polygyrus-infected C57BL/6 mice compared to naïve controls, and mice given antibiotic treatment to diminish these groups were rendered more resistant to H. polygyrus. Infection persistence was prolonged in BALB/c mice which were administered the single species Lactobacillus taiwanensis, a normal component of the microbiota. Next, the impact of a loss of microbiota signalling by immune cells during H. polygyrus infection was examined, through the use of Toll-like receptor (TLR)- and TLR adaptor protein-deficient mice. MyD88-/- mice were more resistant to H. polygyrus than wildtype (Wt) C57BL/6 mice and exhibited increased granuloma formation: phenotypes which were not recapitulated by individual deficiencies in TLR2, TLR4, TLR5 or TLR9, and not seen in TRIF-/- mice. When MyD88-/- mice were additionally deficient in TRIF, the increased granuloma formation phenotype of MyD88-/- mice was lost. Whether MyD88 controls susceptibility to H. polygyrus infection via a TLR-independent mechanism, and how MyD88 and TRIF antagonistically contribute to granuloma formation remains to be resolved. Finally, the importance of TGF-β signalling during H. polygyrus infection was examined, using mice deficient in TGF-β signalling specifically in T cells (TGF-βRII DN mice). These mice were more susceptible to H. polygyrus than Wt C57BL/6 mice, which was explained by an attenuated Th2 response to infection accompanied by exuberant IFN-γ production. The increased susceptibility to H. polygyrus was lost in TGF-βRII DN IFN-γ-/-mice, in which Th2 responsiveness was partly restored. These data highlight the importance of both immune components, particularly IFN-γ, which promotes susceptibility, and the presence of specific intestinal bacterial populations in controlling the persistence of a primary H. polygyrus infection.

Thesis (Ph.D. (Zoology)) --University of Limpopo, 2012 / The diversity of the helminth parasites of fishes in a clear-water, subtropical Lake Tzaneen, in South Africa was investigated. Of the 527 fish specimens sampled approximately 9000 parasites were collected. There are 38 different parasite species discussed comprising 27 Monogenea, 3 Digenea, 4 Cestoda, 3 Nematoda and 1 Acanthocephala. Four new monogenean species are described and these are Dactylogyrus spp. 1 to 4. Three monogenean species are introduced as first records in Africa and these are Actinocleidus fusiformis (Mueller, 1934), Haplocleidus furcatus Mueller, 1937 and Acolpenteron ureteroecetes Fischthal & Allison, 1940. Fourteen monogenean and one acanthocephalan species are discussed as first geographical records for South Africa and these are Gyrodactylus rysavyi Ergens, 1973, Dactylogyrus brevicirrus Paperna, 1973, Dactylogyrus cyclocirrus Paperna, 1973, Dogielius dublicornis Paperna, 1973, Dogielius sp., Schilbetrema quadricornis Paperna & Thurston, 1968, Quadriacanthus aegypticus El Naggar & Serag, 1986, Quadriacanthus clariadis Paperna, 1961, Scutogyrus gravivaginus (Paperna & Thurston, 1969), Cichlidogyrus quaestio Douëllou, 1993, Cichlidogyrus halli Price & Kirk, 1967, Cichlidogyrus sclerosus Paperna & Thurston, 1969, Cichlidogyrus dossoui Douëllou, 1993, Cichlidogyrus tilapiae Paperna, 1960 and Acanthosentis tilapiae Baylis, 1948. Seven species are discussed as first records for their hosts and these are Cichlidogyrus dossoui, Cichlidogyrus halli and Acanthosentis tilapiae on Oreochromis mossambicus; Dactylogyrus sp. 1 on Barbus radiatus and Barbus trimaculatus; Dactylogyrus sp. 2 on Barbus unitaeniatus; Dactylogyrus sp. 3 and Dactylogyrus sp. 4 on Labeo molybdinus. Gyrodactylus rysavyi is the only species with a first site (gills) record. The other monogenean species discussed are Macrogyrodactylus clarii Gussev, 1961, Macrogyrodactylus karibae (Douellou and Chishawa, 1995), Dactylogyrus afrolongicornis afrolongicornis Paperna, 1973, Dactylogyrus allolongionchus Paperna, 1973, Dactylogyrus spinicirrus (Paperna & Thurston, 1968) and Cichlidogyrus philander (Douëllou, 1993). The digeneans discussed are Glossidium pedatum Looss, 1899 and the larvae of Diplostomum van Nordmann, 1832 and Clinostomum Leidy, 1856. The Cestodes discussed are Proteocephalus glanduligerus (Janicki, 1928) Fuhrmann, 1933, Polyonchobothrium clarias Woodland, 1925 and the larvae of Ligula intestinalis Goeze, 1782 and family Gryporhynchidae. The nematodes discussed are Procamallanus laevionchus (Wedl, 1861), Paracamallanus cyathopharynx Baylis, 1923 and larvae of Contracaecum Railliet and Henry, 1912. Monogenea were commonly found on the gills but less on the skin and in the urinary bladder. Digenea were found mainly in the eyes, brain and visceral cavity, with only one species (Glossidium pedatum) present in the intestines of Clarias gariepinus. Cestoda and Nematoda were found in the intestine and body cavity. Only one species of Acanthocephala (Acanthosentis tilapiae) was found in the intestines of Oreochromis mossambicus. No definite seasonal variations of infection and parasite affinities towards the sexes and the sizes of the hosts could be determined. The lake is oligotrophic with the water quality having no influence on the parasite diversity and species richness. / the University of Limpopo Research Office, and the National Research Foundation

The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites to affect the farming of sheep and goats. T.circumcincta infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all currently available classes of anthelmintic drugs, it is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Infection of sheep with T.circumcincta triggers local changes in the abomasum characteristic of a T helper type-2 (Th2) driven immune response, including local eosinophilia, mastocytosis and increased mucus production, which leads to expulsion of the parasite. However, this protective immunity develops slowly during repeated exposure, wanes rapidly, and does not appear to be evident in young lambs. Vaccination to provoke early onset of protective immunity has therefore been suggested as an alternative means of control in the face of spreading anthelmintic resistance. Greater understanding of the development of immunity to T.circumcincta, and why this is delayed in lambs, would be useful in vaccine development. This thesis focuses on cytokine transcription profiling of the ovine abomasal mucosa and local lymphatic tissues. Changes in cytokine transcription over the course of a challenge infection with T.circumcincta were defined in helminth naïve sheep, and in previously infected sheep which have developed a degree of immunity during an eight week trickle infection, to clarify the mechanisms by which this immunity is orchestrated. This work demonstrated a clear Th2 cytokine response in the abomasal mucosa over the course of infection, which developed earlier and was more pronounced in the previously infected sheep; possibly owing to a population of polarised Th2-type cells built up during the previous infection. Suppression of Th1 cytokine transcription was also a prominent finding in the draining lymph node, which likewise occurred earlier in the previously infected sheep. Repetition of this experiment using younger lambs provided a possible explanation for the reduced resistance to T.circumcincta in this age group. While Th2 and proinflammatory cytokine responses in the abomasal mucosa demonstrated similar trends to those found in the older sheep, little suppression of Th1 cytokine transcription was observed in the draining lymph node. It is therefore suggested that the increased susceptibility of young lambs to T.circumcincta is not due to an inability to generate adequate Th2 responses, but an inability to suppress transcription of antagonistic Th1 cytokines.

Solid organ transplantation is the gold standard treatment for a variety of conditions that result in organ failure. However, despite considerable advances in clinical transplantation in recent decades, the almost ubiquitous requirement of life-long immunosuppression of transplant recipients persists and is complicated by graft loss to rejection in the long term and multiple serious adverse effects that are frequently life limiting. Helminths currently infect more than one quarter of the world’s population and it is now well established that their success as parasites is the result of active immunomodulation of the host immune response. Whilst this primarily secures ongoing survival of the parasites, in some cases helminth-induced immunomodulation can be beneficial to the infected host and is not associated with the adverse sequelae of pharmacological immunosuppression. An emerging body of evidence suggests that harmful immune responses to alloantigens can be suppressed by helminths, but little mechanistic data exists and the active immunomodulators involved have remained hitherto unidentified. The hypothesis behind this thesis is that the model intestinal nematode, Heligmosomoides polygyrus, produces immunomodulatory molecules that can suppress responses to allo- and auto-antigens in animal models of transplantation and autoimmunity, and that some of these molecules could potentially be exploited as novel therapeutic agents. Full-thickness skin grafting was performed between fully-allogeneic mouse strains (BALB/c to C57BL/6). Recipient mice infected with H. polygyrus immediately prior to transplantation showed significantly prolonged allograft survival. Likewise, protection from allograft rejection could be replicated in recipient mice in which H. polygyrus excretory-secretory products (HES) (isolated from culture of adult worms) were delivered by continuous infusion via surgically implanted osmotic minipumps. A number of potential mechanisms underlying allograft protection were identified including induction of CD4+CD25+Foxp3+ regulatory T cells (Treg) and suppression of Th1 and Th17 effector CD4+ T cell phenotypes. H. polygyrus and HES were further shown to ameliorate disease in murine (pMOG) experimental autoimmune encephalomyelitis and colitis induced by T cell transfer. In addition to expansion of Treg, H. polygyrus-mediated protection against EAE was found to be almost completely lost in IL-4 receptor deficient mice, indicating a protective role of Th2 immune responses in this context. Finally, the mechanisms of action of the newly-identified TGF-β mimic, TGM, contained within HES were investigated. Despite bearing no sequence homology or structural resemblance to TGF-β, TGM was shown to act through the TGF-β receptor complex to induce Treg in human and mouse CD4+ T cells in vitro and to suppress murine allogeneic skin graft rejection in vivo. TGM may represent the origin of a safe, effective and long-overdue novel alternative to current immunosuppression therapy.

This study was designed to provide the foundation for future work on the ecology and evolution of Necturus-parasite systems by providing vital parasite survey information for the aquatic salamander Necturus alabamensis. During this study 115 N. alabamensis were collected from north Florida. Hosts yielded the cestode Proteocephalus loennbergi, monogeneans from the genus Sphyranura, digeneans from the genus Gorgoderina, and what appears to be several nematode species. Nematodes, digeneans, and cestodes were abundant while monogeneans were rare. Monogenean and cestode parasites were categorized as specialists (parasites specific to one species or the species of a single genus), and digenean and nematode parasites were categorized as generalists (infecting numerous, possibly distantly related hosts). Statistical analysis did not indicate a parasite load difference between sites for digeneans and cestodes but did indicate a difference for nematodes. Helminth populations appear to be overdispersed (the majority of the parasites are found in only a few hosts). This study provides insight into some aspects of the ecological relationships of N. alabamensis and its helminths. However, parasite surveys of the other members of Necturus are necessary for the evaluation of evolutionary relationships within this system.

<p> Helminth parasite infection induces a strong immune response in the host aimed at destroying the parasite and reducing any associated inflammation. In humans and rodents, this response is dominated by the Th2 cytokines and involves the activation of mast cells, B cells and eosinophils, as well as increased production of lgE. There is evidence from both murine and human studies that the helminth-induced anti-inflammatory response is also capable of protecting the host from co-existing autoimmune disease, including asthma, allergies and colitis. My hypothesis is that infection with the parasitic helminth H. diminuta can treat and/or prevent the symptoms of Th1-dominated inflammation. This anti-colitic effect is dependent on IL-10 and involves the recruitment of the alternatively activated macrophages.</p> <p> Using the dinitrobenzene sulfonic acid (DNBS) model of colitis, I examined the ability of the rat tapeworm, Hymenolepis diminuta, to reduce inflammation in a non-permissive mouse host. H. diminuta was chosen as the ideal candidate for treating intestinal disease because it is non-invasive, does not have hooks or teeth which can damage the host, and can be easily maintained in the laboratory and controlled during experimental infection. Mice that received DNBS alone developed colitis within 72 hours. Mice that had been previously infected with five H. diminuta larvae were significantly protected from the colitis, as measured by reduced clinical disease and histological damage scores as well as reduced levels of myeloperoxidase (MPO) from colonic tissue samples. It was also determined that the anticolitic effect was dependent on a viable parasite infection. Infection with H. diminuta induced an increase in colonic IL-10 mRNA and IL-10 secretion by stimulated splenocytes - when IL-10 was blocked by administration of an anti-IL-10 antibody, the anticolitic effect of H. diminuta infection was reduced. H. diminuta infection also induced increased expression of the alternatively activated macrophage (AAM) markers arginase 1 and FIZZ1. Treatment with in vitro-derived AAMs reduced the symptoms of DNBS-colitis.</p> <p> The effect of H. diminuta infection on oxazolone colitis, a chemically induced colitis characterized by increases in IL-4 and IL-13, was also examined. Infection with H. diminuta induced a significant increase in inflammation and inhibited recovery from oxazolone-colitis. Increases in IL-5 and eosinophils were also observed. Further examination revealed that increased IL-5, induced by administration of an adenovirus carrying the IL-5 gene, had a deleterious impact on the oxazolone colitis, exacerbating inflammation and increasing eosinophilia.</p> <p> While the idea of helminth therapy may be unappealing, there is increasing interest in the use of helminth parasites for the treatment of inflammatory disease. There is some concern, however, that the Th2 response induced by H. diminuta infection could exacerbate some disorders involving increases in the Th2 cytokines. Thus, while this therapy may be beneficial for most, careful characterization of the immunological basis of any pre-existing disorders would be necessary in order to avoid any harmful side-effects.</p> / Thesis / Doctor of Philosophy (PhD)

Orientador: João Aristeu da Rosa / Banca: Vera Lucy de Santi Alvarenga / Banca: Vera Lucia Pagliusi Castilho / Resumo: Blastocystis hominis é protozoário causador da infecção intestinal denominada blastocistose humana, cujo diagnóstico é realizado pelo exame coproparasitológico e por meio de técnicas de colorações permanentes que foram utilizadas neste estudo para avaliar a prevalência de Blastocystis hominis nos espécimes fecais de habitantes na região de Araraquara-SP. Foram estudadas 503 amostras de fezes submetidas às técnicas de exame direto a fresco, de Faust e cols., de Lutz e de Rugai, Mattos e Brisola, além das colorações pela hematoxilina férrica, tricrômio e de Kinyoun modificada. Do total das amostras analisadas 174 (34,6%) apresentaram-se positivas para a presença de parasitas intestinais. O protozoário e helminto mais freqüentes foram respectivamente: Entamoeba coli (14,6%) e Strongyloides stercoralis (6,7%). Blastocystis hominis foi observado em 23 (4,6%) amostras fecais com consistência predominantemente pastosa, não caracterizando quadro diarréico. Apesar da baixa prevalência de Blastocystis hominis encontrada na região de Araraquara, comparativamente a outras regiões brasileiras, é importante a realização do diagnóstico laboratorial desse protozoário. O encontro de Blastocystis hominis em material fecal é indicativo de contaminação de alimentos e água de consumo, desde que se admita a rota de transmissão oral-fecal deste parasita, o que implica na orientação da população sobre as medidas de saneamento básico e higiene como meio para se controlar problemas de saúde ocasionados pelos enteroparasitas. / Abstract: Blastocystis hominis is a protozoan which causes an intestinal infection called human blasticistosis. Its diganosis is perfomed by stool examination and permanent staining techniques. Such methodologies were carried out on the present study in order to evaluate the prevalence of Blastocystis hominis in faecal specimens from the Araraquara region inhabitants. A total of 503 faecal samples were evaluated by the following techniques: examination fo fresh specimens, Lutz, Faust et al. and Rugai et al. besides the iron hemotoxylin, trichrome and modified Kinyon staining. Out of 503 stool samples examined 174 (34,6) were found to be positive for intestinal parasites. The most prevalent protozoan and helminth parasites were Entamoeba coli (14,6%) and Strongyloides stercoralis (6,7%) respectively. Balstocystis hominis was present in 23 (4,6%) stool samples, most of all of soft consistence and without diarrheic reports. Blastocystis hominis laboratorial diagnosis is important althought its prevalence has been low in Araraquara region. Blastocystis hominis findings is faecal specimens indicates the food and water contamination and since the transmission of this parasite is iral-faecal it implies that the population needs orientation about hygiene and basic sanitation conditions in order to control health problems caused by enteroparasites. / Mestre