Linking larvae and adults of Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) using molecular data

Because the taxonomy of trematodes is based on adults, the larval stages of most digeneans cannot be identified to species based on morphology alone. Molecular data provide a means of linking larval stages to known adults. We obtained sequences from the barcode region of cytochrome oxidase I (CO1) from adult and larval parasites of fish, frogs, birds, and mammals across North America. Sequences from adult Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) from definitive hosts matched those of meta- and mesocercariae from fish and frogs. These data provided new information on the distributions of all 3 parasite species. Metacercariae of A. cornu, which have not been previously reported in North American hosts, were found in Notemigonus crysoleucas, Pimephales notatus, and Catostomus commersonii in the St. Lawrence River. Metacercariae of H. triloba are reported in Canadian waters and in N. hudsonius for the first time. Alaria mustelae is reported for the first time in frogs from Quebec, Canada, and an additional species of Alaria was detected in California. Sequences of internal transcribed spacer rDNA from a subset of specimens support the same species boundaries indicated by CO1 divergence. There was little divergence in CO1 sequences from an unidentified diplostomid species sampled at a large spatial scale.     

Helminth parasitism in martens (Martes americana) and ermines (Mustela erminea) from Washington, with comments on the distribution of Trichinella spiralis

Helminths are reported for the first time from ermines (Mustela erminea) and martens (Martes americana) in Washington (USA). Among 22 adult ermines, 41% were infected by one or more of five species (Taenia mustelae, Alaria mustelae, Molineus patens, M. mustelae and Trichinella spiralis). Among 78 adult martens from three geographic localities, the prevalence was 83%. Nine species were identified (Mesocestoides sp., T. mustelae and T. martis americana, Euryhelmis squamula, M. patens, Baylisascaris devosi, Physaloptera sp., Soboliphyme baturini and T. spiralis). Trichinella spiralis occurred with a maximum prevalence of 50% in martens, but only occurred in 9% of ermines. Compression and digestion techniques provided a similar estimate of prevalence of T. spiralis, yet neither was entirely accurate in identifying all infected hosts. The species richness of the helminth community of martens in Washington was greater than that reported from other regions of North America.     

Helminth parasites of fisher Martes pennanti (Erxleben) from Manitoba, Canada

Seven species of helminths were recovered during a survey of 162 fisher (Martes pennanti) from four areas of Manitoba: Baylisascaris devosi in 52 fisher; Taenia sibirica in 25; Physaloptera sp. in nine; Alaria mustelae in two; Metorchis conjunctus in one; Trichinella spiralis in one of 81; Molineus sp. in one. B. devosi was the most prevalent parasite and differences in its geographical distribution were possibly related to population density of fisher. The prevalence of other parasites did not appear to be related to density of fisher.     

Parasitic helminths of free-ranging mink (Neovison vison mink) from southern Florida

Five free-ranging mink, Neovison vison mink, from in or near the Fakahatchee Strand Preserve State Park, Collier County, Florida (26 degrees 00'N, 81 degrees 25'W), were examined for parasitic helminths. Nine species of helminths were identified (2 trematodes, 5 nematodes, and 2 acanthocephalans). The most prevalent parasites were Molineus patens (4 of 5 mink), mean intensity 173 (range, 12-342); Strongyloides sp. (4 of 5), mean intensity 48 (range, 1-170); Macracanthorhynchus ingens (immature) (4 of 5), mean intensity 2.5 (range, 1-4); and Alaria mustelae (3 of 5), mean intensity 59.3 (range, 11-127). Polymorphus brevis and Dirofilaria lutrae are reported from the mink for the first time.     

Sexual hybridization experiments and phylogenetic relationships as inferred from rubisco spacer sequences in the genus alaria (phaeophyceae)

Alaria (Alariaceae, Phaeophyceae) is a common genus of kelps generally found in the lower intertidal and shallow subtidal regions of rocky shores subject to strong wave exposure. Fourteen species are currently recognized, of which 11 are found in the cold–temperate North Pacific Ocean. Alaria esculenta (L.) Greville, the type species described from the North Atlantic, exhibits a range of biogeographically correlated morphotypes indicating the possibility of multiple species, subspecies, and/or hybrids. This has led to an unstable taxonomy. We compared five species from the Atlantic and Pacific, including six North Atlantic isolates of A. esculenta. Phylogenetic analyses based on Rubisco spacer sequences resulted in a well-resolved topology of these five species, but did not distinguish between the six biogeographic isolates of A. esculenta. Laboratory hybridization experiments among four A. esculenta isolates showed partial intrafertility. Among five tested Alaria species, interfertility as well as fertility barriers were encountered, inconsistent with reproductive isolation. The data reject both a biological and morphological species concept and support only a phylogenetic species concept for Alaria , demonstrating that morphological variation has evolved independently of molecular variation in the genes under investigation in the species of the genus Alaria.     

NEGATIVE PHOTOTROPISM OF THE HAPTERA OF ALARIA ESCULENTA (LAMINARIALES)1,2

The “root-like” haptera of the holdfast of Alaria esculenta (L.) Grev. display a negative phototropic curvature only when unilaterally irradiated with blue (432 nm) and green (552 nm) wavelengths.     

Genome conservation in Helicobacter mustelae as determined by pulsed-field gel electrophoresis

Abstract Genomic DNA from 15 strains of Helicobacter mustelae was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Pac I and S fi I. H. mustelae genome DNA appeared very similar in all strains examined, whether isolated from ferrets or mink or from animals bred in either the USA or in the UK. The H. mustelae genome size was estimated to be 1.7 Mb, similar in size to that of H. pylori . A minor difference in PacI PFGE pattern and genome size was observed between rifampicin-resistant and rifampicin-susceptible derivatives of H. mustelae F251. Another minor difference in genome pattern based on PFGE with S fi I was observed between an H. mustelae strain used to experimentally infect four ferrets which resulted in loss of an S fi I site in strains obtained from the newly infected ferrets. Thus, although minor differences in PFGE pattern were noted, H. mustelae lacks the genomic diversity observed in H. pylori .     

Characterization and Therapy for Experimental Infection by Helicobacter mustelae in Ferrets

Background. Numerous clinical trials evaluating the efficacy of various antimicrobial compounds against Helicobacter pylori infection have been performed in humans. A convenient animal model for Helicobacter infection would facilitate the evaluation of novel therapies. These experiments were performed to evaluate the use of ferrets as a model of Helicobacter infection.
Materials and Methods. Ferrets were infected experimentally with Helicobacter mustelae and subsequently treated with bismuth subsalicylate (BSS) triple therapy (BSS, metronidazole, and amoxicillin), or left untreated. The status of infection and serology was assessed during treatment and for 8 weeks posttreatment. Seven ferrets successfully treated with triple therapy were challenged with H. mustelae and monitored for infection for an additional 5 weeks.
Results. Infection of ferrets by H. mustelae was accompanied by gastritis and a specific antibody response. Treatment of H. mustelae -infected ferrets with BSS suppressed bacterial growth in four of nine animals but did not eradicate infection. Triple therapy eradicated infection in all nine ferrets with a reduction in gastric inflammation. No relapse of infection occurred up to 8 weeks posttherapy. Challenge with H. mustelae of ferrets successfully treated with triple therapy resulted in a 100% rate of reinfection.
Conclusions. H. mustelae infection can be eliminated by triple therapy, but this does not result in protective immunity against reinfection by H. mustelae. This model, using a strain of Helicobacter indigenous to the host, may be useful for assessing therapeutic efficacy of novel therapies for the treatment of human infection by H. pylori.     

BLUE LIGHT PHOTOREACTIVATION IN ULTRAVIOLET-IRRADIATED YOUNG SPOROPHYTES OF ALARIA ESCULENTA AND LAMINARIA SACCHARINA (PHAEOPHYTA)1

Exposure of ultraviolet (UV)-irradiated young sporophytes of Alaria esculenta (L.) Grev. and Laminaria saccharina (L.) Lamour. to visible light resulted in recovery from UV damage that would otherwise cause much higher mortality. For this photoreactivation, blue light was highly effective, whereas negligible reactivation was produced in green or red light. The blue quantum requirements for a 50% response were 1.9 mol·m^?2 in A. esculenta and 1.2 mol·m^?2 in L. saccharina, which were comparable to those reported for other blue light responses requiring high energy found in brown algae.     

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.     

Random mutagenesis to identify novel Helicobacter mustelae virulence factors

Helicobacter mustelae is a gastric pathogen of ferrets, where it causes disorders similar to those caused by Helicobacter pylori in humans. The H. mustelae ferret model therefore has potential for the in vivo study of Helicobacter pathogenesis in general. In this study a library of 500 individual H. mustelae mutants was generated using an in vitro random insertion mutagenesis technique. Mutants were subsequently tested for motility and adherence, and 43 of the 500 mutants tested were found to be nonmotile in a soft agar assay. Of these 43 mutants, seven were subsequently identified as deficient in their ability to adhere to AGS cells. Insertion had taken place in different positions in the H. mustelae genome, and included mutants in or near to genes involved in motility and urease activity (e.g. the chemotaxis gene cheV and the urease accessory gene ureH). The development of a mutant library for a natural animal model of Helicobacter infection provides the opportunity to study in vivo the role of candidate Helicobacter virulence genes.     

AUXIN,AN ENDOGENOUS REGULATOR OF GROWTH IN ALGAE?1,2

The effects of 5 pairs of synthetic auxins and chemically related, but biologically inactive, derivatives (e.g., 2,4- and 3,5-dichlorophenoxyacetic acid, respectively) on blade growth of the brown alga, Alaria esculenta (L.) Grev.(Laminariales) were studied. The significant result was the inability to divide the growth responses into auxin and non-auxin groups. At high concentrations (10^?3 M) 9 test substances inhibited blade growth; lower concentrations (<10^?3 M) were either slightly inhibitory or showed no effect. The data suggest that a higher plant auxin-specific recognition mechanism is lacking in this alga. As this mechanism is central to higher plant concepts of growth control by auxin, the role of auxin as an endogenous regulator of growth in Alaria, and perhaps other algae as well, seems unlikely. This interpretation is consistent with recent negative biochemical data from auxin binding studies using subcellular preparations from algae.     

Natural and Experimental Helicobacter mustelae Reinfection Following Successful Antimicrobial Eradication in Ferrets

Background. Recrudescence or reinfection may occur after eradication of Helicobacter pylori in humans.
Materials and Methods. We used the ferret Helicobacter mustelae model to investigate the effect of prior infection and eradication on reinfection by experimental and natural routes. Two groups of ferrets with naturally acquired H. mustelae infection were treated with an eradication protocol using amoxicillin, metronidazole, and bismuth subsalicylate. The ferrets were monitored for recrudescence by repeated cultures of endoscopic gastric mucosal biopsies. The ferrets were challenged at 17 months (group I) and 6 months (group II) after eradication with a strain of H. mustelae having a distinctive restriction endonuclease analysis pattern. The eradication protocol was repeated to eliminate the infection produced by experimental challenge. The ferrets were then cohoused intermittently with naturally infected ferrets.
Results. The original H. mustelae infection was successfully eliminated by the eradication protocol. No recrudescence was observed in group I for 12 months nor for 3 months in group II after eradication. All ferrets became persistently reinfected with the challenge strain. The infection from the challenge strain was eradicated successfully. No ferrets in group I and all ferrets in group II became infected through cohousing.
Conclusions. These results suggest that though prior infection with H. mustelae may confer some protection against reinfection, such protection is not universal in all circumstances; that susceptibility to reinfection by contact with infected animals varies between individuals; and that age may be a factor in this individual variability. These results are applicable to studies of reinfection after eradication of H. pylori in humans.     

Growth performance ofAlaria esculenta off helgoland

Alaria esculenta (L.) Grev. sporophytes, raised in culture and originating from the North of Iceland were immersed in the sea near Helgoland (North Sea) for the period from February to August. Notable growth of the thalli was found between February and May, when the water temperature was similar to that of their original habitat in Iceland. Less conspicuous growth but development of sporophylls took place between May and July. The temperature rose to 16.8° C during the first half of July and remained on the same level during August, when the plants deteriorated. The present experiments yield additional evidence that the regional distribution ofA. esculenta is temperature controlled.     

Coprological study on helminth fauna in Eurasian lynx (Lynx lynx) from the Białowieza Primeval Forest in eastern Poland

One hundred fecal samples were collected during research on Eurasian lynx ecology and food habits in the Polish part of the Bia?owieza Primeval Forest (BPF) from 2001 to 2006. Seventy-three percent of samples contained eggs or larvae of helminths. A total of 10 species of helminths was identified, including 3 Cestoda (Diphyllobothrium latum, Spirometra janickii, and unidentified species of Taeniidae), 1 Trematoda (Alaria alata), and 6 Nematoda (Aelurostrongylus abstrusus, Ancylostoma tubaeforme, Eucoleus aerophilus, Metastrongylus sp., Nematodirus sp., and Toxocara cati). Alaria alata has not been reported previously in lynx. A statistical comparison of the 2 techniques used to isolate eggs, i.e., flotation and sedimentation, indicates that sedimentation was more effective.     

24 Diversity of macroalgal epiphytes on Thalassia and Syringodium in tampa bay, florida

Alaria is a common kelp genus generally found in the lower intertidal and shallow subtidal regions of rocky shores in the cold waters of the northern Hemisphere. About 16 species are currently recognized worldwide and, of these, A. fistulosa is distinguished by having hollow midrib and large blades with 10–30 m in length and 30–90 cm in width. It is the only canopy-producing kelp in the northwest Pacific, where it is restricted to the waters of north Hokkaido, Kamchatka, Aleutian Islands, and Alaska. In order to know the phylogenetic position of A. fistulosa , sequences of nr DNA ITS and plastid rbc L including spacer and psaA regions were determined in A. fistulosa and compared with homologous positions of newly sequenced putative relatives and with published sequences of other kelp species. Combined data of ITS and Rubisco spacer show that A. fistulosa was more related to the clade of Lessoniopsis and Pterygophora than to the clade of other species of Alaria , which is supported by the rbc L and psaA sequence data. The topologies from nuclear and plastid DNA sequences lead to phylogenetic independence of A. fistulosa , which is clearly different from the genus Alaria .     

THE RATE OF TRANSLOCATION IN ALARIA ESCULENTA (LAMINARIALES,PHAEOPHYCEAE)12

The rate of translocation of organic carbon in blades of Alaria esculenta (L.) Grev. (Laminariales) was calculated from blade growth data. The cross sectional area of sieve filaments in the midrib medulla was estimated from light microscopic examination of fresh material. Files of these filaments form a perimedullar ring occupying ca. 2/3 of the medulla. Values computed for specific mass transfer of carbon into the blade meristem ranged from 36.2 to 60.8 mg C.wk^?1.0.1 mm^?2 sieve filaments.     

Helminth infections in faecal samples of wolves Canis lupus L. from the western Beskidy Mountains in southern Poland

Eighty-nine samples of grey wolf (Canis lupus L.) faeces were collected between 2002 and 2004 from two areas in the western Beskidy Mts (south Poland). Helminth eggs were observed in 56.2% of faeces examined. These included: Alaria alata (2.2%), taeniid eggs (11.2%), Toxocara canis (5.6%), Toxascaris leonina (1.1%), Eucoleus aerophilus (14.6%), Ancylostoma caninum (12.3%), Uncinaria stenocephala (37%) and unidentified roundworm eggs of the family Strongyloididae (1.1%). Eucoleus aerophilus is recorded for the first time from Poland. The results are compared with the helminth fauna of other wolf populations in Europe.     

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.     

Helminths in the wolf, Canis lupus, from north-western Spain

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.