Ultrastructure and cytochemistry of intrauterine embryonic and larval stages of Ityogonimus lorum (Digenea: Brachylaimidae) involving transitory development of ciliated miracidia.

Results of the present study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs while in the most posterior uterine regions of Ityogonimus lorum, a digenean parasite of an Iberian mole, Talpa occidentalis (Eulipotyphla, Talpidae). Using transmission electron microscopy (TEM), the ultrastructural characteristics of diverse cell types and their organelles of these developing embryos and fully formed miracidia within the eggshell were examined. The eggshell and embryonic envelopes are similar to those described previously by many authors for other digeneans. However, the developing miracidia are unique among previously described digeneans in possessing transitory cilia during larvigenesis, but completely lacking cilia in fully formed miracidium larvae. The evidence for completion of miracidial maturation in intrauterine eggs is based on the presence of the following structures: (1) transitional stage of ciliated differentiating miracidial epithelium; (2) apical and lateral glands, characteristic for digenean miracidia; and (3) fully developed germinative cells grouped together in the germinative sac localized in the posterior region of the miracidium. The protonephridial system with its characteristic flame cells and the nervous system with diverse types of neurons and nerve centers, which are characteristic for other digenean species reported until now, are absent from all these developmental stages of I. lorum. Based on these observations, we hypothesize that the life cycle of I. lorum is entirely terrestrial, involving passive transmission by ingestion of eggs containing unciliated miracidia to the first intermediate host.

Functional ultrastructure of eggs and cellular organization of hexacanths of the cyclophyllidean cestode Thysanotaenia congolensis: a phylogenetic implication of obtained results.

The functional ultrastructure of eggs and cellular organization of hexacanths from gravid proglottids of Thysanotaenia congolensis, from black rats from Cape Verde, were examined by transmission electron microscopy. Mature eggs with fully formed hexacanths are grouped within parenchymatous capsules of gravid proglottids. Oncospheral envelopes surrounding mature hexacanths are reduced to a very thin membranous embryophore as their protective function is taken over by the parenchymatous capsules originating from the medullary parenchyma of immature proglottids and composed of three layers. Six major cell types are present: a bi-nucleate medullary centre; a six-nucleate U-shaped penetration gland; a second type of penetration gland; two neurosecretory-type nerve cells; about 30 somatic cells; and about 12 germinative cells. Present results on the functional ultrastructure of eggs and cellular organization of hexacanths support the phylogenetic distinction between T. congolensis and cestodes of the subfamily Anoplocephalinae.

Ultrastructural study of the egg wall surrounding the developing miracidia of the digenean Prosotocus confusus (Looss, 1894) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope.

Helminth eggs play a critical role in movement of the parasite from definitive to intermediate host. Eggs of the pleurogenid digenean trematode Prosotocus confusus (Looss, 1894), a parasite of naturally infected frogs Pelophylax lessonae (Amphibia: Ranidae) in Europe, are described here for the first time. Particular emphasis is placed on the ultrastructure on the egg wall and on the detailed description of a unique cocoon-like envelope. Each embryonating egg is composed of an early embryo surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate eggshell; (3) not fully formed, a differentiating outer embryonic envelope containing large nuclei of macromeres; and (4) situated below, an undifferentiated layer of the future inner embryonic envelope containing mesomere nuclei. Layers enveloping the egg apparently play an important role in the protection, metabolism, and storage of nutritive reserves for the developing miracidium. The outer anucleate layer, or cocoon, is situated externally to the eggshell and composed of an electron-lucent substance with numerous electron-dense islands attached to its peripheral membrane. A cocoon envelope such as this has never been seen in previous TEM studies of the eggs of parasitic platyhelminths, with the exception of another pleurogenid Brandesia turgida. The origin, formation, functional ultrastructure, and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. Numerous lysosome-like structures observed in some eggs may be involved in the autolysis of both the embryonic envelopes (particularly the early degeneration of macromere nuclei of the outer envelope, characteristic for this species) and in the disintegration of several early micromeres. The inner envelope, which forms later from mesomeres, persists longer during embryogenesis.

Functional ultrastructure of the parenchymatic capsules of the cestode Thysanotaenia congolensis (Cyclophyllidea, Anoplocephalidae, Inermicapsiferinae).

Proglottids from adult Thysanotaenia congolensis from naturally infected black rats Rattus rattus from Santiago Island, Cape Verde, were examined by transmission electron microscopy (TEM). The uterus in mature proglottids is composed of an ephemeral transverse tube or sac that breaks down, releasing eggs into the parenchyma where they are sequestered in groups and encapsulated by parenchymatous layers. In gravid proglottids, eggs accumulate in groups of 6-12 at the distal end of sac-like uterine ducts. As eggs accumulate, the end of the uterine ducts expands until it pinches off, releasing groups of eggs into the parenchyma surrounded by remnants of uterine epithelium. These epithelium-bound groups of eggs remain in the parenchyma until they are encapsulated with several parenchymatous layers, forming parenchymatic egg capsules, typical for mature and gravid proglottids of Inermicapsiferinae. The parenchymatic capsules originate from the medullary parenchyma of immature proglottids, which undergoes differentiation into the three layers of gravid proglottids: (1) an outer connective tissue layer composed of long delicate filaments of unknown chemical nature embedded in a granular extracellular matrix; (2) a middle layer appearing as an accumulation of large closely packed PAS-positive mucous goblets that are intensely metachromatic after toluidine blue staining and (3) an inner compact layer composed of lipid-containing cells, muscle cells with elongated muscle fibres and cells of various sizes and shapes forming or containing calcareous corpuscles. The mature hexacanths of T. congolensis are surrounded by reduced oncospheral envelopes consisting of remnants of a very thin membranous layer of degenerating embryophore with long, irregularly shaped cytoplasmic processes and by remnants of uterine epithelium extending as numerous apical microlamellae into the parenchymatic capsule lumen.

Ultrastructural evidence for completion of the entire miracidial maturation in intrauterine eggs of the digenean Brandesia turgida (Brandes, 1888) (Plagiorchiida: Pleurogenidae).

Results of this TEM study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs situated in the most posterior uterine regions of the pleurogenid trematode Brandesia turgida (Brandes, 1888). The ultrastructural characteristic of different larval organelles and cell types of these eggshell-enclosed, but fully formed, cilated miracidia is described. The body wall of the pyriform mature miracidium of B. turgida is composed of ciliated epidermis and underlying peripheral body musculature. Two miracidial flame cells of the protonephridial excretory system are localized in the central region of the ciliated larvae. Three types of miracidial glands were observed: a single apical gland, two lateral glands, and several small vesiculated glands; each gland type contains characteristic, but different types of secretory granules. The anterior end of each miracidium consists of an apical papilla on which are situated the exits of the three main larval glands: an exit of a single apical gland as well as the individual exits of two lateral glands. The exits of vesiculated glands, containing characteristic spherical membrane-bound and highly electron-dense granules, evidently different from the two other types of secretory granules of apical and lateral glands, were not identified. Germinative cells, grouped together in a sac-like germinative follicle, are situated in the medioposterior part of the larva, the germatophore. The germinative cells contain numerous electron-dense heterochromatin islands arranged in the form of a network or chain-like pattern and distributed mainly in the karyoplasm adjacent to the nuclear membrane. The thin layer of granular cytoplasm is rich in free ribosomes and contains a few small mitochondria. Both nuclear and cytoplasmic features if these cells indicate their great developmental potential for further growth and multiplication in postembryonic stages of the life cycle. In the mature eggs, the areas of focal cytoplasmic degradation were frequently observed and may be involved in the autolysis of some embryonic structures. Obtained results are compared with available literature data on the functional ultrastructure of the miracidia of other digeneans.

ULTRASTRUCTURE OF EGG ENVELOPES AND EARLY EMBRYOS OF ROHDELLA AMAZONICA (TREMATODA: ASPIDOGASTREA) PARASITIC IN BANDED PUFFER FISH, COLOMESUS PSITTACUS.

Egg structure and early embryonic development of the aspidogastrean, Rohdella amazonica, a basal trematode, were studied by transmission electron microscopy (TEM) to gain insight into functional, developmental, and phylogenetic characteristics. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard TEM methods. By the time of pronuclear fusion, the fertilized zygote was already enclosed in a thick, electron-dense pre-operculate eggshell and an underlying layer of vitellocytes that fused into a vitelline syncytium as they were still secreting their shell granules. When cleavage commenced, a small number of macromeres moved to the area just underneath the eggshell, where they fused to form a single syncytial embryonic envelope. Simultaneously, the smaller blastomeres continued to divide as they maintained contact with each other, but remained separate from the vitelline syncytium. Concurrent with these cellular changes, a thickened knob expanded at one pole of the eggshell and began to form an opercular suture. By the time the operculum was fully formed, the vitelline syncytium had mostly degenerated, while the smaller blastomeres had become cohesive as a single mass that preceded the differentiation and morphogenesis of the cotylocidium larva. The general pattern of cleavage and eggshell formation resembles that of other trematodes and polylecithal cestodes, but the single embryonic envelope has been reported only in a few basal taxa. The only other aspidogastrean studied in detail to date is very similar, indicating close phylogenetic affinity and conservatism within this basal neodermatan and neoophoran group.

ULTRASTRUCTURE AND CYTOCHEMISTRY OF LATE EMBRYOS AND COTYLOCIDIUM LARVAE OF ROHDELLA AMAZONICA (TREMATODA: ASPIDOGASTREA), FROM THE TROPICAL ESTUARINE FISH, COLOMESUS PSITTACUS.

Developmental ultrastructure of late embryos and cotylocidium larval morphogenesis of Rohdella amazonica, an aspidogastrean parasite of fish, were studied to reveal the functional aspects of larvigenesis within the egg as well as phylogenetically relevant characteristics of the embryos and larvae in this basal trematode group. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard methods of transmission electron microscopy (TEM) and cytochemistry. During late cleavage and rearrangement of the blastomeres, the vitelline syncytium that plays a role in eggshell formation and nutrient provision to the embryo completes its apoptotic degeneration as the embryonic mass grows substantially. Early larval morphogenesis involves cellular positioning that defines the anteroposterior polarity of the differentiating larva. Progressing through larvigenesis, the anterior end forms a muscular oral sucker surrounding the mouth, which leads inward into the pharynx and expanding digestive cavity. At the posterior end, a large disc forms as a precursor to the eventual ventral disc. The fully formed cotylocidium, still within the eggshell, is flexed ventrally, bringing the 2 poles into near juxtaposition. The neodermatan tegument with outwardly projecting small microvilli becomes fully formed, as myocytons, a protonephridial system, and 2 glandular regions occupy the body's interior. The ultrastructural features described here are very similar to those reported for Aspidogaster limacoides from fish and somewhat similar to those reported for Cotylogaster occidentalis from molluscs, but differ from the more diverse larvae of neodermatan taxa that have been studied more extensively.

Tannic Acid-Modified Silver Nanoparticles in Conjunction with Contact Lens Solutions Are Useful for Progress against the Adhesion of Acanthamoeba spp. to Contact Lenses.

Acanthamoeba spp. are amphizoic amoebae that are widely distributed in the environment and capable of entering the human body. They can cause pathogenic effects in different tissues and organs, including Acanthamoeba keratitis (AK), which may result in a loss of visual acuity and blindness. The diagnostics, treatment, and prevention of AK are still challenging. More than 90% of AK cases are related to the irresponsible wearing of contact lenses. However, even proper lens care does not sufficiently protect against this eye disease, as amoebae have been also found in contact lens solutions and contact lens storage containers. The adhesion of the amoebae to the contact lens surface is the first step in developing this eye infection. To limit the incidence of AK, it is important to enhance the anti-adhesive activity of the most popular contact lens solutions. Currently, silver nanoparticles (AgNPs) are used as modern antimicrobial agents. Their effectiveness against Acanthamoeba spp., especially with the addition of plant metabolites, such as tannic acid, has been confirmed. Here, we present the results of our further studies on the anti-adhesion potential of tannic acid-modified silver nanoparticles (AgTANPs) in combination with selected contact lens solutions against Acanthamoeba spp. on four groups of contact lenses. The obtained results showed an increased anti-adhesion activity of contact lens solutions in conjunction with AgTANPs with a limited cytotoxicity effect compared to contact lens solutions acting alone. This may provide a benefit in improving the prevention of amoebae eye infections. However, there is still a need for further studies on different pathogenic strains of Acanthamoeba in order to assess the adhesion of the cysts to the contact lens surface and to reveal a more comprehensive picture of the activity of AgTANPs and contact lens solutions.

Infection of Mammary Glands of Small Mammals in Eastern North America by Helminths.

To determine whether small mammals living in natural settings harbor helminth infections in their mammary glands, we conducted a survey of helminths infecting rodents and soricimorphs in three widespread locations in the eastern United States: states of New York, Tennessee, and Georgia. We examined all the primary organs in all hosts, and identified all helminths. We also excised the complete mammary glands within their subcutaneous fat pads, then stained and mounted each whole mammary gland set for microscopical examination. A total of 53 individual hosts were examined, including 32 Peromyscus spp., 11 Mus musculus, 5 Sigmodon hispidus, 4 Clethrionomys gapperi, and 1 Blarina carolinensis. Helminths collected included Heligmosomoides sp., Hymenolepisdiminuta, Hymenolepis nana, Pterygodermatites peromysci, Schistosomatium douthitti, Syphacia obvelata, Syphacia sigmodontis, and Trichostrongylus sigmodontis. Four S. hispidus were infected by T. sigmodontis in the small intestine; in all four, we also found nematode larvae in lactiferous duct lumen and lactogenic tissue of the mammary glands. We were unable to identify the species of nematode larvae, but the co-occurrence with T. sigmodontis in all cases may suggest an association. Future studies should seek to identify such larvae using molecular and other methods, and to determine the role of these mammary nematode larvae in the life cycle of the identified species. No other host species harbored helminths in the mammary glands. Overall, our results suggest that mammary infections in wild small mammals are not common, but warrant inclusion in future surveys.

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.

Ultrastructure of the early gravid uterus of Corallobothrium fimbriatum (Cestoda: Proteocephalidea).

The ultrastructure of the uterine epithelium and underlying extracellular matrices has been studied in several cyclophyllideans, but relatively little has been published on uterine fine structure in other cestode orders. To develop comparative data, we initiated a study focusing on a common and widespread genus of the order Proteocephalidea. Adult specimens of Corallobothrium fimbriatum were removed from the small intestine of brown bullhead catfish, Ameiurus nebulosus, from the St. Lawrence River in North America. Early gravid proglottids were examined by light microscopy as whole mounts or diced in buffered glutaraldehyde, processed by standard techniques and examined by as stained sections by light microscopy and by transmission electron microscopy. Voucher specimens were deposited in the Museum of Comparative Zoology at Harvard University. The uterus consisted of an epithelial wall supported basally by an extracellular matrix and parenchyma cells, which together formed epitheliomesenchymal villi at many points. The uterine epithelium consisted of a single thin syncytial layer, folded distally into long epithelial villi that protruded into the lumen at some points. The epithelium's numerous nuclei were very close to the lumen, but bulged along with the perinuclear cytoplasm proximally into the surrounding medullary parenchyma. Each nucleus contained small amounts of evenly dispersed heterochromatin and euchromatin, with a central nucleolus. Numerous mitochondria occurred in the cytoplasm, which was densely filled with free ribosomes but contained scant endomembrane components and showed little evidence of secretory activity. The uterine cells possessed few of the apical microvilli that are typical of some cyclophyllidean species, but the apical plasma membrane and cortical cytoplasm were extensively developed into long microlamellae similar to other species. The basic syncytial nature of the epithelium is similar to that of other cestodes studied previously, but the specific combination of features is unlike any previously described. Based on this one proteocephalidean species, we discerned no pattern that would distinguish between Proteocephalidea and Cyclophyllidea based on uterine structure.

Examination of oral biofilm microbiota in patients using fixed orthodontic appliances in order to prevent risk factors for health complications.

INTRODUCTION AND OBJECTIVE: In recent decades the use of orthodontic appliances in Poland has increased; however, data on their influence on changes of components of the microbiome connected with oral biofilm are scarce. The objective of this study was to evaluate oral microbiota in terms of their role as risk factors for health complications. MATERIAL AND METHODS: The study included 100 patients treated with removable or fixed appliances. Oral hygiene and gingival health were determined, and periodontal swabs taken from each patient for parasitological, bacteriological and mycological microscopic and in vitro examinations. RESULTS: Oral protists and various pathogenic and opportunistic bacterial and fungal strains were identified in the superficial layer of biofilm. A higher prevalence of bacteria, Enterococcus faecalis, E. faecium, Staphylococcus aureus and Escherichia coli, and various strains of yeast-like fungi from the Candida albicans group, occurred in patients treated with the fixed appliance than in those using a removable appliance or not treated orthodontically. In some periodontal samples from patients treated with fixed appliances, cysts of the Acanthamoeba spp. were found. CONCLUSIONS: The use of orthodontic appliances alters the status of the oral cavity; it has impact on the colonization of oral biofilm by opportunistic/pathogenic strains, and increases the risk of their dissemination to various human tissues and organs. Pretreatment examination of oral microbiome, its monitoring particularly during treatment with fixed appliances, and preventive elimination of the potentially pathogenic strains to avoid health complications, are highly recommended, especially in patients with impaired immunity.

Developmental and functional ultrastructure of Ornithodiplostomum ptychocheilus diplostomula (Trematoda: Strigeoidea) during invasion of the brain of the fish intermediate host, Pimephales promelas.

We examined tegumental development of the diplostomulum of Ornithodiplostomum ptychocheilus, with respect to structural transformations that have functional relevance to the invasion, migration, and site establishment processes in the brain of the fish second-intermediate host, Pimephales promelas. Using a combination of brightfield, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy (CM), we demonstrated that the diplostomula become established in the outer region of the optic lobes within 24-48 hr of penetration and continue to grow and transform over a period of 4-14 days. During this period, the J-shaped body consists of 2 distinct regions: (1) a highly motile prosoma with distinctive tegumental spines and (2) an opisthosoma, the tegument of which is elaborated into a dense uniform layer of long, thin microvilli. The prosoma is alternately invaginated into and everted from the opisthosoma, thus constituting a protrusible proboscis. By day 14 postinfection (PI), the body has lost this bipartite structure and has taken on the uniformly flattened form characteristic of metacercariae. The transitory complex structure of the diplostomula appears to be well suited to burrowing through host tissues (primarily by action of the prosoma), followed by rapid dissociation of host tissue and nutrient accumulation (primarily by action of the opisthosoma) in preparation for metacercaria encystment.

A standardised terminology of the embryonic envelopes and associated developmental stages of tapeworms (Platyhelminthes: Cestoda).

Over the past 40 years, much has been published on the ultrastructure and cellular development of embryonic structures in a wide range of cestodes. However, the literature contains many discrepancies in both terminology and interpretations because of the facts that these organisms are phylogenetically diverse within their respective orders and families, the habitats that affect embryonic envelope structure are diverse, and the work has been done in various laboratories around the world. This review and synthesis was initiated by a working group of biologists from around the world convened at the Fifth International Workshop on Cestode Systematics and Phylogeny in Ceské Budejovice, at the Institute of Parasitology of the Biology Centre, Academy of Sciences of the Czech Republic. It brings together the data from published work and establishes a uniform terminology and interpretation based on the data as they are presented. A consensus was reached for standardised definitions of the oncosphere, hexacanth, coracidium, embryonic envelopes, outer envelope, inner envelope, embryophore, vitelline capsule, shell, and outer coat. All of these are defined as components of the embryo or its vitellocyte-derived or uterine-derived coatings.

Ultrastructure of digenean trematode eggs (Platyhelminthes: Neoophora): A review emphasizing new comparative data on four European Microphalloidea.

Despite their tremendous diversity and their medical and veterinary importance, details of egg ultrastructure among the digenean trematodes has been studied rather little. The available literature is spread over several decades and several species, but has not been adequately reviewed to reveal patterns of similarity and divergence. We present this review to synthesize and analyse what is known from the available literature reporting studies using both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To support our general review of existing literature, we also have synthesized our own previously published descriptions, and present herein our new previously unpublished data. From these new electron micrographs, we provide a comparative analysis of the intrauterine eggs of four digenean species, representing four genera and three families of the superfamily Microphalloidea, collected from four different host wildlife species in four European countries: 1) Mediogonimus jourdanei (Prosthogonimidae) from Myodes glareolus (Mammalia: Rodentia), collected in France; 2) Maritrema feliui (Microphallidae) from Crocidura russula (Mammalia: Soricimorpha), collected in Spain; 3) Brandesia turgida (Pleurogenidae) from Pelophylax ridibundus (Amphibia: Anura: Ranidae), collected in Russia; and 4) Prosotocus confusus (Pleurogenidae) from Rana lessonae (Amphibia: Anura: Ranidae), collected in Belarus. All were studied by preparing whole worms by various techniques for TEM, so that eggs could be studied in situ within the uterus of the parent worm. Based on the literature review and the new data presented here, we describe basic similarities in patterns of embryogenesis and egg formation among all trematode species, but substantial variations in timing of larvigenesis, sculpturing of egg shell surfaces, and some other features, especially including accessory cocoon coverings outside the egg shells of B. turgida and P. confusus. In the future, many more studies are needed to explore egg ultrastructure in other digenean taxa, to explore potential phylogenetic patterns in egg development and structure, and to correlate structure with function in the life cycle.

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.

Effect of povidone iodine, chlorhexidine digluconate and toyocamycin on amphizoic amoebic strains, infectious agents of Acanthamoeba keratitis - a growing threat to human health worldwide.

INTRODUCTION: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. MATERIAL AND METHODS: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. RESULTS: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. CONCLUSIONS: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.

Immunoproteomics and Surfaceomics of the Adult Tapeworm Hymenolepis diminuta.

In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.

Synanthropic flies as vectors of Cryptosporidium and Giardia among livestock and wildlife in a multispecies agricultural complex.

The capacity of synanthropic flies belonging to the families Calliphoridae, Sarcophagidae, and Muscidae to serve as mechanical vectors of Cryptosporidium and Giardia among livestock and wildlife in a multispecies agricultural complex in northwest Georgia (USA) was studied using fluorescent in situ hybridization (FISH) and immunofluorescent antibody (IFA) techniques. Flies from all three families were carrying viable Cryptosporidium oocysts and Giardia cysts internally and externally, and deposited them on trap surfaces. Furthermore, the contaminated flies and trap surfaces occurred in association with each of the four domestic animal units (beef, dairy, equine, and sheep), and in wildlife areas dominated by deer (Odocoileus virginianus) and Canada Geese (Branta canadensis). Cryptosporidium was isolated from 55.56% of the fly samples, whereas Giardia was isolated from only 7.94%. The highest numbers of Cryptosporidium were associated with cattle and wildlife areas, while Giardia occurred in greatest numbers in the sheep area. Cryptosporidium was isolated comparably from exoskeletal elutants and fly homogenates, but Giardia was isolated primarily from homogenates, indicating primarily internal transport in the latter. Surfaces visited by the flies (i.e., traps) became contaminated by both Cryptosporidium (18.89%) and Giardia (1.11%). In conclusion, these flies can serve as mechanical vectors of both Cryptosporidium and Giardia in all of these settings, and undoubtedly play a role in movement of these pathogens among the various host species.

Mesocestoides lineatus (Goeze, 1782) (Mesocestoididae): new data on sperm ultrastructure.

Spermiogenesis and the ultrastructural characters of the spermatozoon of Mesocestoides lineatus are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a golden hamster (Mesocricetus auratus) after experimental infection with tetrathyridia metacestodes obtained from naturally infected lizards (Anolis carolinensis) from Louisiana. Spermiogenesis in M. lineatus is characterized by the orthogonal growth of a free flagellum, a flagellar rotation, and a proximodistal fusion. The zone of differentiation contains 2 centrioles associated with striated rootlets and a reduced intercentriolar body. The mature spermatozoon of M. lineatus lacks a mitochondrion, and it is characterized by the presence of (1) a single, spiraled, crested body 150 nm thick; (2) a single axoneme of the 9+'1' pattern of trepaxonematan Platyhelminthes; (3) a parallel and reduced row of submembranous cortical microtubules; (4) a spiraled cordon of glycogen granules; and (5) a spiraled nucleus encircling the axoneme.