The forgotten exotic tapeworms: a review of uncommon zoonotic Cyclophyllidea.

As training in helminthology has declined in the medical microbiology curriculum, many rare species of zoonotic cestodes have fallen into obscurity. Even among specialist practitioners, knowledge of human intestinal cestode infections is often limited to three genera, Taenia, Hymenolepis and Dibothriocephalus. However, five genera of uncommonly encountered zoonotic Cyclophyllidea (Bertiella, Dipylidium, Raillietina, Inermicapsifer and Mesocestoides) may also cause patent intestinal infections in humans worldwide. Due to the limited availability of summarized and taxonomically accurate data, such cases may present a diagnostic dilemma to clinicians and laboratories alike. In this review, historical literature on these cestodes is synthesized and knowledge gaps are highlighted. Clinically relevant taxonomy, nomenclature, life cycles, morphology of human-infecting species are discussed and clarified, along with the clinical presentation, diagnostic features and molecular advances, where available. Due to the limited awareness of these agents and identifying features, it is difficult to assess the true incidence of these 'forgotten' cestodiases as clinical misidentifications are likely to occur. Also, the taxonomic status of many of the human-infecting species of these tapeworms is unclear, hampering accurate species identification. Further studies combining molecular data and morphological observations are necessary to resolve these long-standing taxonomic issues and to elucidate other unknown aspects of transmission and ecology.

Prevalence survey of gastrointestinal and respiratory parasites of shelter cats in northeastern Georgia, USA.

The goal of this study was to assess the prevalence of gastrointestinal and respiratory parasites of shelter cats from northeast Georgia, thus promoting a more targeted approach in parasite diagnosis and treatment. Fecal samples of cats kept in a shelter located in Lavonia, northeastern Georgia, USA, were processed for the presence of parasites using double centrifugation sugar flotation (n = 103) and Baermann techniques (n = 98). Flotation revealed eggs of Toxocara cati (17.5%), Ancylostoma sp. (11.7%), Taeniidae (3.9%), Spirometra mansonoides (2.9%), Mesocestoides sp. (1%), Dipylidium caninum (1%), and Eucoleus aerophilus (1%), and oocysts of Cystoisospora felis (16.5%), and Cystoisospora rivolta (8.7%). Baermann diagnosed Aelurostrongylus abstrusus larvae in 5 cats (5.1%), while fecal flotation alone identified only 2 of these infections. Taeniidae eggs were identified to species-level by PCR and sequencing targeting the cytochrome oxidase c subunit 1 (cox1) of the mitochondrial DNA. All isolates belong to Hydatigera taeniaeformis sensu stricto, which is the first unequivocal report of the species in North America. Overall, 45.6% of the cats were infected with at least one parasite. This prevalence of infection is much higher than what is generally reported in client owned animals, highlighting the importance of using appropriate fecal diagnostic techniques to detect gastrointestinal and respiratory parasites on newly adopted cats. Correct diagnosis may direct appropriate treatment and control strategies, which would mitigate the risk of infection of other animals in household, and human exposure to zoonotic parasites.

Species delimitation based on mtDNA genes suggests the occurrence of new species of Mesocestoides in the Mediterranean region.

BACKGROUND: This study is the first contribution to the molecular taxonomy of Mesocestoides spp. from domestic and wild carnivores in the Mediterranean area. A total of 13 adult worms and 13 larval stages of Mesocestoides spp. were collected from domestic and wild carnivore hosts in Italy and Tunisia. Samples collected in the Slovak Republic were used as comparative samples from outside the Mediterranean. The genes cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) of the mitochondrial genome were used as molecular markers to investigate the presence of cryptic Mesocestoides species in the area analysed. RESULTS: Results were consistent in showing three well-supported clusters of Mesocestoides spp. in southern Italy and Tunisia, which were strongly divergent from Mesocestoides litteratus, M. corti and M. lineatus. High levels of genetic variation and no evidence of geographical structuring was found between the clusters. CONCLUSIONS: Considering the low dispersal capability of the intermediate hosts of Mesocestoides spp., the lack of geographical structuring among the Mediterranean regions could be due to a high potential for dispersion of the definitive hosts. This study provides a foundation for future formal descriptions of new species of the genus Mesocestoides in the Mediterranean area.

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.

Environmental determinants of the spatial distribution of Mesocestoides spp. and sensitivity of flotation method for the diagnosis of mesocestoidosis.

Mesocestoides spp. are zoonotic cestodes of wild and domesticated carnivores. Although the adult stages are relatively harmless intestinal parasites, the metacestode stages (tetrathyridia) can be responsible for life-threatening peritonitis and pleuritis in several species including dogs, cats, non-human primates and probably man. The aim of the present study was to reveal the spatial distribution pattern of Mesocestoides spp. in the most important final hosts, red foxes (Vulpes vulpes), to analyse the relationship of these patterns with landscape and climate by geographical information systems and to evaluate faecal flotation method for the detection of infection in the final host. Fox carcasses, representing 0.5% of the total fox population were randomly selected out of all the foxes of Hungary. The intestinal tract was examined by sedimentation and counting technique. The sensitivity of the flotation method was evaluated by the testing of the faecal samples of 180 foxes infected with Mesocestoides spp. The prevalence of infection was high in foxes (45.8%; 95% CI=41.0-50.6%), and the parasite was detected in all areas of Hungary. The high prevalence of the parasite in foxes suggests that the infection might also be common in outdoor dogs and cats. Mesocestoides infection could not be detected in any of the foxes by flotation method indicating that the sensitivity of the method is less than 0.6%. Therefore, almost all canine and feline infections remain undetected in the veterinary practice. Based on the statistical analysis, the altitude was the only determinant of the spatial distribution of Mesocestoides spp. indicating that infections in carnivores including dogs and cats can be expected mainly in midland regions (150-750 m above sea level). It might be attributed to the altitude-dependent species richness and abundance of the intermediate and final hosts of the parasite.

The first report of peritoneal tetrathyridiosis in squirrel monkey (Saimiri sciureus).

This report describes a case of peritoneal larval cestodiasis caused by tetrathyridia of Mesocestoides sp. in an adult female squirrel monkey. The monkey had lived in a zoological garden in Japan and had a clinical history of wasting. At necropsy, numerous whitish oval masses were found in the liver and peritoneal cavity. These masses contained larval cestodes. Morphological observation and molecular analyses of the mitochondrial 12S ribosomal RNA gene and cytochrome c oxidase subunit 1 gene sequences allowed us to identify the larva as the tetrathyridium of Mesocestoides sp. This is the first report of Mesocestoides larvae in a squirrel monkey in Japan.

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.

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.

First case of canine peritoneal larval cestodosis caused by Mesocestoides lineatus in Germany.

A 13-year-old Dalmatian dog was presented with a history of abdominal enlargement and reduced appetite for several months. Acute clinical signs were anorexia, vomiting and diarrhoea. During exploratory laparotomy, acute intestinal perforation due to a foreign body and peritonitis was diagnosed. In addition, the abdominal cavity was filled with multiple small (0.5 cm), white, cyst-like structures. Histopathology revealed typical cestode structures of the cyst walls but no protoscolices were found. PCR was performed with cestode specific primers of the mitochondrial 12S rDNA. The sequence showed a 99.75% identity with a Mesocestoides lineatus isolate published in the NCBI GenBank. This is the first case of canine peritoneal larval cestodosis (CPLC) in Germany and the first evidence of M. lineatus as causal agent for CPLC.

First larval record of Mesocestoides in carnivora of Tenerife (Canary Islands).

Larvae of Mesocestoides sp. were recovered in Tenerife (Canary Islands) in 2004 from the peritoneal cavities of 2 domestic dogs and a domestic cat. Morphological and molecular identification were carried out. Mesocestoides litteratus from Vulpes vulpes was sequenced for the first time using the ITS-2 region (18S rDNA), and was included in the phylogenetic analysis to compare the sequence variability among these and other Mesocestoides spp. belonging to different carnivores. Phylogenetic studies were carried out based on maximum parsimony and neighbor-joining analysis. The results showed the relationships between these and other previously published Mesocestoides species. Moreover, it is demonstrated that Mesocestoides sp. from Tenerife comprises a previously unreported sequence. This is the first larval record of Mesocestoides sp. in domestic animals from Tenerife, Canary Islands, Spain.

Proliferative peritoneal and pleural cestodiasis in a cat caused by metacestodes of Mesocestoides sp. Anatomohistopathological findings and genetic identification.

A 10-year-old female cat was brought to Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana for post-mortem examination. The animal used to live, together with 26 other cats, in the big terrace of an apartment at the 8th floor in Rome; and was always fed with industrial pet food. Anamnesis referred balance troubles, vomit and convulsions, during a couple of days, followed by sudden death. At necropsy, the cat presented mucoid rhinitis, purulent tracheitis, small areas of pneumonia, dark spots in the liver, catarrhal-hemorrhagic gastritis, fibrinous enteritis and meningeal hyperemia. Thoracic and abdominal cavities were completely invaded by hundreds of larval stages of cestodes. The same parasites were also included in nodules in pancreatic, lung and kidney parenchyma. Microscopic examination of parasites allowed their identification as larval stages (metacestodes) of cestodes of the genus Mesocestoides. The molecular genotyping of the metacestodes indicates a close relationship with members of the genus Mesocestoides, although a significant variation was found with respect to the available sequences of other species of the genus.

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.

Mesocestoides corti: a LIM-homeobox gene upregulated during strobilar development.

To understand the molecular processes regulating morphological changes during cestode life histories we focused on homeodomain (HD) proteins, a family of transcription factors essential for pattern formation during development. In this study we report the isolation of the partial sequence of MvLim, a LIM-HD gene of Mesocestoides corti. Other members of this gene family, characterized in Drosophila melanogaster, Caenorhabditis elegans and vertebrates contribute to cell fate determination of various neuronal subtypes. Phylogenetic analyses showed that MvLim clusters with members of the LIN-11 group and that platyhelminths have at least two different LIM-HD genes. By real time PCR we determined that MvLim expression is 20-fold greater in segmented worms than in tetrathyridia. The enhancement of MvLim expression during strobilation could be associated to changes in the innervation pattern occurring in proglottids development.

Molecular systematics of Mesocestoides sPP (cestoda: mesocestoididae) from domestic dogs (Canis familiaris) and coyotes (Canis latrans).

The genus Mesocestoides Vaillant, 1863 includes tapeworms of uncertain phylogenetic affinities and with poorly defined life histories. We previously documented 11 cases of peritoneal cestodiasis in dogs (Canis familiaris L.) in western North America caused by metacestodes of Mesocestoides spp. In the current study, DNA sequences were obtained from metacestodes collected from these dogs (n = 10), as well as proglottids from dogs (n = 3) and coyotes (Canis latrans Say, 1823 [n = 2]), and tetrathyridia representing laboratory isolates of M. corti (n = 3), and these data were analyzed phylogenetically. Two nuclear genetic markers, 18S ribosomal DNA and the second internal-transcribed spacer (ITS 2), were sequenced. Phylogenetic analysis of the 18S rDNA data recovered a monophyletic group composed of all samples of Mesocestoides spp., distinct from closely related outgroup taxa (Amurotaenia Akhmerov, 1941 and Tetrabothrius Rudolphi, 1819). Initial analysis of the ITS 2 data resolved 3 clades within Mesocestoides. Two proglottids from dogs formed a basal clade, a second clade was represented by tetrathyridial isolates, and a third clade included all other samples. Interpretation of these data from an apomorphy-based perspective identified 6 evolutionary lineages. We also assessed whether metacestodes from dogs (n = 4) are capable of asexual proliferation in laboratory mice. One tetrathyridial and 2 acephalic isolates from dogs proliferated asexually. Further investigation is warranted to determine which of the lineages represent distinct species and to determine the life history strategies of Mesocestoides spp.

Ultrastructure of spermiogenesis and the spermatozoon of Mesocestoides litteratus (Cestoda, Mesocestoididae).

This paper constitutes the first ultrastructural study of spermiogenesis and the spermatozoon of a cestode belonging to the family Mesocestoididae, Mesocestoides litteratus. Spermiogenesis in M. litteratus is characterised by a flagellar rotation and a proximodistal fusion. The zone of differentiation presents striated roots associated with the centrioles and also an intercentriolar body. The most interesting ultrastructural feature found in the mature spermatozoon of M. litteratus is the presence of parallel cortical microtubules. The spermatozoon also exhibits a single crest-like body and granules of glycogen. The pattern of spermiogenesis and the parallel position of cortical microtubules reveal the lack of concordance between M. litteratus and cyclophyllidean species studied to date in spermiogenesis and in the ultrastructural organisation of spermatozoon. This study provides new spermatological data and calls into question the validity of the current systematic position of mesocestoidids.

Shedding of gravid proglottids and destrobilation in experimental infections of foxes with Mesocestoides leptothylacus Loos-Frank, 1980 (Cestoda).

The implications of the confused taxonomy of the genus Mesocestoides and the misuse of the name M. lineatus are described. In Southwest Germany rodents are intermediate hosts and red foxes are definitive hosts of M. leptothylacus. The shedding patterns of experimentally infected foxes showed that destrobilation occurs frequently and that there are long periods during which no gravid proglottids are shed at all. Lengths of worms can be taken as a measure of a possible crowding effect only when worms with gravid segments are present, i.e. at the end of the prepatent period (11 to 13 days) or at the beginning of a shedding period.