Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

Distribution, transformation, and long-term accumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans in different tissues of fish and piscivorous birds

The present study monitored 10-year-old fish and piscivorous birds from sites contaminated for many Stars. The data reflected the results of actual, long-term environmental exposures, The results demonstrate that different tissues of fish have quite different concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F), The concentration order of PCDD/F within fish is liver congruent to egg congruent to intestines kidney congruent to hearts gill congruent to bladders > muscle > brain. The concentration order of PCDD/F within piscivorous birds was livers egg congruent to hearts muscle congruent to stomachs brain, The results obtained also demonstrate that the accumulation patterns of piscivorous birds and fish are quite different. The tissues of fish and piscivorous birds have different capacities for bioaccumulation and biotransformation of PCDD/F; variable proportions of TEQs were also found throughout their bodies. In fish, toxic equivalency quotient (TEQ): PCDD/F ratios in various tissues ranged from 0.01 to 0.07, whereas in birds the ratios ranged from 0.07 to 0.43. If the concentrations are normalized with lipid content, the results vary less. The effect of different lipid properties is obvious in the case of brain tissue, which is richer in phospholipids. (C) 2000 Academic Press.

Gut contents of silver carp, Hypophthalmichthys molitrix, and the disruption of a centric diatom, Cyclotella, on passage through the esophagus and intestine

The digestibility of algae by stomachless filter-feeding fish has been debated for decades. Results from gut contents and digestive enzyme analysis suggest poor utilization, while the measurement of food assimilation using radiolabeled isotope techniques indicate reasonable assimilation efficiency. Phytoplankton in the gut contents of the planktivorous filter-feeding silver carp were studied during March-May. The fish were cultured in a large net cage in a shallow, hypertrophic subtropical Chinese lake, Lake Donghu. In terms of biomass, the dominant phytoplankton in the fore-gut contents were Cyclotella (average 77.8%, range 69.7-93.5%) and Cryptomonas (average 9.57%, range 0-20.4%). The Ivlev's electivity index E of silver carp was much higher for Cyclotella (1.54) than for Cryptomonas (0.56). The majority of the phytoplankton found in the intestines of silver carp were 8-20 mu m, but they were also able to collect particles as small as 4.5-10 mu m, smaller than their filtering net meshes, suggesting that the secretion of mucus may play an important role in collecting such small particles. We conclude that disruption of cell walls is principally by the pharyngeal teeth, explaining previous contradictory conclusions. (C) 1999 Elsevier Science B.V. All rights reserved.

Evidence of host blood feeding by the monogenean, Ancyrocephalus mogurndae (Monogenea : Ancyrocephalidae) from the gills of the mandarin fish, Siniperca chuatsi

By examining iron contents, it is demonstrated that the monogenean Ancyrocephalus mogurndae (Yamaguti, 1940) feeds on the blood of its host, the mandarin fish Siniperca chuatsi (Basilewsky). The iron content and then the quantity of blood necessary to produce this amount of iron are found different in young and fully-matured worms. Young worms contain higher levels of iron and estimated amount of blood. It is suggested that A. mogurndae may start to feed on host blood as attached on gills, and the amount of blood ingested by young worms may vary from 0.01 to 1.00 mu l before reproduction. The difference between young and fully-matured worms may be accounted for by the elimination of haematin and change of food composition in matured worms and may also be affected by reproduction. Experimental infections of the monogenean may provide supportive information for explaining the difference, and further studies should also examine the effect of immune components in host blood ol mucus on the intestines of the parasite.

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Microsporidia-nematode associations in methane seeps reveal basal fungal parasitism in the deep sea

The deep sea is Earth’s largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at Pacific Ocean methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over two years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes’ intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host’s body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep sea biosphere.

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models

Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully crossreacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19%., of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24-71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (

Apc(MIN) modulation of vitamin D secosteroid growth control

A central paradox of vitamin D biology is that 1alpha,25-(OH)(2) D(3) exposure inversely relates to colorectal cancer (CRC) risk despite a capacity for activation of both pro- and anti-oncogenic mediators including osteopontin (OPN)/CD44 and E-cadherin, respectively. Most sporadic CRCs arise from adenomatous polyposis coli (APC) gene mutation but understanding of its effects on vitamin D growth control is limited. Here we investigate effects of the Apc(Min/+) genotype on 1alpha,25-(OH)(2) D(3) regulation of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated Apc(Min/+) versus Apc(+/+) intestines, expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1alpha,25-(OH)(2) D(3) or rationally designed analogues (QW or BTW) enhanced OPN but inhibited expression of CD44, the OPN receptor implicated in cell growth. These treatments also enhanced E-cadherin tumour suppressor activity, characterized by inhibition of beta-catenin nuclear localization, T-cell factor 1 and c-myelocytomatosis protein expression in Apc(Min/+) intestine. All secosteroids suppressed Apc(Min/+)-driven tumourigenesis although QW and BTW had lower calcium-related toxicity. Taken together, these data indicate that the Apc(Min/+) genotype modulates vitamin D secosteroid actions to promote functional predominance of E-cadherin tumour suppressor activity within antagonistic molecular networks. APC heterozygosity may promote favourable tissue- or tumour-specific conditions for growth control by vitamin D secosteroid treatment.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

Role of cytokines in secondary lymphoid organ development

Summary Secondary lymphoid organs are sites of antigen presentation, clonal expansion of B and lymphocytes, and affinity maturation of B lymphocytes. In the intestine, these immune functions occur mainly in Peyer's patches (PP). PP develop through the interplay of two main cell types, haematopoietic cells and meserichyrnal cells. One particular haematopoietic cell type was identified as the inductive cell type in the formation of both PP and lymph nodes and was therefore designated as lymphoid tissue inducer cell. For a successful PP organogenesis, the crucial molecular components involved in the crosstalk of inducer cells and their mesenchymal target cells are adhesion molecules, lymphotoxin (LT) family members, and cytokines. In particular, the interleukin 7 receptor (IL-7R) expressed on inducer cells is absolutely required. To investigate the contribution of the ligand for the IL-7R. the cytokine IL-7, in the process of PP formation, we analyzed double transgenic (TG) mice. These mice resulted from an interbreeding of an IL-7TG mouse strain where the transgene is under the control of the MHC class II promoter with a second transgenic mouse strain, which overexpresses a transactivator for MHC class II genes. Double TG offsprings revealed higher levels of IL-7 mRNA occuring earlier in embryogenesis. Consequently, double TG mice showed a striking phenotype with a 3- to 5-fold increase in PP numbers compared to single IL-7TG or control littermates. Analysis of embryonic double TG intestines demonstrated that the process of PP development was already elevated during development as early as the embryonic day 16.5. Importantly, inducer cells were significantly increased in numbers in these embryonic intestines. Furthermore, the expression of LT? mRNA, which at this early time point is exclusively expressed by inducer cells, was also increased in double TG animals. These data clearly indicate a direct influence of IL-7 on the expansion of lymphoid tissue inducer cells and on the availability of LT? leading to a higher frequency of developing PP in fetal life. Interestingly, in addition to an enhanced frequency of PP development, in double TG mice, three additional phenotypic differences were observed. i) Lymphocyte infiltration in various non-lymphoid organs, such as stomach, salivary gland, and liver. Subsequent analysis demonstrated that B lymphocytes were predominant within these tertiary lymphoid structures. ii) Ectopic lymph node-like structures containing both B and T lymphocytes were found near the inguinal lymph node. iii) Double TG mice had a severe bone resorption syndrome most likely as a consequence of the pro-osteoclastic effect of IL-7. Taken together, these results show that IL-7 plays a key role in the homeostasis of inducer cells, in the generation of PP in the gut, in the formation of ectopic lymphoid tissue, and in bone resorption. Résumé Les organes lymphoïdes secondaires sont les lieux de présentation des antigènes aux lymphocytes, permettant l'expansion des lymphocytes B et T et la maturation d'affinité des lymphocytes B. Dans l'intestin, ces fonctions immunitaires se déroulent dans les plaques de Peyer (PP). Ces plaques se développent grâce à l'interaction des cellules hématopoïétiques avec des cellules mésenchymales. Un type particulier de cellules hématopoïétiques a été identifié comme cellule inductrice dans la formation des PP et des ganglions lymphatiques et de ce fait a été désigné cellule inductrice des tissus lymphoïdes. Durant l'organogénèse des PP, les composants moléculaires cruciaux impliqués dans l'interaction des cellules inductrices et des cellules mésenchymales sont les molécules d'adhésion, les membres de la famille des lymphotoxines (LT) et les cytokines. En particulier, le récepteur de l'interleukine 7 (IL-7R) exprimé par les cellules inductrices est absolument nécessaire. Pour étudier le rôle du ligand de l'IL-7R, l'interleukine IL-7, dans la formation des PP, nous avons croisé une lignée de souris transgénique (TG) surexprimant IL-7 sous contrôle du promoteur MHC class Il avec une lignée de souris transgénique surexprimant un transactivateur des genes MHC class II. Les souris doubles TG présentent une concentration élevée d'ARNm de l'IL-7 durant l'embryogénèse, ce qui résulte en une augmentation du nombre de PP de 3 à 5 fois en comparaison aux souris ayant seul le transgène IL-7 et aux souris contrôles. L'analyse des intestins des souris doubles TG démontre que le processus de développement des PP était élevé dès le jour 16.5 du développement embryonnaire. L'augmentation du nombre des cellules inductrices dans ces intestins embryonnaires est signilicative. De plus l'expression de l'ARNm LT?, qui à ce stade précoce est exclusivement exprimé dans les cellules inductrices, est également augmenté dans les doubles TG. Ces résultats indiquent clairement une influence directe d'IL-7 sur l'expansion des cellules inductrices des tissues lymphoïdes et sur la synthèse de LT? induisant une augmentation des PP se développant durant la vie foetale. En plus du développement accru des PP dans les souris doubles TG, trois différences phénotypiques ont été observées. i) L'infiltration lymphocytaire dans différents organes non-lymphoïdes, comme l'estomac, les glandes salivaires et le foie. Des analyses complémentaires ont demontré que les lymphocytes B étaient prédominants dans ces structures lymphoïdes tertiaires. ii) Des structures de ganglions lymphatiques ectopiques contenant des lymphocytes B et T ont été trouvées près des ganglions lymphatiques inguinaux. iii) Les souris doubles TG présentent un syndrome de résorption osseuse sévère probablement dû à l'effet pro-osteoclaste d'IL-7. Globalement, ces résultats montrent que IL-7 joue un rôle clé dans l'homéostasie des cellules inductrices dans la génèse de PP de l'intestin, dans la formation des tissus lymphoïdes ectopiques et dans la résorption osseuse.

GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins secreted in response to oral glucose ingestion by intestinal L and K cells, respectively. The molecular mechanisms responsible for intestinal cell glucose sensing are unknown but could be related to those described for beta-cells, brain and hepatoportal sensors. We determined the role of GLUT2, GLP-1 or GIP receptors in glucose-induced incretins secretion, in the corresponding knockout mice. GLP-1 secretion was reduced in all mutant mice, while GIP secretion did not require GLUT2. Intestinal GLP-1 content was reduced only in GIP and GLUT2 receptors knockout mice suggesting that this impairment could contribute to the phenotype. Intestinal GIP content was similar in all mice studied. Furthermore, the impaired incretins secretion was associated with a reduced glucose-stimulated insulin secretion and an impaired glucose tolerance in all mice. In conclusion, both incretins secretion depends on mechanisms involving their own receptors and GLP-1 further requires GLUT2.

Identification of a novel herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti).

On December 8, 2008, a male fisher (Martes pennanti) housed in a quarantine enclosure at the St-Félicien Zoo was found dead with multiple skin ulcers on the muzzle and plantar pads. At necropsy, no major findings were found, and a specific cause of death was not determined microscopically. However, at the borders of ulcerated sites, there were increased numbers of koilocytes, with perinuclear vacuolation and nuclear enlargement. A pan-herpesvirus nested polymerase chain reaction (PCR) assay was conducted, and an expected PCR product of 230 nucleotides was obtained within tissues collected from around the skin ulcers. Other tissues, including intestines and pool of lung, liver, and kidney, tested negative. The obtained PCR amplicon was sequenced and was highly related to the partial viral DNA polymerase (DPOL) gene of Mustelid herpesvirus 1. Virus isolation was negative, and no virion was detected by electron microscopy. The pathogenic potential of this novel herpesvirus and its role in the death of the fisher are unknown.

Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish

Motile aeromonads isolated from the intestines of farm-raised freshwater fish such as Catla catla, Labeo rohita and Ctenopharyngodon idella have been characterized to species level. Morphological and physiological grouping revealed 61% Aeromonas hydrophila, 30% Aeromonas caviae, 7% Aeromonas sobria and 2% which remained unidentified. Hemolytic activity was detected mostly in A. hydrophila, while only half of the A. sobria and A. caviae showed this activity. Antibiotic resistance patterns of the strains revealed that they had acquired a relatively higher resistance to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B, implicating possible use of these antibiotics in the aquaculture systems.