Tegument Ultrastructure and Morphology of the Capsule Surrounding the Tetrathyridia of the Genus Mesocestoides Vaillant, 1863 in the Liver of the Root Vole.

The ultrastructure of the tegument of encapsulated tetrathyridia of the genus Mesocestoides Vaillant, 1863 (Cestoda, Cyclophyllidea, Mesocestoididae) from the liver of root voles Microtus oeconomus (Pallas, 1776) and the structure of the three-layered capsule surrounding them were studied for the first time. Several types of extracellular structures were noted on the surface of the tetrathyridia tegument: vesicles, fine granular material, and vacuoles. In addition, the phenomenon of shedding microtriches, which have expanded parts, was found. Host cells in contact with extracellular material show signs of destruction. A characteristic feature of the capsules surrounding the tetrathyridia is the reticular structure of the fibrous layer containing both native and degenerating inflammatory cells.

The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs.

BACKGROUND: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 µM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed. CONCLUSION, SIGNIFICANCE: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.

Morphological and Molecular Characterization of Post-Larval Pre-Tetrathyridia of Mesocestoides sp. (Cestoda: Cyclophyllidea) from Ground Skink, Scincella lateralis (SAURIA: SCINCIDAE), FROM SOUTHEASTERN OKLAHOMA.

Free pre-tetrathyridia of Mesocestoides sp. are described, for the first time, from samples obtained from the coelomic cavity of a ground skink, Scincella lateralis, from Oklahoma. Closer examination of these early-stage tapeworms revealed they were transitional metamorphosis stages between a post-hexacanth procercoid form to the full metacestode of Mesocestoides. A series of transitional stages was found that span the full period of sucker and apical organ development. However, we did not see any fully developed tetrathyridia, i.e., having classic Mesocestoides morphology but with the apical sucker absent following developmental atrophy. This is the first time that metamorphic pre-tetrathyridial stages of a Mesocestoides sp. have been reported in vivo from a natural infection. These observations corroborate earlier reports of such stages of Mesocestoides vogae developed in vitro, though the previously reported isolate of M. vogae is asexually proliferative, and the species from the present study showed no sign of asexual proliferation. The fact that these immediately post-hexacanth stages can occur in a single lizard intermediate host may suggest that Mesocestoides spp. might develop through a simple 2-host life cycle rather than an obligate 3-host cycle that has been speculated to occur by most previous authors. DNA sequence comparisons and phylogenetic analyses based on mitochondrial 12S, cox1, and nad1 genes have demonstrated that our specimens from S. lateralis represent a species clearly distinct from all previously sequenced Mesocestoides and closely related to 2 forms from domestic dogs and Channel Island fox in California previously published as Mesocestoides sp. C.

Morphological characteristics of Mesocestoides canislagopodis (Krabbe 1865) tetrathyridia found in rock ptarmigan (Lagopus muta) in Iceland.

Necropsies of 1010 rock ptarmigans (Lagopus muta) sampled in autumn 2006-2015 in northeast Iceland revealed Mesocestoides canislagopodis tetrathyridia infections in six birds (0.6 %), two juvenile birds (3 month old), and four adult birds (15 months or older). Four birds had tetrathyridia in the body cavity, one bird in the liver, and one bird both in the body cavity and the liver. There were more tetrathyridia in the body cavity of the two juveniles (c. 50 in each) than in three adults (10-40), possibly indicating a host-age-related tetrathyridia mortality. Approximately, half of tetrathyridia in the body cavity were free or loosely attached to the serosa, the other half were encapsulated in a thin, loose connective tissue stroma, frequently attached to the lungs and the liver. Tetrathyridia in the liver parenchyma incited variably intense inflammation. Tetrathyridia from the juvenile hosts were whitish, heart-shaped, and flattened, with unsegmented bodies with a slightly pointed posterior end. In the adult hosts, tetrathyridia were sometimes almost rectangular-shaped, slightly wider compared to those in the juveniles, but more than twice as long as the younger-aged tetrathyridia. Tetrathyridia infections are most likely acquired during the brief insectivorous feeding phase of ptarmigan chicks, and the tetrathyridia persist throughout the lifespan of the birds.

Tetrathyridia of Mesocestoides lineatus in Chinese snakes and their adults recovered from experimental animals.

Morphological characteristics of Mesocestoides lineatus tetrathyridia collected from Chinese snakes and their adults recovered from experimental animals were studied. The tetrathyridia were detected mainly in the mesentery of 2 snake species, Agkistrodon saxatilis (25%) and Elaphe schrenckii (20%). They were 1.73 by 1.02 mm in average size and had an invaginated scolex with 4 suckers. Adult tapeworms were recovered from 2 hamsters and 1 dog, which were orally infected with 5-10 larvae each. Adults from hamsters were about 32 cm long and those from a dog were about 58 cm long. The scolex was 0.56 mm in average width with 4 suckers of 0.17 by 0.15 mm in average size. Mature proglottids measured 0.29 by 0.91 mm (av.). Ovaries and vitellaria bilobed and located in the posterior portion of proglottids. The cirrus sac was oval-shaped and located median. Testes were follicular, distributed in both lateral fields of proglottids, and 41-52 in number per proglottid. Gravid proglottids were 1.84 by 1.39 mm (av.) with a characteristic paruterine organ. Eggs were 35 by 27 µm in average size with a hexacanth embryo. These morphological characteristics of adult worms were identical with those of M. lineatus reported previously. Therefore, it has been confirmed that the tetrathyridia detected in 2 species of Chinese snakes are the metacestodes of M. lineatus, and 2 snake species, A. saxatilis and E. schrenckii, play the role of intermediate hosts.

A high resolution analysis of the structure and chemical composition of the calcareous corpuscles from Mesocestoides corti.

Mesocestoides corti (syn. vogae), similar to many other cestode platyhelminthes, contains abundant calcium carbonate structures called calcareous corpuscles. These concretions that may constitute as much as 40% of the dry weight of the body, and were proposed to form intracellularly in certain parenchymal cells. As an approach to elucidate the biological role of calcareous corpuscles in cestodes, our aim was to characterize more precisely the structure and topological composition of the corpuscles from M. corti. Employing a variety of high resolution technical approaches, we found that the calcareous corpuscles are spheroid or ovoid layered concretions. They are formed by topographically homogeneous but compositionally heterogeneous layers, suggesting a cyclic process of biomineralization. The layers are composite structures, with granules of tens of nanometers, each surrounded by a cortex of about eight nanometers.

Tetrathyridia of Mesocestoides lineatus in Chinese Snakes and Their Adults Recovered from Experimental Animals

Morphological characteristics of Mesocestoides lineatus tetrathyridia collected from Chinese snakes and their adults recovered from experimental animals were studied. The tetrathyridia were detected mainly in the mesentery of 2 snake species, Agkistrodon saxatilis (25%) and Elaphe schrenckii (20%). They were 1.73 by 1.02 mm in average size and had an invaginated scolex with 4 suckers. Adult tapeworms were recovered from 2 hamsters and 1 dog, which were orally infected with 5-10 larvae each. Adults from hamsters were about 32 cm long and those from a dog were about 58 cm long. The scolex was 0.56 mm in average width with 4 suckers of 0.17 by 0.15 mm in average size. Mature proglottids measured 0.29 by 0.91 mm (av.). Ovaries and vitellaria bilobed and located in the posterior portion of proglottids. The cirrus sac was oval-shaped and located median. Testes were follicular, distributed in both lateral fields of proglottids, and 41-52 in number per proglottid. Gravid proglottids were 1.84 by 1.39 mm (av.) with a characteristic paruterine organ. Eggs were 35 by 27 microm in average size with a hexacanth embryo. These morphological characteristics of adult worms were identical with those of M. lineatus reported previously. Therefore, it has been confirmed that the tetrathyridia detected in 2 species of Chinese snakes are the metacestodes of M. lineatus, and 2 snake species, A. saxatilis and E. schrenckii, play the role of intermediate hosts.

Normal and aberrant Mesocestoides tetrathyridia from Crocidura spp. (Soricimorpha) in Corsica and Spain.

Tetrathyridia of Mesocestoides sp. were collected from the body cavities of the shrews (Insectivora), Crocidura russula, in Valencia, Spain and Crocidura suaveolens on the Mediterranean island of Corsica, France. Specimens were processed by routine microscopic and histological techniques, including examination with brightfield, phase-contrast, and differential-interference-contrast optics. Most tetrathyridia were clustered together inside host-derived fibrotic capsules, but some occurred free in the body cavity. All specimens examined from both locations had solid hindbodies, i.e., lacking a primary lacuna, thus conforming to the plerocercoid metacestode type; all possessed a single normal tetra-acetabulate scolex. All metacestodes from C. russula in Valencia were normal tetrathyridia. Those from C. suaveolens in Corsica were either normal tetrathyridia or had aberrant deep convolutions of an unusually elongated hindbody. No tetrathyridium from either location or host showed tegumental or excretory duct anomalies such as those reported by several authors from aberrant tetrathyridia and spargana in some other locations. No definitive evidence of asexual proliferation was visible in any of the tetrathyridia, but those with abnormally convoluted hindbodies from a single C. suaveolens in Corsica suggest the potential for asexuality by fission of the hindbody. These results add to our understanding of morphological and developmental variation among metacestodes in this widespread and variable genus.

Molecular and morphological circumscription of Mesocestoides tapeworms from red foxes (Vulpes vulpes) in central Europe.

Here we examine 3157 foxes from 6 districts of the Slovak Republic in order to determine for the first time the distribution, prevalence and identity of Mesocestodes spp. endemic to this part of central Europe. During the period 2001-2006, an average of 41.9% of foxes were found to harbour Mesocestoides infections. Among the samples we confirmed the widespread and common occurrence of M. litteratus (Batsch, 1786), and report the presence, for the first time, of M. lineatus (Goeze, 1782) in the Slovak Republic, where it has a more restricted geographical range and low prevalence (7%). Using a combination of 12S rDNA, CO1 and ND1 mitochondrial gene sequences together with analysis of 13 morphometric characters, we show that the two species are genetically distinct and can be differentiated by discrete breaks in the ranges of the male and female reproductive characters, but not by the more commonly examined characters of the scolex and strobila. Estimates of interspecific divergence within Mesocestoides ranged from 9 to 18%, whereas intraspecific variation was less than 2%, and phylogenetic analyses of the data showed that despite overlapping geographical ranges, the two commonly reported European species are not closely related, with M. litteratus more closely allied to North American isolates of Mesocestoides than to M. lineatus. We confirm that morphological analysis of reproductive organs can be used to reliably discriminate between these often sympatric species obtained from red foxes.

Interactions between anomalous excretory and tegumental epithelia in aberrant Mesocestoides tetrathyridia from Apodemus sylvaticus in Spain.

Tetrathyridia of Mesocestoides sp. (Platyhelminthes: Cestoda) were collected from the body cavities of Apodemus sylvaticus (Rodentia: Muridae) in Murcia, Spain, and processed by routine microscopical and histological techniques, including examination with brightfield, phase-contrast, and differential-interference-contrast optics. All specimens examined had solid hindbodies (i.e., lacked a primary lacuna), thus conforming to the plerocercoid metacestode type. The vast majority of tetrathyridia were highly aberrant, often lacking a scolex, varying greatly in body size, and exhibiting buds or duplexed body forms indicative of asexual proliferation. All of the aberrant forms, including those with normal scoleces and apparent absence of proliferation, possessed anomalous multi-lobed invaginations of the hindbody tegument, which were attached at many points to abnormally dilated excretory ducts. The tegumental and excretory epithelia were joined basally, but did not share a common lumen. These abnormal connections between these aberrant tegumental modifications and the abnormally dilated excretory ducts have not been described previously for any metacestode. This report contributes to understanding previously published reports of abnormalities in other plerocercoid metacestodes, including Mesocestoides spp. and diphyllobothriidean spargana.

Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates.

A hypothesis-based framework was used to test if 3 genetic strains of Mesocestoides (clades A, B, and C) are distinct evolutionary lineages, thereby supporting their delimitation as species. For comparative purposes, 3 established cestode species, Taenia pisiformis, Taenia serialis, and Taenia crassiceps were assessed using the same methods. Sequence data from mitochondrial rDNA (12S) and the second internal transcribed spacer of nuclear rDNA (ITS-2) revealed derived (autapomorphic) characters for lineages representing clade A (n = 6 autapomorphies), clade B (n = 4), and clade C (n = 9) as well as T. pisiformis (n = 15) and T. serialis (n = 12). Furthermore, multivariate analysis of morphological data revealed significant differences among the 3 genetic strains of Mesocestoides and between T. pisiformis and T. serialis. The level of phenotypic variation within evolutionary lineages of Mesocestoides and Taenia spp. tapeworms was similar. Results from this study support recognizing Mesocestoides clades A, B, and C as separate species, and provide evidence that clade B and Mesocestoides vogae are conspecific.

The spatial organisation of the central nervous system of Mesocestoides corti as revealed by microinjection of carbocyanine dye.

The carbocyanine dyes DiI, DiA and DiO were microinjected into the cerebral ganglion of intact Mesocestoides corti tetrathyridia to determine the spatial organisation and connectivity patterns of the CNS. Of the dyes tested, DiI proved to be the most effective, giving highly fluorescent and persistent staining of even very fine calibre afferent and efferent nerve fibres. DiI labelling, in conjunction with transmission electron microscopy, revealed the nervous system to consist of sensory endings, directly connected to the cerebral ganglion by elongated cellular tracts, efferent nerve fibres which innervated the suckers, and longitudinal nerve cords which travelled along the remainder of the body.

The effect of the tetrathyridia of Mesocestoides corti on the livers and peripheral blood of three different strains of mice.

Three strains of male and female mice, CFLP, BALB/c and CBA/ca, were infected i.p. with the tetrathyridia of Mesocestoides corti and examined in groups of 5 at 0, 7, 14, 21, 28 and 42 days post-infection. At post-mortem the numbers of parasites both loose in the peritoneal cavity and in the liver tissue were counted, the livers weighted fresh and sections of liver stained to examine the inflammatory response, encapsulation of the tetrathyridia and for eosinophils, neutrophils, mast cells and plasma cells. BALB/c mice had significantly more parasites loose in the peritoneal cavity than CFLP and CBA/ca mice. Infected livers of all three strains were significantly heavier than control livers; the heaviest livers were those of the CFLP followed by the CBA/ca mice which also showed the greatest rate of weight increase. The tetrathyridia from the peritoneal cavities of the CBA/ca and CFLP mice were covered with a 'mucilage-like' substance. Tetrathyridia within the host capsules of the CBA/ca mice contained host cells. There was no strain difference with regard to numbers of tetrathyridia in the liver but male mice harboured significantly more parasites than females. Differences in the numbers of cell types within the liver were detected between the strains but no one strain showed any consistent pattern. There was an overall increase in total white blood counts as well as an increase in the number of eosinophils, monocytes, neutrophils and lymphocytes during the course of the infection.