Characterisation of cuticular inflation development and ultrastructure in Trichuris muris using correlative X-ray computed tomography and electron microscopy.

The parasitic nematode Trichuris trichiura is a significant burden on public health in developing countries, and currently available drugs exhibit a poor cure rate. Worms live within a specialised tunnel of host intestinal epithelial cells and have anterior-ventral projections of the cuticle termed "cuticular inflations", which are thought to be involved in host-parasite interactions. This work aimed to characterise structure and suggest a function of cuticular inflations in the most tractable and widely-used model of trichuriasis, Trichuris muris. Using scanning electron microscopy, we show for the first time that most cuticular inflations develop between the second and third larval moults. Correlative X-ray computed tomography (CT)-steered Serial Block Face Scanning Electron Microscopy (SBF-SEM) and transmission electron microscopy enabled ultrastructural imaging of cuticular inflations, and showed the presence of an additional, web-like layer of cuticle between the median and cortical layers of the inflation. Additionally, we characterised variation in inflation morphology, resolving debate as to the inflations' true shape in situ. Cells underlying the inflations had many mitochondria, and we highlight their potential capacity for active transport as an area for future investigation. Overall, insights from the powerful imaging techniques used provide an excellent basis for future study of cuticular inflation function.

First paleoparasitological study of micromammal coprolites from the holocene of the Somuncurá Plateau Protected Natural Area (Patagonia Argentina).

The Somuncurá Plateau is a Protected Natural Area located in the middle of the northern extra-Andean arid Patagonia. Inhabited by at least 20 small mammal species, is the place with the uppermost species richness in Patagonia. The aim of this study was to examine the parasite remains from micromammal coprolites collected in association with a bone sequence recovered at the east of the Somuncurá Plateau (site "Alero Las Lechuzas"). Coprolites came from the four temporal units previously defined: unit I (4790 ±â€¯100 yrs. 14C B.P.), unit II, unit III (7840 ±â€¯120 yrs. 14C B.P.) and unit IV. Each coprolite was processed, rehydrated, homogenized, processed by spontaneous sedimentation and examined using a light microscope. Coprolites and eggs were described, measured and photographed. Samples were positive for two nematode species: Helminthoxys caudatus Freitas, Lent & Almeida, 1937 (Oxyurida, Oxyuridae) and Trichuris spp. (Trichinellida: Trichuridae). This is the first paleoparasitological study developed for the Somuncurá Plateau Protected Area. Moreover, this is the first time that the genus Helminthoxys is reported from ancient times worldwide. Coprolites were attributed to the mountain cavy Microcavia australis (Rodentia, Caviidae).The presence of H. caudatus for the Middle Holocene of northern Patagonia contributes to the study of the history of the histricomorphs and pinworms relationships.

Morphological-molecular characterization and phylogenetic relationships of a new Trichuris species (Nematoda: Trichuridae) parasitic on Holochilus chacarius (Cricetidae: Sigmodontinae) from the Chaco ecoregion (Argentina).

A new Trichuris species isolated from Holochilus chacarius (Cricetidae: Sigmodontinae) from the Chaco ecoregion in Argentina is described based on morphological characteristics and mitochondrial (cox1, cob) and nuclear (ITS2) markers. The new species is distinguished from 27 species of Trichuris from North and South American rodents based on morphological and biometrical features, such as the absence of a spicular tube, presence of a cylindrical spicular sheath, non-protrusive vulva, length of spicule, and proximal and distal cloacal tube. In addition, the results based on three molecular markers of the new species and three Trichuris species previously analysed from sigmodontine rodents: Trichuris pardinasi, Trichuris navonae and Trichuris bainae confirmed that the specimens here studied belong to a different species. Molecular data are further used to discuss the phylogenetic relationships among the Trichuris species of rodents from Argentina. The combined analysis of mitochondrial genes (cox1 and cob) revealed four clades corresponding with four different species of Trichuris. T. navonae (Akodontini rodents) and T. massoiai n. sp. as a sister group related to T. bainae (Oryzomyini rodents) and separated of T. pardinasi (Phyllotini rodents).

A new species of Trichuris Roederer, 1761 (Nematoda: Trichuridae) from Heteromys gaumeri Allen & Chapman (Rodentia: Heteromyidae) in Yucatan, Mexico.

In Mexico, four species of Trichuris Roederer, 1761 have been recorded in wild rodents belonging to the family Heteromyidae. In the present paper, we describe a new species based on specimens collected from Heteromys gaumeri Allen & Chapman (Heteromyidae: Heteromyinae) in the tropical forests of the Yucatan Peninsula, Mexico. Trichuris silviae n. sp. can be differentiated from the congeners described in North and South American rodents by morphological and morphometric features, such as the possession of a wide spicular tube, a thicker proximal cloacal tube, a shorter distal cloacal tube and a cylindrical spicular sheath. This is the first description of a Trichuris spp. from heteromyid rodents in Mexico and the fourth in North America. Despite the broad distribution of Heteromys spp., few cases of Trichuris infection have been reported. Further studies are necessary to verify if the new species is present in other heteromyid rodents in order to increase our knowledge about its geographical and host distribution.

Trichuris trichiura in a post-Colonial Brazilian mummy.

Trichuris trichiura is a soil-transmitted helminth which is prevalent in warm, moist, tropical and subtropical regions of the world with poor sanitation. Heavy whipworm can result either in Trichuris dysenteric syndrome - especially in children - or in a chronic colitis. In heavy infections, worms can spread proximally and may cause ileitis. Here we provide first microscopic evidence for a T. trichiura adult worm embedded in the rectum of a post-Colonial Brazilian adult mummy. During Colonial and post-Colonial times, many European chroniclers described a parasitic disease named Maculo whose symptomatology coincides with heavy helminthiasis. Based on our findings and on comparison of ancient textual evidence with modern description of heavy whipworm, we feel confident in considering that the two syndromes are expressions of the same pathological condition.

Secretion of RNA-Containing Extracellular Vesicles by the Porcine Whipworm, Trichuris suis.

Trichuris suis is a common parasitic helminth of pigs. As with many other parasites, T. suis ensures its own survival by evading host immune responses, but little is known about how this is achieved. MicroRNAs (miRNA) have been shown to be involved in various immunological processes by post-transcriptional regulation of specific genes, and the potential of using these molecules as biomarkers of disease is currently being examined. It has recently been shown that parasites may secrete extracellular structures such as exosomes and microvesicles, containing proteins and miRNA. The fusion of these structures with host cells has been demonstrated, and a role of exosome-derived miRNA in host gene regulation has been suggested. In the present study, we show that exosome- and microvesicular-like structures are secreted by T. suis L1 larvae and also demonstrate the presence of miRNA-sized RNA inside these structures. A potential role of these molecules in host-parasite interactions is suggested. In addition, an electron-dense layer covering the surface of the larvae was observed, which may play a function in host immune evasion.

Trichuris colobae n. sp. (Nematoda: Trichuridae), a new species of Trichuris from Colobus guereza kikuyensis.

In the present work, a morphological and biometrical study of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae) parasitizing Colobus guereza kikuyensis has been carried out. Biometrical and statistical data showed that the mean values of individual variables between Trichuris suis and Trichuris sp. from C. g. kikuyensis differed significantly (P < 0.001) when Student's t test was performed: seven male variables (width of esophageal region of body, maximum width of posterior region of body, width in the place of junction of esophagus and the intestine, length of bacillary stripes, length of spicule, length of ejaculatory duct, and distance between posterior part of testis and tail end of body) and three female variables (width of posterior region of body, length of bacillary stripes, and distance of tail end of body and posterior fold of seminal receptacle). The combination of these characters permitted the discrimination of T. suis with respect to Trichuris sp. from C. g. kikuyensis, suggesting a new species of Trichuris. Furthermore, males of Trichuris sp. from C. g. kikuyensis showed a typical subterminal pericloacal papillae associated to a cluster of small papillae that were absent in males of T. suis, while females of Trichuris from Colobus appeared with a vulval region elevated/over-mounted showing a crater-like appearance. The everted vagina showed typical triangular sharp spines by optical microscopy and SEM. Thus, the existence of a new species of Trichuris parasitizing C. g. kikuyensis has been proposed.

Comparative analysis of Trichuris muris surface using conventional, low vacuum, environmental and field emission scanning electron microscopy.

The whipworm of the genus Trichuris Roederer, 1791, is a nematode of worldwide distribution and comprises species that parasitize humans and other mammals. Infections caused by Trichuris spp. in mammals can lead to various intestinal diseases of human and veterinary interest. The morphology of Trichuris spp. and other helminths has been mostly studied using conventional scanning electron microscopy of chemically fixed, dried and metal-coated specimens, although this kind of preparation has been shown to introduce a variety of artifacts such as sample shrinking, loss of secreted products and/or hiding of small structures due to sample coating. Low vacuum (LVSEM) and environmental scanning electron microscopy (ESEM) have been applied to a variety of insulator samples, also used in the visualization of hydrated and/or live specimens in their native state. In the present work, we used LVSEM and ESEM to analyze the surface of T. muris and analyze its interaction with the host tissue using freshly fixed or unfixed hydrated samples. Analysis of hydrated samples showed a set of new features on the surface of the parasite and the host tissue, including the presence of the secretory products of the bacillary glands on the surface of the parasite, and the presence of mucous material and eggs on the intestinal surface. Field emission scanning electron microscopy (FESEM) was also applied to reveal the detailed structure of the glandular chambers in fixed, dried and metal coated samples. Taken together, the results show that analysis of hydrated samples may provide new insights in the structural organization of the surface of helminth parasites and its interaction with the infected tissue, suggesting that the application of alternative SEM techniques may open new perspectives for analysis in taxonomy, morphology and host-parasite interaction fields.

SEM studies on Trichurus muris from Psammomys obessus from Sinai Peninsula, Egypt.

The whipworm Trichuris muris was recovered from the caecum of the wild rodent Psammomys obessus trapped from Sinai Peninsula, Egypt. The cuticular surface ultrastructure is described using SEM. T. muris is closely related to other Trichuris species but can be distinguished from them mainly by differences in the posterior end of males. Details of the surface such as the bacillary gland, cuticular inflations and several morphological details obtained by scanning electron microscopy confirmed the characteristics that differentiate the species. P. obessus (Cretzschmar, 1828) is considered a new host record and Sinai is considered a new locality for the genus. This may through light on the spread of T. muris between Asia and Africa.

New species of Trichuris (Nematoda:Trichuridae) from Akodon montensis, Thomas, 1913, of the Paranaense Forest in Argentina.

Species of Trichuris (Nematoda:Trichuridae) parasitize a broad range of mammalian hosts. To date, 21 Trichuris species infecting nine families of rodents have been found in North and South America. Trichuris navonae n. sp. is described on the basis of specimens recovered from a species of forest-dwelling mice, Akodon montensis (Cricetidae: Sigmodontinae), from nine localities of Misiones Province, Argentina. A comparison with all the species of Trichuris from North and South American rodents is given. The separation of the new species of Trichuris is based on morphologic and morphometrica features, such as the absence of a spicular tube, the presence of a cylindrical spicular sheath with sharp spines, a non-protrusive vulva, a long anterior-posterior portion of the body, a lengthy spicule, and a proximal and distal cloacal tube. This is the third record of this genus in rodents of the Sigmodontinae from Argentina and the fifth record from South American rodents. Despite the large number of potential host species, only about 1.9% of sigmodontine rodent species have been reported as hosts of Trichuris spp. It is suggested that this number represents but a small fraction of Trichuris spp. that occur in sigmodontine rodents, and that additional survey of this group should yield additional species.

Comparative analysis of destruction of the infective forms of Trichuris trichiura and Haemonchus contortus by nematophagous fungi Pochonia chlamydosporia; Duddingtonia flagrans and Monacrosporium thaumasium by scanning electron microscopy.

The present study aimed to demonstrate by scanning electron microscopy (SEM) the in vitro predatory activity of nematophagous fungi Pochonia chlamydosporia (VC1 and VC4 isolates) Duddingtonia flagrans (AC001 isolate) and Monacrosporium thaumasium (NF34a isolate) on eggs of Trichuristrichiura and infective larvae (L3) of Haemonchus contortus. The work was divided into two experimental tests (A and B). In tests A and B, the predatory activity of nematophagous fungi P. chlamydosporia, D. flagrans and M. thaumasium on eggs of T. trichiura and H. contortus L3 was observed. After 6h, in test A, isolates P. chlamydosporia (VC1 and VC4) had a role in destroying eggs of T.trichiura. For fungi D. flagrans and M. thaumasium the ovicidal activity on T. trichiura eggs was not observed. Test B showed that D. flagrans (AC001) and M. thaumasium (NF34a) were capable of predating H. contortus L3, but no predation by the fungus P. chlamydosporia was seen. These fungi can offer potential for the biological control of nematodes.

Scanning electron microscope study of ancient parasite eggs recovered from Korean mummies of the Joseon Dynasty.

We have previously shown that parasite eggs have been identified in the coprolites of Korean mummies. These eggs have shed light on parasitic infection patterns in Korean populations living several hundred years ago. We conducted a scanning electron microscopy (SEM) study on ancient Trichuris trichiura, Ascaris lumbricoides, Metagonimus yokogawai, Paragonimus westermani, and Gymnophalloides seoi eggs recovered from Korean mummies of the Joseon Dynasty. We anticipated that the taphonomic conditions of mummification would alter the eggs of certain species but not of others. Our SEM data show that each species of ancient egg exhibited different degrees of preservation. Thus, some of them, for example, M. yokogawai, exhibited a better preservation status than others, suggesting that they should be the first candidates considered when choosing subjects for future paleoparasitological studies.

What are the infectious larvae in Ascaris suum and Trichuris muris?

In the present study, larvae of Ascaris suum and Trichuris muris were investigated by light and electron microscopy after incubation in a hatching medium containing 89% phosphate-buffered saline (pH 7.4), 10% RPMI-1640 and 1% sodiumhypochlorite at 40 and 37 degrees C, respectively. The larvae were obtained from fertilised eggs of the worms during defined phases of development (A. suum, 36th-50th day of development; T. muris, once a week from week 16 to 20). Light and electron micrographs of the larvae gave evidence that the third larval stage of A. suum is probably the infectious stage. The first moult of the larvae had already taken place before the 36th day of incubation starting at day 1. After 36 days of incubation, only the second larval stage was found within eggs. Some of these larvae were coated by a separated sheath so that a second moult of the larvae is reasonable. On the other hand, no sheathed larvae of T. muris were found in the eggs incubated for 20 weeks in distilled water. No signs of moult were seen for 20 weeks neither on light nor on the electron micrographs. Therefore, in T. muris, the first larval stage is the infectious stage, which was proven by means of re-infections of mice 16, 18 or 20 weeks after incubation of the eggs.

Light and electron microscopic studies on two nematodes, Angiostrongylus cantonensis and Trichuris muris, differing in their mode of nutrition.

The morphological characteristics of the adult heteroxenous blood nematode Angiostrongylus cantonensis and the adult monoxenous intestinal nematode Trichuris muris were compared with special regard to the ultrastructure of their digestive systems. The small circular mouth of A. cantonensis appears sucker like. The very narrow mouth of T. muris is surrounded by three lips covered by the cuticle that extends into the buccal space. In the buccal cavity of A. cantonensis, a single tooth occurs opposite to a cutting plate, while no teeth are present in T. muris. The lumen of the well-developed muscular pharynx of A. cantonensis shows a trifurcated star-like cross-section. The anterior segment of the bipartite pharynx presumably functions as a pump. The lumen of the bipartite pharynx and esophagus of T. muris exhibits a very narrow oval cross-section and possesses no musculature. It is composed of a long column of stichosome cells. The esophagus region is lined inside by bands of bacillary cells as well as outside by two longitudinal rows of funnel-like papillae. These structures may be involved in the uptake of nutrients by T. muris. The gland cells might excrete digestive exoenzymes, while the bacillary cells take up the predigested nutrients. The presence of many vesicles suggests a vesicular transport of the material into the pharynx. The intestinal epithelium of A. cantonensis is densely covered with short microvilli. The lumen itself is filled with red blood cells originating from host blood. The intestine of T. muris has a thick epithelium being placed on a basal lamina and shows long thin microvilli. The intestinal lumen is very narrow and free from particles or granules. This again suggests that T. muris lives on low molecular nutrients resorbed from the environment. The epithelium cells of the intestine of T. muris contain glycogen and electron light granules but are lacking mitochondria. This finding may indicate that the epithelium cells have an anaerobic energy metabolism. This statement fits with the fact that the habitat of the worm, the cecum, is largely anaerobic.

Examen paleoparasitológico de sedimentos de un sitio arqueológico, Río Mayo, Chubut, Argentina / Paleoparasitological analysis of sediments of an archaeological site, Río Mayo, Chubut, Argentina

En este trabajo, que tiene como objetivo el estudio paleoparasitológico, se analizan sedimentos procedentes del rescate arqueológico de un entierro múltiple en un alero rocoso, cercano a la localidad de Río Mayo, al sudoeste del Chubut. Se obtuvieron sedimentos extraídos de la región pélvica de un esqueleto humano procedente de Patagonia y vinculado al período de contacto europeo-aborigen. Mediante técnicas cuantitativas y cualitativas se registraron huevos de anoplocefálidos y de Trichuris sp. La inspección macroscópica previa de la muestra evidenció fecas de roedor y su examen paleoparasitológico evidenció huevos de anoplocefálidos, Trichuris sp, Capillaria sp y un ascadidido no identificado. Por lo tanto, se advierte la posible contaminación del depósito con parásitos de roedores.

New weapons in the war on worms: identification of putative mechanisms of immune-mediated expulsion of gastrointestinal nematodes.

Parasitic nematode infections of humans and livestock continue to impose a significant public health and economic burden worldwide. Murine models of intestinal nematode infection have proved to be relevant and tractable systems to define the cellular and molecular basis of how the host immune system regulates resistance and susceptibility to infection. While susceptibility to chronic infection is propagated by T helper cell type 1 cytokine responses (characterised by production of IL-12, IL-18 and interferon-gamma), immunity to intestinal-dwelling adult nematode worms is critically dependent on a type 2 cytokine response (controlled by CD4+T helper type 2 cells that secrete the cytokines IL-4, IL-5, IL-9 and IL-13). However, the immune effector mechanisms elicited by type 2 cytokines in the gut microenvironment that precipitate worm expulsion have remained elusive. This review focuses on new studies that implicate host intestinal epithelial cells as one of the dominant immune effector cells against this group of pathogens. Specifically, three recently identified type 2 cytokine-dependent pathways that could offer insights into the mechanisms of expulsion of parasitic nematodes will be discussed: (i) the intelectins, a new family of galactose-binding lectins implicated in innate immunity, (ii) the resistin-like molecules, a family of small cysteine-rich proteins expressed by multiple cell types, and (iii) cytokine regulation of intestinal epithelial cell turnover. Identifying how the mammalian immune response fights gastrointestinal nematode infections is providing new insights into host protective immunity. Harnessing these discoveries, coupled with identifying what the targets of these responses are within parasitic nematodes, offers promise in the design of a new generation of anti-parasitic drugs and vaccines.

A new species of Trichuris (Nematoda: Trichuridae) from Phyllotini rodents in Argentina.

A new species of Trichuris is described. Trichuris pardinasi n. sp. was recovered from Phyllotis xanthopygus Waterhouse (Rodentia: Muridae: Phyllotini) in Sierra de la Ventana, Buenos Aires Province, and Pampa de Achala, Córdoba Province (Argentina). This is the first record of Trichuris parasitizing Phyllotini rodents. The new species can be differentiated from the other 10 species parasitizing rodents from South America by the absence of the spicular tube, spicular sheath with spines uniformly distributed, the length of spicule, the J-shaped proximal cloacal tube, and the nonprotrusive vulva. Also, a description of the bacillary band is provided. The present and the future findings of shared parasite fauna from both populations of P. xanthopygus in these disjunct areas will support the hypothesis of a continuous distribution of this host species at a past time.

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris.

Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.

Adaptation of a nematode parasite to living within the mammalian epithelium.

Trichuris muris is a large metazoan pathogen that has been proposed to live intracellularly within living host intestinal epithelial cells. We sought to determine how Trichuris bores its way through the mucosal epithelium and to elucidate the parasite strategies for taking advantage of this intracellular niche. Since the apical surface of the mucosal epithelium is stabilized by the actin cytoskeleton and cell junctions, it remains intact over the worm following its entry into cells. In contrast, non-stabilized lateral membranes of the host epithelial cells are ruptured and cells are killed to form an inert syncytial tunnel. The ventral surface of the nematode worm is studded by pores that overlie bacillary cells; these pores penetrate through the cuticle and are in direct contact with host cytoplasm. From scanning electron micrographs of isolated worms, we calculate that each adult contains approximately 50,000 bacillary cells. The apical surface of the bacillary cells is extensively folded into plicae 40 nm in diameter, thereby increasing the surface area many-fold. Bacillary cells lack organelles for enzyme synthesis and secretion and fail to export protons. However, by confocal light microscopy it was observed that fluorescent macromolecules in excess of 100,000 Da can penetrate into the pores. Taken together, we conclude that the bacillary cells are essential for living inside host epithelium and function predominantly in absorption of soluble molecules from the host mucosal cytoplasm, in essence behaving as an external gut epithelium that is protected from abrasion by the cuticle that surrounds the openings of the bacillary cells.

Scanning electron microscopy of Trichuris trichura.

Scanning electron microscopy (SEM) studies have been carried out on adult and ova of T. trichura to provide the additional topographical details which are not obtainable by the light microscopy. The egg of T. trichura has a chitinous shell which consists of layers of dense lamellae and bordered by a limiting membrane. An operculum and a collar made of chitin form the opercular area. The lamallae of the egg shell are diffuse with numerous micropores. Cuticular pores are found scattering on the surface of the adult T. trichuira. The spicule sheath in male is cylindrical and covered with spines of different shape, size and distribution along its length. Spines are compact proximally, reduced in number on the middle part and lacking distally. SEM of the cloacal part showed paracloacal papillae.