A Comparison of Low-Temperature and Ambient-Temperature SEM for Viewing Nematode Faces

Faces of lesion nematodes Pratylenchus teres (populations RTB and JK) and P. zeae or the bacterivore Distolabrellus veechi were observed on frozen specimens with low-temperature scanning electron microscopy and as chemically fixed, critical-point dried specimens with conventional scanning electron microscopy. Amphidial secretions were preserved in chemically fixed but not cryofixed lesion nematodes. Overhanging liplets of chemically fixed D. veechi may be artifactual because they appeared as variably filled, mostly empty membranes when cryofixed. The diagnostically useful lips of the frozen lesion nematodes exhibited six sectors of variable prominence that were absent in chemically fixed specimens. This variability may be due to different degrees of muscle contraction captured during cryofixation, which occurs in milliseconds. This is the first evidence that rarely observed lip sectors in Pratylenchus may be something other than an artifact of shrinkage.     

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.     

Anisakis spp. in fishery products from Japanese waters: Updated insights on host prevalence and human infection risk factors

The nematodes of the genus Anisakis are among the most relevant parasitic hazards in fishery products since they are responsible for human infection and allergy cases. In a food safety and epidemiological perspective, several marine hosts from different locations around Japan were examined to characterize the parasitism of Anisakis larvae. Chum salmon (Oncorhynchus keta) and Alaska pollock (Gadus chalcogrammus) showed the highest overall prevalence (100%), followed by blue mackerel (Scomber australasicus) (97.5%), Pacific cod (Gadus macrocephalus) (80%), chub mackerel (Scomber japonicus) (60.1%), Japanese flying squid (Todarodes pacificus) (17%) and Japanese pilchard (Sardinops sagax melanostictus) (2%). In Pacific krill (Euphausia pacifica), apart from one Hysterothylacium aduncum larva, no Anisakis specimens were detected. Anisakis simplex sensu stricto was molecularly identified (PCR-RFLP) for the first time in Japanese flying squid and Japanese pilchard distributed in the Northwestern Pacific ocean. That was the most frequent parasitic species detected followed by A. pegreffii, mostly in the western areas of Japan, hybrid genotypes between the two sibling species as well as A. typica and A. berlandi. Surprisingly, A. simplex s.s. was the most abundant species in one batch of chub mackerel from the East China Sea and A. pegreffii was the main species found in one batch from the Pacific coast of Aomori, which seems to indicate that the ranges of these two sibling species might be more variable than previously thought.     

长江口和杭州湾凤鲚胃含物与海洋浮游动物的种类组成比较

2009年6月—8月期间在长江口及杭州湾水域收集了1355尾的凤鲚（Coilia mystus）标本,对其胃含物进行分析,期间采用出现频率（F）、个数百分比（N）、重量百分比（W）、相对重要性指标（IRI）、优势度（Y）公式计算,并进行种类的相似性分析,同步采集了取样点附近的浮游动物,比较了凤鲚胃含物食谱与该海域浮游动物种类组成。结果表明：河口性的长额刺糠虾IRI 为703.25,%IRI为62.94%,远远超过其他种类,甚至超过任何一个桡足类优势种,因而是凤鲚食谱中的最重要的种类。凤鲚摄食的其他主要种类包括：火腿许水蚤（Schmackeria poplesia）（IRI = 261.04）,虫肢歪水蚤（Tortanus vermiculus）（IRI = 107.53）、真刺唇角水蚤（Labidocera euchaeta）（IRI = 27.27）。以上四种浮游动物重要性合计%IRI为98.36%。在主要食物种类占绝对优势。因而是凤鲚饵料中最重要的优势种。种类组成相似性分析结果显示：胃含物和海域浮游动物之间的相似度值大致在0.363—0.365之间;而两者桡足类之间的相似值在0.521—0.575之间。推测凤鲚对个体较小的桡足类摄食方法是过滤性摄食,对种类的选择性较低。而对个体较大的糠虾则是有选择性追逐摄食。     

GASTROINTESTINAL HELMINTHS OF THE DUSKY DOLPHIN,LAGENORHYNCHUS OBSCURUS (GRAY, 1828), OFF PATAGONIA,IN THE SOUTHWESTERN ATLANTIC

The stomachs and intestines of 23 dusky dolphins incidentally caught in a trawl fishery off Patagonia were surveyed for helminths. All the dolphins were parasitized, with a total of 3,936 helminth individuals. Only five species occurred, of which three were common (prevalence ± 10%): Anisakis simplex, Braunina cordiformis and Hadwenius sp. A. simplex was present in all the dolphins and showed the highest abundance and mean intensity (104.9 individuals/infected host). B. cordiformis was next most common (87%), and Hadwenius sp. third (52.2%). A. simplex was found mostly in the stomach (94.8%), B. cordiformis in the duodenal ampulla (51.4%), and Hadwenius sp. in the stomach (64.5%). The rare species Corynosoma australe (immature specimens) and Pholeter gastrophilus occurred in the stomach. A. simplex and B. cordiformis showed a clumped distribution along the intestine. Brillouin's diversity and evenness indices for the intestinal helminth community were 0.329 and 0.393, respectively. The diversity values and the number of parasite species were within the range for other small cetaceans.     

Parasites as Biological Tags for Stock Discrimination of Beaked Redfish (Sebastes mentella): Parasite Infra-Communities vs. Limited Resolution of Cytochrome Markers

The use of parasites as biological tags for discrimination of fish stocks has become a commonly used approach in fisheries management. Metazoan parasite community analysis and anisakid nematode population genetics based on a mitochondrial cytochrome marker were applied in order to assess the usefulness of the two parasitological methods for stock discrimination of beaked redfish Sebastes mentella of three fishing grounds in the North East Atlantic. Multivariate, model-based approaches demonstrated that the metazoan parasite fauna of beaked redfish from East Greenland differed from Tampen, northern North Sea, and Bear Island, Barents Sea. A joint model (latent variable model) was used to estimate the effects of covariates on parasite species and identified four parasite species as main source of differences among fishing grounds; namely Chondracanthus nodosus, Anisakis simplex s.s., Hysterothylacium aduncum, and Bothriocephalus scorpii. Due to its high abundance and differences between fishing grounds, Anisakis simplex s.s. was considered as a major biological tag for host stock differentiation. Whilst the sole examination of Anisakis simplex s.s. on a population genetic level is only of limited use, anisakid nematodes (in particular, A. simplex s.s.) can serve as biological tags on a parasite community level. This study confirmed the use of multivariate analyses as a tool to evaluate parasite infra-communities and to identify parasite species that might serve as biological tags. The present study suggests that S. mentella in the northern North Sea and Barents Sea is not sub-structured.     

Can habitat modelling for the octopus Eledone cirrhosa help identify key areas for Risso’s dolphin in Scottish waters?

Marine protected areas (MPAs) are becoming increasingly important tools in the conservation of cetaceans. This has led to an interest in the use of species distribution models to predict where cetacean species are likely to occur based on the distribution of environmental variables. However, relationships between cetacean distribution and environmental variables are generally assumed to reflect the environmental preferences of their prey. Thus, understanding the distribution of prey may increase our ability to identify important areas for cetaceans. Here, we describe the diet of Risso’s dolphin (Grampus griseus) by analysing stomach contents of individuals stranded in Scotland over the last twenty years. Next, we use habitat modelling to test whether Rissos’s dolphin distribution in Scottish waters, as inferred from sightings, is related to the distribution of its main prey, the octopus Eledone cirrhosa. While good models of the relationship between the distribution of E. cirrhosa and environmental variables were obtained, there was no evidence of a relationship between modelled octopus distribution and the occurrence of Risso’s dolphins. These results suggest that identifying key areas for its main prey species is unlikely to help identify potential MPAs for Risso’s dolphin, at least at the spatial resolution used in this study.     

The metazoan parasites of swordfish from Atlantic tropical–equatorial waters

The metazoan parasite fauna composition of the swordfish Xiphias gladius is reported from 145 (72 females and 73 males) specimens caught in Atlantic tropical–equatorial waters during June to July 2006 and August 2007. Seventeen metazoan taxa (two monogeneans, two digeneans, one acanthocephalan, four cestodes, seven nematodes and one copepod) were identified by morphological and genetic methodologies. Among those identified, Anisakis brevispiculata, Anisakis paggiae and the acanthocephalan Bolbosoma vasculosum are new host records. Dominant species were: the anisakid nematode larvae Anisakis physeteris, Anisakis brevispiculata, Hysterothylacium incurvum and Hysterothylacium corrugatum (s.l.), the adult cestode Fistulicola plicatus, the plerocercoid Hepatoxylon squali and the monogeneans Tristoma integrum and Tristoma coccineum, which showed high prevalences and intensities. The relationship between metazoan parasites and host size and sex was investigated for the first time. No statistically significant correlation was found between host sex and parasite burden. Infection levels with T. integrum and T. coccineum were observed to be significantly negatively correlated to host size. Infection levels by endoparasites observed in X.gladius from Atlantic tropical‐equatorial area may be related to host feeding behaviour and the availability of the parasites’ intermediate and final hosts in the area studied. Anisakids of the genus Anisakis, were identified genetically to species level, and were considered to be promising biological tags for the characterization of X.gladius stocks.     

Anisakis spp. in toothed and baleen whales from Japanese waters with notes on their potential role as biological tags

In this study, Anisakis nematodes isolated from toothed and baleen whales from localities around Japan were molecularly (PCR-RFLP) identified. In Wakayama, common bottlenose dolphins (Tursiops truncatus) were infected with A. simplex sensu stricto (s.s.), A. typica and A. pegreffii, while A. typica was the only species found in pantropical spotted dolphin (Stenella attenuata) and striped dolphin (S. coeruleoalba). Offshore common minke whales (Balaenoptera acutorostrata) and sei whales (B. borealis) were almost exclusively infected with A. simplex s.s.. However, in common minke whales from two Hokkaido localities, mature worms mostly consisted of A. simplex s.s. in some individuals and of A. pegreffii in others, but immature worms were mainly A. simplex s.s.. Gross and histopathological examination on gastric mucosa attached by anisakids resulted in mild and superficial reactions by the two baleen whale species in contrast to severe inflammatory reaction associated with ulcer formations by common bottlenose dolphin. Host specificity and adaptability of Anisakis spp. in these baleen and toothed whales were discussed from the points of view of adult worm size, worm population and pathological reactions by hosts. Interestingly, most of the common minke whales predominantly harboring mature A. pegreffii adults belonged to the Yellow Sea – East China Sea stock (J stock), which migrates through the Sea of Japan, whereas most of those mainly parasitized by mature A. simplex s.s. adults were from the Okhotsk Sea – West Pacific stock (O stock), mostly inhabiting the Pacific side, suggesting that these sibling species may have utility as biological tags to differentiate whale stocks. These results represent the first definitive host records for A. pegreffi in the Northwestern Pacific Ocean.     

Parasitism in the bathypelagic fish, Melanostigma pammelas

Three hundred and thirty-seven specimens of the zoarcid fish, Melanostigma pammelas, collected between December, 1950 and December, 1969 from bathypelagic waters off southern California, were examined for their parasites. Incidences of infection were: digentic Trematoda 61%, Myxosporida 39%, larval Nematoda 9%. Two adult trematodes are described: Fellodistomum melanostigmum nov. sp. from the intestine, and Aponurus pacificus nov. sp. from the stomach. This parasite record is markedly unlike the typical record reported for eastern Pacific mesopelagic fishes in which many larval nematodes and few, if any, adult trematodes were found. Our long-term objective is to explain parasite pattern differences in the deep ocean in terms of their biological and physical environments. Our initial purpose is to determine the numbers and kinds of parasites in M. pammelas.     

Anisakid parasites of the pouting (Trisopterus luscus) from the Cantabrian Sea coast, Bay of Biscay, Spain

An epidemiological survey was undertaken of anisakids in 139 specimens (length: 13.2-24.5 cm) of pouting or bib (Trisopterus luscus) captured off the coast of northern Spain in the Cantabrian Sea. Third-stage larvae of two species of nematodes, Anisakis larvae type I and Hysterothylacium aduncum, were isolated. One adult female H. aduncum was also detected in the intestine of one pouting. Total prevalence of anisakids was 88.5%. Hysterothylacium aduncum and Anisakis showed, respectively, prevalence of 87.8% and 22.3%, mean intensity of 19.7 and 3.5, and mean abundance of 17.3 and 0.8. Analysis of infection parameters as a function of host length revealed a much higher prevalence in pouting specimens with length < 20 cm (94.4% for H. aduncum; 28.0% for Anisakis) than in those with length > or = 20 cm (65.6% for H. aduncum; 3.1% for Anisakis). The high mean intensity of Anisakis in muscle of parasitized pouting (5.9) may pose human health risks, although these are minimized by eating only thoroughly cooked pouting, as is the custom in Spain.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Helminth parasites found in faecal samples of phocids from the Antarctic Peninsula

Information on helminth parasites in Antarctic phocids is scarce and fragmented. Anisakidae nematodes and Diphyllobothriidae cestodes have been reported in Antarctic and subantarctic phocids, although the prevalence and health significance remain unclear. In the present study, the presence of helminth parasites in faeces of Leptonychotes weddellii, Hydrurga leptonyx and Mirounga leonina has been investigated. Faecal samples were collected from different locations of the Antarctic Peninsula. Macroscopical inspection and standard flotation and migration techniques were used for faecal examination. Eggs, larvae and adult parasites of nematodes and cestodes were found in 76.9 % of samples analysed. Positive samples were recorded from all locations surveyed and species investigated. The prevalence was 71.3 % for M. leonina, 95.4 % for L. weddellii and 100 % for H. leptonyx. Anisakidae (eggs and worms), Metastrongyloidea (larvae) and Diphyllobothriidae (eggs) were identified in M. leonina and L. weddellii. Metastrongyloidea (larvae) and Diphyllobothriidae (eggs) were found in H. leptonyx. Molecular characterisation of some of the adult parasites found was useful for the identification of Anisakis simplex and Pseudoterranova sp. in M. leonina, and Contracaecum sp., Contracaecum osculatum, and Pseudoterranova sp. in L. weddellii.     

Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

Background

Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.

Methodology/Principal Findings

We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4).

Conclusion/Significance

Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.     

Anisakis simplex and Terranova sp.: Inhibition by larval excretory-secretory products of mitogen-induced rodent lymphoblast proliferation

Execretory-secretory products were collected from supernatants of in vitro cultures of larval nematodes, Anisakis simplex (type I) and Terranova sp. (Hawaii type A). These materials were found to be more potent inhibitors of rodent lymphocyte blast transformation induced by concanavalin A and bacterial lipopolysaccaride than whole worm extracts of the same parasites. Inhibition of blast transformation was a result of cytostatic rather than toxic effects on proliferating lymphoid cells. The material(s) responsible for suppression are greater than 10,000 MW and are heat labile.     

The parasite fauna of <Emphasis Type="Italic">Arctogadus glacialis</Emphasis> (Peters) (Gadidae) from western and eastern Greenland

In all 155 specimens of the high Arctic codfish Arctogadus glacialis examined for metazoan parasites, 55 specimens were from southern and northern Baffin Bay, western Greenland, and 100 specimens from north-eastern Greenland and Scoresby Sound. A total of 20 parasite taxa were recorded. A new myxozoan Gadimyxa arctica was found in southern Baffin Bay and Scoresby Sound. The gadid myxozoan Zschokkella hildae, the digeneans Gonocerca phycidis and Lecithaster gibbosus, the gill copepod Haemobaphes cyclopterina and third-stage larvae of the nematodes Anisakis simplex and Hysterothylacium aduncum were found in Scoresby Sound only. The digenean Hemiurus levinseni and third-stage larvae of the nematode Contracaecum sp. were found at all four stations. The nematodes Ascarophis spp. were found at three stations. The parasite fauna of A. glacialis from Scoresby Sound was very similar to that of 50 specimens of the closely related Boreogadus saida from the same area.     

Identification of the secreted neutral proteases from Anisakis simplex

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.     

Intrahepatic growth and maturation of Gnathostoma turgidum in the natural definitive opossum host,Didelphis virginiana

Gnathostoma turgidum is a gastric nematode parasite of opossums found in the Americas. We recently found that G. turgidum juveniles appear in the liver of the opossums where they become mature adults and almost synchronously move to the stomach during certain months of the year, suggesting the importance of the liver for the growth and maturation of this species in the final hosts. In this study we attempted to detect G. turgidum larvae in the liver of opossums, Didelphis virginiana that are the natural final hosts. The results show that tiny (< 3 mm in length) third stage larvae (L3) appeared in the liver of opossums around November and December. Also in the liver, we found large L3 of up to about 10 mm in length together with juveniles and mature adults from February to March. In spite of their length, large L3 have 4 rows of hooklets, and their gonads remained undeveloped. Morphological features of the small and large L3 of G. turgidum are described including scanning electron microscope images. The seasonal switching of the several growth stages of G. turgidum from small L3 to adult worms in the liver and eventual migration to the stomach in opossums suggests the unique feature of G. turgidum utilizing the liver as the maturation site.     

Gastrointestinal helminths of Cuvier's beaked whales, Ziphius cavirostris, from the western Mediterranean

We examined the gastrointestinal helminth fauna of 2 Cuvier's beaked whales, Ziphius cavirostris, stranded on the Spanish Mediterranean coast. Information regarding intestinal parasites of this species is provided for the first time. Six helminth taxa were identified. Thirty type II larvae of the nematode Anisakis sp. were found in the stomach and the intestine of both hosts; 2 type I larvae of Anisakis sp. were found in the intestine of 1 host. One juvenile of the acanthocephalan Bolbosoma vasculosum was found in the intestine; the metacestode Scolex pleuronectis was found mainly in the terminal colon and the anal crypts of both hosts; adult cestodes of Tetrabothrius sp., which may represent a new species, were collected from the duodenum of 1 host. Composition of the intestinal parasitic community is similar to that of other oceanic cetaceans, which mostly include species of Bolbosoma and tetrabothriids (Cestoda).     

Cookie-cutter shark <Emphasis Type="Italic">Isistius brasiliensis</Emphasis> eats Bryde’s whale <Emphasis Type="Italic">Balaenoptera brydei</Emphasis>

We examined the stomach contents of cookie-cutter sharks, Isistius brasiliensis, collected by drift-net surveys in the western North Pacific in the early summer. The stomachs contained fresh flesh plugs and cephalopod beaks. Approximately half of the flesh plugs were identified as Bryde’s whale, Balaenoptera brydei, based on genetic analysis, and several of the lower beaks were identified as boreal clubhook squid, Onychoteuthis borealijaponica. The range of estimated dorsal mantle lengths for the squids was 60–172 mm. These results are evidence of predation by of cookie-cutter sharks on cetaceans and of an overlap in the vertical distribution of both species.