Parasitic flatworms infecting thorny skate, Amblyraja radiata: Infection by the monogeneans Acanthocotyle verrilli and Rajonchocotyle emarginata in Svalbard.

Parasite diversity above the Arctic circle remains understudied even for commercially valuable host taxa. Thorny skate, Amblyraja radiata, is a common bycatch species with a growing commercial value. Its natural range covers both sides of the North Atlantic including the Arctic zone. Svalbard is a Norwegian archipelago located on the northwest corner of the Barents Shelf which sustains a spectacular species diversity. So far, several monogenean species have been reported infecting thorny skate across the Atlantic Ocean. In the present study, we intend to fill in the knowledge gap on monogenean parasites infecting thorny skate in the northern part of its range and thus indirectly assess the connectivity between the thorny skate populations off the Svalbard coast and from previously studied locations. 46 monogenean individuals were recovered from 11 specimens of thorny skate. Following morphological and molecular assessment, two species of monogeneans, Acanthocotyle verrilli and Rajonchocotyle emarginata, were identified. The results serve as the northernmost record for both parasite genera and the first record of monogenean species off Svalbard. Detailed morphometric evaluation revealed a relatively high level of morphological variation in A. verrilli compared to its congeners. Phylogenetic reconstruction placed A. verrilli in a well-supported clade with A. imo. Our study also suggests high diagnostic significance of sclerotised structures in the identification of Rajonchocotyle. Even though the occurrence of two directly transmitted parasite species supports the previously suggested long-distance migration of A. radiata, future studies employing highly variable genetic markers are needed to assess the ongoing and historical migration patterns.

Gastrointestinal parasites of arctic foxes (Vulpes lagopus) and sibling voles (Microtus levis) in Spitsbergen, Svalbard.

The arctic fox (Vulpes lagopus), an apex predator with an omnipresent distribution in the Arctic, is a potential source of intestinal parasites that may endanger people and pet animals such as dogs, thus posing a health risk. Non-invasive methods, such as coprology, are often the only option when studying wildlife parasitic fauna. However, the detection and identification of parasites are significantly enhanced when used in combination with methods of molecular biology. Using both approaches, we identified unicellular and multicellular parasites in faeces of arctic foxes and carcasses of sibling voles (Microtus levis) in Svalbard, where molecular methods are used for the first time. Six new species were detected in the arctic fox in Svalbard, Eucoleus aerophilus, Uncinaria stenocephala, Toxocara canis, Trichuris vulpis, Eimeria spp., and Enterocytozoon bieneusi, the latter never found in the arctic fox species before. In addition, only one parasite was found in the sibling vole in Svalbard, the Cryptosporidium alticolis, which has never been detected in Svalbard before.

Population co-divergence in common cuttlefish (Sepia officinalis) and its dicyemid parasite in the Mediterranean Sea.

Population structure and biogeography of marine organisms are formed by different drivers than in terrestrial organisms. Yet, very little information is available even for common marine organisms and even less for their associated parasites. Here we report the first analysis of population structure of both a cephalopod host (Sepia officinalis) and its dicyemid parasite, based on a homologous molecular marker (cytochrome oxidase I). We show that the population of common cuttlefish in the Mediterranean area is fragmented into subpopulations, with some areas featuring restricted level of gene flow. Amongst the studied areas, Sardinia was genetically the most diverse and Cyprus the most isolated. At a larger scale, across the Mediterranean, the population structure of the parasite shows co-diversification pattern with its host, but a slower rate of diversification. Differences between the two counterparts are more obvious at a finer scale, where parasite populations show increased level of fragmentation and lower local diversities. This discrepancy can be caused by local extinctions and replacements taking place more frequently in the dicyemid populations, due to their parasitic lifestyle.

Heterolobosean amoebae from Arctic and Antarctic extremes: 18 novel strains of Allovahlkampfia, Vahlkampfia and Naegleria.

The diversity of heterolobosean amoebae, important members of soil, marine and freshwater microeukaryote communities in the temperate zones, is greatly under-explored in high latitudes. To address this imbalance, we studied the diversity of this group of free-living amoebae in the Arctic and the Antarctic using culture dependent methods. Eighteen strain representatives of three heterolobosean genera, Allovahlkampfia Walochnik et Mulec, 2009 (1 strain), Vahlkampfia Chatton et Lalung-Bonnaier, 1912 (2) and Naegleria Alexeieff, 1912 (15) were isolated from 179 samples of wet soil and fresh water with sediments collected in 6 localities. The Allovahkampfia strain is the first representative of the genus from the Antarctic; 14 strains (7 from the Arctic, 7 from the Antarctic) of the highly represented genus Naegleria complete the 'polar' cluster of five Naegleria species previously known from the Arctic and Sub-Antarctic regions, whereas one strain enriches the 'dobsoni' cluster of Naegleria strains of diverse origin. Present isolations of Naegleria polarisDe Jonckheere, 2006 from Svalbard, in the Arctic and Vega Island, in the Antarctic and N. neopolarisDe Jonckheere, 2006 from Svalbard and Greenland in the Arctic, and James Ross Island, the Antarctic demonstrate their bipolar distribution, which in free-living amoebae has so far only been known for Vermistella Morand et Anderson, 2007.

Vermistella arctica n. sp. Nominates the Genus Vermistella as a Candidate for Taxon with Bipolar Distribution.

A new amoebozoan species, Vermistella arctica n. sp., is described from marine habitats in the central part of Svalbard archipelago. This is the first report on Arctic amoebae belonging to the genus Vermistella Moran and Anderson, 2007, the type species of which was described from the opposite pole of the planet. Psychrophily proved in the new strains qualifies the genus Vermistella as a bipolar taxon. Molecular phylogenetic analyses based on 18S rDNA and actin sequences did not show any affinity of the genus Vermistella to Stygamoeba regulata ATCC(®) 50892(™) strain. A close phylogenetic relationship was found between Vermistella spp. and a sequence originating from an environmental sample from Cariaco basin, the largest marine permanently anoxic system in the world. Possible mechanisms of bipolar distribution are discussed.

Spathebothriidea: survey of species, scolex and egg morphology, and interrelationships of a non-segmented, relictual tapeworm group (Platyhelminthes: Cestoda).

Tapeworms of the order Spathebothriidea Wardle et McLeod, 1952 (Cestoda) are reviewed. Molecular data made it possible to assess, for the first time, the phylogenetic relationships of all genera and to confirm the validity of Bothrimonus Duvernoy, 1842, Diplocotyle Krabbe, 1874 and Didymobothrium Nybelin, 1922. A survey of all species considered to be valid is provided together with new data on egg and scolex morphology and surface ultrastructure (i.e. microtriches). The peculiar morphology of the members of this group, which is today represented by five effectively monotypic genera whose host associations and geographical distribution show little commonality, indicate that it is a relictual group that was once diverse and widespread. The order potentially represents the earliest branch of true tapeworms (i.e. Eucestoda) among extant forms.

Myxozoa in high Arctic: Survey on the central part of Svalbard archipelago.

Myxosporeans (Myxozoa), microscopic metazoan parasitic organisms, are poorly studied in the Arctic region. Our survey of benthic and pelagic fish (n = 234) collected in Isfjorden (Svalbard, Norway) together with detailed morphological and molecular examination revealed the presence of nine myxosporean species. We compared observed myxosporean diversity with diversity documented in regions close to the Arctic and revealed that water depth rather than geographic distribution is an important factor influencing myxosporean fauna. WE DESCRIBE THREE NEW MYXOSPOREAN SPECIES: Zschokkella siegfriedi n. sp. from kidney of Boreogadus saida, Parvicapsula petuniae n. sp. from the urinary bladder of Gymnocanthus tricuspis, and Sinuolinea arctica n. sp. from the urinary bladder of Myoxocephalus scorpius. We characterise Latyspora-like organism from kidney of Clupea harengus. We provide new data for Ceratomyxa porrecta, Myxidium gadi, Myxidium finnmarchicum, Schulmania aenigmatosa, and Parvicapsula irregularis comb. nov. The phylogenetic analyses including the newly obtained SSU and LSU rDNA data revealed that most of the species studied cluster in the marine urinary clade within the marine myxosporean lineage. Newly obtained sequences including the first molecular data for the member of the genus Schulmania, substantially enriched the Zschokkella subclade. C. porrecta and the two Myxidium species cluster within the Ceratomyxa and marine Myxidium clade, respectively. Newly described species, Z. siegfriedi n. sp., was revealed to be morphologically indistinguishable but genetically diverse from Zschokkella hildae known from numerous gadid fish. Therefore, we consider Z. siegfriedi to be a cryptic myxosporean species that might be misidentified with Z. hildae. A Latyspora-like organism was found to be taxonomically problematic due to its suture line and its distant phylogenetic position from the type species Latyspora scomberomori did not allow us to assign it to the genus Latyspora. Based on an increased taxon sampling and SSU + LSU rDNA-based phylogeny, evolutionary trends within the marine urinary clade are investigated.

Detection of ancient DNA of Encephalitozoon intestinalis (Microsporidia) in archaeological material.

Ancient DNA (aDNA) of Encephalitozoon intestinalis (Microsporidia, Fungi) was detected in archaeological material originated from New Town of Prague (Czech Republic) with the use of molecular methods. Microsporidial aDNA was found in 3 samples originating from 2 objects, in a well/cesspit (samples from layers from the 18th century) and in a well from the 18th/19th century. The ability to use molecular methods to detect microsporidia extends the range of paleoparasitological inquiry, and could contribute to a better understanding of parasites shared between human and animals.

Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus).

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24×3.69µm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49×3.90 µm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.

The first reported cases of human cryptosporidiosis caused by Cryptosporidium hominis in Slovak Republic.

Cryptosporidiosis belongs to the important parasitic infections with zoonotic potential and the occurrence in European countries is rare. The first cases of cryptosporidiosis caused by Cryptosporidium hominis detected in the Slovak republic were described here. Collection of examined humans consisted of five family members. Faecal specimens were examined by formalin sedimentation, by the Sheather's sugar flotation and by immunochromatography and visualised by the Ziehl-Neelsen acid fast stain. A fragment of the Cryptosporidium small subunit ribosomal RNA gene was amplified by nested polymerase chain reaction and species was determined by restriction fragment length polymorphism analysis with the endonucleases SspI and VspI. C. hominis was found in faeces of two immunocompetent siblings (a 7-year-old boy and a 2-year-old girl). The symptoms occurred only in the boy as gastrointestinal disorders lasting 5 days, and manifested by abdominal pain, an elevated body temperature (37.2 °C), mild diarrhoea, accompanied by lassitude, depression and anorexia. Ultrasonic scan revealed enlarged spleen and mezenteric lymph nodes. Microscopic examination of the stool sample revealed numerous Cryptosporidium oocysts. The DNA typing identified C. hominis subtype IbA10G2. Cryptosporidium was also detected in the boy's sister without any complications and symptoms. Their father, mother and grandmother were parasitologically negative. The source of infection remained unknown. Human cases in present study reflect necessity of systematic attention on intestinal parasites diagnostic inclusive of cryptosporidia.

Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa).

We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81-5.96 µm (mean=5.16)×4.23-5.29 µm (mean=4.83) with a length to width ratio of 1.07±0.06 (n=400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4-6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250-4000 oocysts per gram of feces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB/c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species.

Encephalitozoon cuniculi genotype I as a causative agent of brain abscess in an immunocompetent patient.

A brain abscess caused by Encephalitozoon cuniculi genotype I together with Streptococcus intermedius occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole.

Microsporidia in exotic birds: intermittent spore excretion of Encephalitozoon spp. in naturally infected budgerigars (Melopsittacus undulatus).

Nine naturally infected asymptomatic budgerigars (Melopsittacus undulatus) were screened daily for microsporidia spore excretion during a 30-day period and the localization of infection was evaluated using microscopy and molecular methods. While the microscopic examination revealed 2.4% positivity out of all fecal samples, using PCR the positivity was 10x higher (24.6%). All nine budgerigars excreted microsporidial spores intermittently in irregular intervals with 1-11-day long interruptions. Most of the birds were infected simultaneously with Encephalitozoon cuniculi and Encephalitozoon hellem. While histological and TEM examination failed to confirm the presence of microsporidial spores in tissues, the PCR detected microsporidial DNA mostly in the small intestine, liver and lungs of four selected budgerigars dissected. Despite the chronic infection proved using molecular methods, no clinical signs of disease were observed during monitoring and no pathological findings were found during dissection.

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants.

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.

Viability staining and animal infectivity of Cryptosporidium andersoni oocysts after long-term storage.

Preservation of biological properties of oocysts during the storage is critical for experimental work. Stomach species of cryptosporidia are less resistant against external influences, and their infectivity decreases rapidly in comparison with intestinal cryptosporidia. Cryptosporidium andersoni oocysts lost their infectivity for gerbil (Meriones unguiculatus) after 7 months storage in deionised water (DW) or in 2.5% potassium dichromate solution (PDS). Evaluation of oocyst viability by flow cytometry indicates higher percentage of viable oocysts stored in PDS than in DW, particularly after 6 months of storage. However, direct counting using fluorescent microscope revealed that these results are false and are influenced by the change of staining properties during the storage in PDS. Moreover, the examination of oocyst integrity by flow cytometry revealed that oocysts preserved in PDS kept their wall integrity longer than those stored in DW, and this fact should be taken into consideration during quantification of oocyst survival.

Humoral intestinal immunity against Encephalitozoon cuniculi (Microsporidia) infection in mice.

Three strains of mice, BALB/c, IL-12 knock-out (KO) and INF-gamma knock-out, were chosen as an experimental model for the study of intestinal immunity induction against Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 infection. Mice were infected perorally with 10(7) spores and re-infected with the same dose 70 days after the first infection. The anti-E. cuniculi IgA, IgG and IgM responses in sera and extracts of stool samples were determined by ELISA. Results have shown specific antibody production in the sera and intestinal secretions of all three strains of mice induced orally by E. ciniculi spores. BALB/c mice developed a stronger humoral immune response than IL-12 KO mice. The lowest antibody response developed in INF-gamma KO mice that succumbed to the infection within 28 days post infection.

Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic.

To study the diversity of Cryptosporidium spp. in various hosts, we used the variability of the small-subunit rRNA gene and the Cryptosporidium oocyst wall protein genes. Oocysts from humans, cattle, horses, dogs, field mice, chickens, reptiles, deer, goat, cat, antelope and from a sample of water reservoir were assayed. The zoonotic C. parvum bovine genotype sequence was found to be present in the most of isolates. This study shows a complex epidemiology pattern for C. parvum bovine genotype infections. The identification of cattle, horse, and deer isolates emphasizes a transmission route for C. parvum via these hosts, and identifies a potential source for human infection in the Czech Republic. Furthermore, C. andersoni from a cow, C. baileyi from a chicken, C. felis from a cat, C. meleagridis from a dog, and C. saurophilum and C. serpentis from reptiles were also identified in the isolates from the Czech Republic.

Failed attempt of Cryptosporidium andersoni infection in lambs.

Four lambs 4 months old were inoculated with Cryptosporidium andersoni oocysts (bovine isolate) in a dose of 5 x 10(6) per animal followed later by 1 x 10(7) oocysts per animal. No animal shed Cryptosporidium oocysts during 56 days post infection, no lamb showed clinical symptoms of cryptosporidiosis and no macroscopic changes were detected in the abomasum immediately after autopsy. Histological examinations did not demonstrate the occurrence of Cryptosporidium in the abomasum and other selected organs.

Role of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon intestinalis infection.

Severe combined immunodeficient (SCID) mice reconstituted with spleen cells from naive adult BALB/c mice were completely resistant to peroral infection with Encephalitozoon intestinalis (Calli, Kotler and Orenstein, 1993) Canning, Field, Hing and Marriott, 1994, whereas control, non-reconstituted SCID mice succumbed to the infection. The role of T-lymphocyte subpopulations in the protection against peroral E. intestinalis infection was studied in adoptive transfer experiments using SCID mice. SCID mice reconstituted with both CD4+ and CD8+ T-lymphocyte-depleted splenocytes succumbed to the peroral route of infection. In contrast, SCID mice reconstituted with either CD4+-depleted or CD8+ T-lymphocyte-depleted splenocytes completely resolved the infection. This indicates that CD4+ and CD8+ T-lymphocyte subpopulations play a substantive role in protection against peroral infection with the microsporidian, E. intestinalis.