Intrinsic properties of anisakid nematode larvae as a potential tool for the detection in fish products.

Anisakid nematode larvae (NL) in fish products comprise a risk to human health and, if visible, lead to the rejection of these products by consumers. Therefore, great efforts are being made for the identification of these anisakid larvae to estimate the potential consumer health risk as well as to develop effective detection methods in order to prevent the introduction of heavily infected fish products into the market. The tasks of national reference laboratories include the improvement of detection methods and to promote their further development. As a prerequisite for improved detection, it is important to understand the structural properties of anisakid NL and compounds produced during host-parasite interactions. This review provides an overview of the intrinsic properties of anisakid NL and reports the latest detection methods in published literature. First, in order to define the potentially interesting intrinsic properties of anisakid nematodes for their detection, anatomy and compounds involved in host-parasite interactions are summarised. These can be used for various detection approaches, such as in the medical field or for allergen detection in fish products. In addition, fluorescence characteristics and their use as both established and promising candidates for detection methods, especially in the field of optical sensing technologies, are presented. Finally, different detection and identification methods applied by the fish processing industries and by control laboratories are listed. The review intends to highlight trends and provide suggestions for the development of improved detection and identification methods of anisakid NL in fish products.

Studies on the influence of sampling on the levels of dioxins and PCB in fish.

Beaked redfish (Sebastes mentella) and Greenland halibut (Reinhardtius hippoglossoides) were collected in waters around Svalbard (Barents Sea) to study the influence of different muscle separation/filleting techniques at small and medium fishes on the dioxin/PCB content. Sampling of both species included preparation techniques such as fillets with or without belly flaps, commercially trimmed fillets and cutting into anterior and posterior cutlets. In case of Greenland halibut also the whole edible muscle part and middle cutlets were studied. All samples analysed were far below the maximum level of 6.5 pg/g wet weight (ww) WHO-PCDD/F-PCB-TEQ and 75 ng/g ww ndl-PCB. Trimmed fillets of beaked redfish had the lowest fat content and the lowest level of dioxins and PCB (1.70%, WHO-PCDD/F-PCB-TEQ = 0.320 pg/g ww). The respective posterior cutlets showed the highest fat content and highest levels of dioxins and PCB (2.66%, WHO-PCDD/F-PCB-TEQ = 0.729 pg/g ww). Levels of dioxins and PCB in Greenland halibut samples were generally higher and ranged between WHO-PCDD/F-PCB-TEQ = 0.784 pg/g ww (fillets without bells flaps, fat content = 8.83%) and WHO-PCDD/F-PCB-TEQ = 2.022 pg/g ww (edible part whole muscle, fat content = 8.62%). The results show a considerable influence of the different sampling methods on the dioxin and PCB levels of the species analysed.

Viability Test Device for anisakid nematodes.

Up to now the visual inspection of mobility of isolated anisakid larvae serves as a measure of viability and possible risk of infection. This paper presents a new method to rule out unreliability - caused by the temporary immobility of the larvae and by the human uncertainty factor of visual observation. By means of a Near infrared (NIR) imaging method, elastic curvature energies and geometric shape parameters were determined from contours, and used as a measure of viability. It was based on the modelling of larvae as a cylindrical membrane system. The interaction between curvatures, contraction of the longitudinal muscles, and inner pressure enabled the derivation of viability from stationary form data. From series of spectrally signed images within a narrow wavelength range, curvature data of the larvae were determined. Possible mobility of larvae was taken into account in statistical error variables. Experiments on individual living larvae, long-term observations of Anisakis larvae, and comparative studies of the staining method and the VTD measurements of larvae from the tissue of products confirmed the effectiveness of this method. The VTD differentiated clearly between live and dead nematode larvae isolated from marinated, deep-frozen and salted products. The VTD has been proven as excellent method to detect living anisakid nematode larvae in fishery products and is seen as useful tool for fish processing industry and control authorities.

Mitigation strategies for ester bound 2-/3-MCPD and esterified glycidol in pre-fried breaded and frozen fish products.

Pre-frying of chloride-containing raw materials (e.g., breaded frozen fish products) can lead to the formation of fatty acid esters of 2-monochloropropane-1,3-diol, 3-monochloropropane-1,2-diol (MCPD-E), and glycidol (G-E). The aim of the present study was to identify relevant parameters for the formation of these process contaminants during the pre-frying. Secondly, several mitigation approaches have been investigated. The major proportion of the MCPD-E and G-E in the fish products resulted from the pre-frying oil absorbed, while the temperature and the heating period of the pre-frying oil showed the strongest impact. A significant reduction of the MCPD-E content in the pre-frying oil was achieved by filtering-off solid breading particles. Additionally, the G-E content decreased resulting from the use of adsorbent materials. Moreover, the analyses of total polar material and the color intensity of the pre-frying oil are suggested as screening methods for estimating the MCPD-E and G-E contents in the fish products.

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.

Large scale distribution of dioxins, PCBs, heavy metals, PAH-metabolites and radionuclides in cod (Gadus morhua) from the North Atlantic and its adjacent seas.

Regarding cod as sea food for human consumption and as bio indicator of the marine eco system, this study is the first approach to combine the analysis of organic and inorganic contaminants and radionuclides in cod muscle as well as PCDD/Fs and dl-PCBs in its livers from the same fishing areas. Concentrations of 1-hydroxypyrene, PCDD/Fs, PCBs, cesium-137 (Cs-137), cadmium and lead were determined in individual or pooled samples over a wide geographic area, including Greenland Seas, Barents Sea, North and Baltic Sea. Highest concentrations were found in samples from the Baltic Sea, lowest in the pristine areas of the Barents Sea and Greenland. Levels of contaminants in cod muscle were found to be far below the established EU maximum levels (ML), regardless of which fishing grounds. In contrast to this, most cod liver samples from the North and Baltic Sea showed PCDD/F and PCB contents exceeding the ML. In addition, new background assessment criteria (BAC) for 1-hydroxypyrene in cod of 4.6 ng mL(-1) bile and for Cs-137 a BAC of 0.16 Bq kg(-1) wet weight are proposed to be included in the European Marine Strategy Framework Directive for cod from the Northeast Atlantic.

Anisakid nematodes in beaked redfish (Sebastes mentella) from three fishing grounds in the North Atlantic, with special notes on distribution in the fish musculature.

Parasitic anisakid nematodes commonly occur in the musculature and visceral organs of many fish species from the North Atlantic. In this respect, the presence of the third stage larvae of Anisakis spp. in the fish musculature may pose a potential consumer hazard due to the parasite's ability to cause anisakidosis. Thus, knowledge on the occurrence and distribution of these potentially zoonotic parasites in the commercially important North Atlantic fish species is crucial in order to evaluate and consequently prevent human infections. In the present study, 300 Sebastes mentella from three North Atlantic fishing grounds (Northern North Sea: Tampen; Barents Sea: off Bear Island; Irminger Sea: off SE Greenland) were examined for anisakid nematodes, with emphasis on occurrence and distribution in the musculature. Overall larval prevalence and mean intensity were significantly higher in redfish from Tampen (94%; 13.5±20.0) and Bear Island (94%; 14.5±19.4) than in fish from SE Greenland (75%; 6.0±5.8; p<0.01). The same trend was observed for larval infection in the musculature showing prevalence and mean intensities of 79%, 73%, and 55%, and 5.9±6.6, 5.8±6.5, and 3.2±2.4, in the musculature of redfish from Tampen, Bear Island, and Greenland, respectively. Conventional microscopy and rDNA ITS-gene sequencing of various subsamples of muscle-dwelling nematode larvae of redfish from every catching locality revealed the presence of two anisakid species; Anisakis simplex sensu stricto and the non-zoonotic Hysterothylacium aduncum. Since the larvae of H. aduncum typically occur in or on the viscera of fish, our findings of two specimens in the belly flaps of redfish were unusual. Additionally, more than 92% of the muscle-dwelling larvae occurred in the belly flaps, i.e. the hypaxial part of the musculature surrounding the visceral organs on either fish side. Thus, trimming the fillets of beaked redfish by removal of most of the belly flaps would significantly reduce the probability of anisakid nematode larvae to be present in the final product.

Effects of feeding diets containing increasing proportions of bunt-infected wheat (Tilletia caries) on performance and health of pigs.

Tilletia caries is a fungus that mainly infects wheat, causing the disease bunt. Wheat bunt had been one of the most dangerous plant diseases before seed dressing was a common practice. However, it is still of importance in organic farming, since seed dressing is not permitted. Trimethylamine (TMA) is a metabolic product of T. caries known to cause a fishy odour of highly contaminated wheat. Therefore, contamination of feed is supposed to decrease feed intake. However, systematic studies on the effect of practically relevant proportions of bunt-infected wheat on performance and health of pigs are not available. Therefore, the aim of the present study was to investigate the effects of increasing proportions of bunt-infected wheat (0%, 10%, 20% and 30% of the diet) corresponding to a total spore content of 0%, 0.2%, 0.4% and 0.6% on the performance and health of piglets during a 5-week experiment starting after weaning. TMA was not detectable in the contaminated wheat and the feed intake of piglets remained unaffected. However, live weight gain decreased linearly and feed-to-gain ratio significantly increased as the proportion of contaminated wheat increased. Haematological and serum clinical-chemical characteristics as well as the viability of peripheral blood mononuclear cells were not influenced by dietary treatments. Based on the limited data of the present experiment it might be concluded from the adverse effects of bunt-infected wheat in piglet diets on the feed-to-gain ratio that a spore content of 0.2% of the complete diet should not be exceeded.

Sealworm Pseudoterranova decipiens s.s. infection of European smelt Osmerus eperlanus in German coastal waters: ecological implications.

European smelt Osmerus eperlanus (n = 501) from the German Wadden Sea (North Sea) near the city of Cuxhaven were examined for their infestation with parasitic anisakid nematodes, especially with sealworms of the genus Pseudoterranova. The distribution of third-stage larvae (L3) in the musculature and viscera of the fish was analyzed. In total, we isolated 543 L3 from the hosts' body cavity and musculature. A subsample of 105 larvae were identified as the (sibling) species P. decipiens s.s. using direct sequencing of the highly variable ribosomal ITS1-5.8S-ITS2 genetic marker. The mean abundance was 1.1, the mean intensity was 2.3 P. decipiens s.s. and the prevalence was 47.3%. Total length and total weight, but not Fulton's condition factor (K), were significantly different in infected compared to uninfected smelt. No correlation was found between the total length of infected fish and the intensity of anisakid nematodes. The vast majority of P. decipiens s.s. was found in the musculature of the smelt. More than half (55.7%) of all nematodes were located in the 3 parts of the epaxial musculature, whereas 18.4 and 26.0% were found in the hypaxial musculature and the compartments of the tail muscles, respectively.

WEFTA interlaboratory comparison on total lipid determination in fishery products using the Smedes method.

Lipid determination by the Smedes method was tested in an interlaboratory trial performed by nine laboratories from seven countries belonging to the West European Fish Technologists Association Analytical Methods Working Group. Five samples of fish and fishery products with different lipid contents, including two blind duplicates, were distributed among the participants. All laboratories applied a slightly modified Smedes method, which included extraction of lipids by cyclohexane and isopropanol, transfer of lipids to the cyclohexane phase by addition of water, phase separation by centrifugation, and gravimetric lipid determination. The results indicate that the RSD for reproducibility (RSD(R)) was between 4.11 and 6.31% for samples with moderate (7%) and high (14%) lipid content, depending on the sample. Larger SDs among the laboratories were obtained for a cod sample with low lipid content of 0.5%. The method is judged to be suitable as a routine method for lipid determination in fish and fishery products.

Anisakis simplex (s.s.) larvae in wild Alaska salmon: no indication of post-mortem migration from viscera into flesh.

The prevalence, mean intensity and distribution of Anisakis nematode third-stage larvae (L3) in the muscle and viscera of wild-caught chum salmon Oncorhynchus keta, pink salmon O. gorbuscha and sockeye salmon O. nerka were compared immediately after catch. Salmon were collected during the fishing season in July 2007 in Bristol Bay and Prince William Sound close to Cordova, Alaska (USA). All fish were infected, and more than 90% of the nematode larvae were found in the edible muscle meat. The isolated anisakid L3 were genetically identified as A. simplex (s.s.). The distribution of nematodes in the muscle meat of fresh-caught salmon was examined in 49 O. keta, 50 O. nerka and 12 O. gorbuscha from Cordova. Most of the larvae were detected in the muscle parts around the body cavity, but nematodes were also found in the tail meat and epaxial muscle (loins). The mean intensity of Anisakis larvae in the edible part was 21 individuals for O. gorbuscha, 62 individuals for O. keta and 63 individuals for O. nerka. No difference in the intensity of Anisakis larvae in the hypaxial muscle was found between fresh-caught and immediately gutted salmon and individuals stored ungutted for 24 h either on ice or in refrigerated sea water.

Genetic variability of Anisakis simplex s.s. parasitizing European hake (Merluccius merluccius) in the Little Sole Bank area in the Northeast Atlantic.

In this study, we researched the presence of anisakids in specimens of Merluccius merluccius caught in the area of Little Sole Bank, in the Northeast Atlantic, and found that 100% of the European hake examined were infected and showed high average values of abundance (976.88) and intensity (976.88). The larvae were identified in morphological terms as morphotype type I and in molecular terms as Anisakis simplex s.s via polymerase chain reaction (PCR) restriction fragment length polymorphism of the rDNA. The genetic variability of the A. simplex s.s population in the North Atlantic is notable, with at least two ribosomal and three mitochondrial haplotypes which are different from the specimen used as control, reflecting the diversity of this species, an aspect which has scarcely been studied to date. The cox-2 gene appears to be an interesting candidate for generating new genetic markers which can be applied to differentiate between A. simplex s.s and Anisakis pegreffii. We detected 11 fixed differences in this gene, and it also offers the advantage of being easily amplified by PCR. The high prevalence of infection by A. simplex s.s and the extremely high average intensity and abundance values can have significant repercussions on public health, especially among populations which regularly eat insufficiently cooked or raw fish and have a certain genetic predisposition; the genetic variability of the parasite could be another factor to take into account.

Electron microscopy reveals fungal cells within tumor tissue from two African patients with AIDS-associated Kaposi sarcoma.

Electron microscopic investigation of biopsy materials from Kaposi sarcoma (KS) skin lesions of 2 African AIDS patients occasionally revealed fungal cells within the tumor tissue. The spherical cells were surrounded by a triple-layered cell wall and were not encapsulated. The ultrastructural characteristics of the cells resemble those of Candida albicans organisms. Neither patient showed clinical signs of a generalized fungal infection. The presence of Candida albicans in the KS tissue specimens seems to represent an early and asymptomatic stage of cutaneous candidiasis in the 2 severely immunocompromised AIDS patients.

EAU guidelines for the management of urogenital schistosomiasis.

More than 100 million people worldwide are affected by bilharziasis, caused by Schistosoma haematobium. For travellers precaution is most important. For the population in endemic areas, an integrated approach including health education is necessary. Effective pharmacologic treatment is available.

Occurrence of tubuloreticular structures and intracisternal paracrystalline inclusions in endothelial cells of tissue from different epidemiological types of Kaposi's sarcoma.

Biopsied tissue specimens from 40 cases of classic, atypical classic, endemic, and AIDS-associated Kaposi's sarcoma (KS) were investigated by electron microscopy. To search for ultrastructural differences between non-AIDS-associated KS and AIDS-associated KS, the occurrence of the following 2 ultrastructural abnormalities of the rough-surfaced endoplasmic reticulum in KS cells was evaluated semi-quantitatively: tubuloreticular structures (TRS) and intracisternal paracrystalline inclusions (IPI). These peculiar structures were found in 23 of the 40 KS cases. Two types of TRS could be distinguished: loose TRS (LTRS) and compact ones (CTRS). LTRS were observed in endothelial cells of tissue from all the different epidemiological types of KS. CTRS were confined to AIDS-associated KS. IPI were present in endothelial tumor cells of only 3 non-AIDS-associated KS cases. The study shows that in cells of KS tissue only CTRS, but not LTRS, are an ultrastructural marker for AIDS-associated KS.

Heterogeneity in prostate cancer: prostate specific antigen (PSA) and DNA cytophotometry.

BACKGROUND: The heterogeneity in prostate cancer is the reason for the difficult diagnosis and prognosis of this tumor. In this study, we looked for a correlation between prostate specific antigen (PSA), tumor staging and DNA cytophotometry. MATERIALS AND METHODS: Twenty-two prostates (pT1-T4) from patients with prostate cancer, who underwent radical prostatectomy, were examined. Preoperative PSA and postoperative DNA image cytometry, after 2-8 needle biopsies out of each organ, were evaluated. RESULTS: The prostate cancer tissues showed, in DNA stemline-interpretation according to Fu, in homogenous diploid tumors an average PSA level of 3.8 ng/ml, and, in homogenous aneuploid tumors, a level of 14.0 ng/ml. Tumors with heterogeneous DNA patterns with a majority of aneuploidy had an average PSA level of 85.6 ng/ml, and heterogeneous tissues with a majority of diploidy a level of 10.9 ng/ml. CONCLUSION: Only the stemline-interpretation of Fu after DNA cytophotometry is efficient for diagnosis of prostate cancer, and allows prognostic statements of the disease.

Examination of tumorigenesis of precursor lesions in bladder cancer by in situ hybridization.

Through examinations using fluorescence in situ hybridization (FISH) of chromosomes 1 and 9, we tried to obtain more information on dysplasia and carcinoma in situ (Cis) in relation to the oncogenesis of bladder cancer. 63 paraffin sections (dysplasia grades I-III and Cis) were evaluated, and 8 negative sections functioned as a control group. For FISH, DNA samples of CEP 1 and 9 (alpha satellites) were chosen. Gains (aneuploidy) or losses (monosomy) of chromosomal material were determined microscopically. Dysplasia grades I-III showed a 5-18% aberration in chromosome 1 aneuploidy and a 19-29% aberration in monosomy 9. Cis revealed 27% aneuploidy of chromosomes 1 and 9. Although at present dysplasia grade III and Cis of the bladder are viewed as histopathologically identical, we examined both molecular genetic differences in chromosome 9. As referred to in the literature we found the same genetic aberrations for dysplasias (grades I-III) and noninvasive papillary bladder tumors as well as for Cis and solid invasive bladder cancer.

Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B.

Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.

Accuracy of the ImmunoCyt assay in the diagnosis of transitional cell carcinoma of the urinary bladder.

The ImmunoCyt assay (Diagnocure Inc., Québec, Canada) is a new immunocytological fluorescence test for identifying two different mucins and a high-molecular-weight glycosylated carcinoembryonic antigen (CEA) present in tumours originating from transitional epithelial cells. The test promises a higher diagnostic sensitivity in transitional cell carcinoma (TCC) of the bladder than voided urine cytology. Our study was designed to evaluate this test especially for TaG1 carcinomas, which are characterised by a low detection rate in urinary cytology. A total of 121 spontaneous urine samples of 92 patients (age range 28 to 86, mean 62.5 years) were examined. The samples were taken from patients suspected of having TCC (41 out of 121) or tumor recurrence (46 out of 121), or who were part of a follow-up protocol (34 out of 121). Cystoscopy was practiced in all patients. The ImmunoCyt test was carried out according to the manufacturer's protocol. For cytology cytospins were made from the same urine samples and stained according to the method of Papanicolaou. One hundred and thirteen specimens could be evaluated. In 87 cystoscopy and/or histology were negative. There was histological evidence of 7 pTaG1, 4 pTaG2, 8 pT1G2/G3 and 7 pT2G2/G3 TCC. As for ImmunoCyt and cytology, specificity was 83.9% and 91.9%, respectively. A combination of either test indicated 81.6% specificity. The sensitivity amounted to 38.5% and 34.6%, respectively, and the combined sensitivity to 53.8%. The sensitivity for TaG1 carcinomas was 14.3% each, for TaG2 carcinomas 25% and 50%, for T1G2/G3 carcinomas it amounted to 37.5% each, while for T2G2/G3 carcinomas it was 71.4% and 42.9%, respectively. The higher sensitivity of the ImmunoCyt test as compared to urinary cytology renders improved identification of exfoliated tumour cells in bladder cancer possible. In our study, however, the expected increase in detecting TaG1 carcinomas was not found. Because of its lower specificity, the test should only be used in combination with voided urine cytology. On account of its low sensitivity, the ImmunoCyt test cannot replace cystoscopy (with biopsy) in the diagnosis and monitoring of bladder cancer.