In situ synthesis and characterization of hydroxyapatite/natural rubber composites for biomedical applications.

In this work, a biomimetic synthesis reaction for the production of hydroxyapatite (HA)/natural rubber (NR) composites is presented. HA was synthesized in the presence of solubilized NR in tetrahydrofuran (THF), which permits that negatively charged domains in proteins and lipids in NR work as nucleation sites for hydroxyapatite. The mechanical and physicochemical properties of composites containing 10, 20 and 30wt% HA were studied. NR influenced HA crystallite morphology, shape and size and was able to disperse the HA particles in THF. The dispersion of HA in the composites promoted the enhancement of mechanical properties and loss of solubility of composites, which is believed to be macroscopic manifestations of the physical adsorption of NR to HA particles, known as the bound rubber phenomenon. Preliminary cell studies indicated that HepG2 cells presented viabilities ranging from 71.6 to 91.3%, with cell proliferation positively influenced by HA content at shorter culture times. The good dispersion of HA in the composites is expected to improve NR bioactivity and direct its use towards bone applications.

Residency and movement patterns of Arctic charr Salvelinus alpinus relative to major estuaries.

Estuarine residency and marine movements of 43 anadromous Arctic charr Salvelinus alpinus (mean ± s.d. fork length = 523 ± 97 mm) were examined using acoustic tracking in inner Frobisher Bay (IFB; 63° N; 68° W), Canada, from July to September 2008 and 2009. A mean ± s.d. migration duration of 63 ± 7 days occurred from late June to early September. Detected S. alpinus were either continuously (maximum 34 days) or intermittently present in estuarine zones, on average residing approximately one third of time tracked and returning once every 9 days. Significantly higher estuarine residency during the final 15 migration days suggested that a transition phase may occur prior to freshwater re-entry. Low travel rates during flood tide suggested individuals staged before accessing intertidal and estuarine zones. Although the two main estuaries were c. 22 km apart, 19% of tagged individuals used both. Individuals remained relatively close to freshwater overwintering systems, although late-migration inter-estuarine movements may have indicated natal homing. Approximately half of the individuals exhibited extra-estuarine travel, mostly during mid-migration, but remained within 3 km of shore ranging < 30 km straight line distance (SLD) of either estuary. It was concluded that IFB S. alpinus (1) spent a significant portion of their migration within or adjacent to the estuaries and (2) had a restricted marine distribution within 30 km SLD of the river mouths.

Altered distribution of lipid-soluble antioxidant vitamins in juvenile sturgeon exposed to waterborne ethynylestradiol.

Studies in mammals have shown that exposure to estrogenic compounds can affect lipid metabolism and plasma concentrations of lipid-soluble vitamins. However, the potential for estrogenic contaminants to induce these effects in fish has not yet been examined. The ability of the estrogen analog ethynylestradiol (EE2) to alter concentrations of the lipid-soluble vitamins A and E in plasma, liver, and kidney was investigated in juvenile lake sturgeon (430 +/- 20 g). The EE2 was delivered to the sturgeon in the tank water at nominal concentrations of 0 (control), 15, 60, or 125 ng EE2/L for a period of 25 d. Concentrations of the egg yolk precursor, vitellogenin, increased dose dependently in plasma. Plasma vitamin E (tocopherol), A1 (retinol), and A2 (dehydroretinol) were elevated by the two highest EE2 treatments compared with the controls. Esterified storage forms of vitamin A were marginally lower in the livers of fish from the highest EE2 dose group, but vitamin E levels in the liver were not significantly affected. Concentrations of vitamin E and vitamin A were significantly depleted in the kidney of fish from the two highest EE2 dose groups. Total lipid content was elevated in the gonad of fish treated with the highest dose of EE2 compared with the controls. Altered lipid and vitamin distribution may be induced by estrogen to facilitate gonadal maturation in sturgeon. Results from these studies indicate that an examination of the implications for vitamin depletion by estrogenic contaminants in juvenile fish is warranted.

Diphyllobothriasis: update on human cases, foci, patterns and sources of human infections and future considerations.

Diphylobothriasis is a well documented disease of humans. On a world scale new infections are reported regularly, especially from Russia and parts of Japan. Globally, new species have been discovered and the etiology of the disease may be changing. Human infections appear to be in decline but it is not clear if the sources of infection are also in decline or if public health awareness has improved. In North America there has been a decline in human cases while in South America an increase in reports from fish, especially salmonids suggests high levels in these fish species. The history of human infections of Diphyllobothrium latum is primarily associated with the consumption of the northern circumpolar distributed pike and percids and is often considered a parasite of humans only. Indeed some researchers believe that D. latum was introduced to North America by northern European immigrants. The more benign human infections of D. dendriticum appears to be primarily associated with salmonids and coregonid fishes and fish eating birds. Although the early cases of diphyllobothriasis in the 1930s in North America came from fish originating in Lake Winnipeg, Manitoba, there was general belief that it was declining in fish populations and therefore of little significance to humans in the area. However, high levels of a plerocercoid in the flesh of walleyes and pike led to rejection of commercially harvested walleye and pike in Manitoba and northern Ontario, Canada, and a financial loss to Aboriginal fishers. D. latum is widely distributed in fishes of Manitoba and is infective to humans where it is not pathogenic and has a life span up to 4.5 years. The distribution and potential infection routes has not changed in a century and is still well established in natural hosts in the boreal regions of North America. Evidence is building for an old pre-European presence in North America, involving the Beringian land bridge and later involvement of susceptible hosts (northern European immigrants).

Enteric helminths of perch (Perca fluviatilis L.) and yellow perch (Perca flavescens Mitchill): stochastic or predictable assemblages?

Component communities of perch (Perca fluviatilis L) in Eurasia and the North American yellow perch (Perca flavescens Mitchill) were examined to determine the nature of their parasite communities. The scale of this investigation is continental and includes data collected across the distribution of each host species. Data were compiled from the literature and from 5 sample sites in North America. Four parasite species were found to occur frequently in the helminth component communities of P. flavescens. The cestodes Bothriocephalus cuspidatus and Proteocephalus pearsei, the digenean Crepidostomum cooperi, and the nematode Dichelyne cotylophora comprised a suite of species of which some or all occurred in most samples. Similarly, a group of 4 predictable parasite species was identified for P. fluviatilis in Eurasia, the digenean Bunodera luciopercae, the nematode Camallanus lacustris, the cestode Proteocephalus percae, and the acanthocephalan Acanthocephalus lucii. Specificity was not a requirement for predictability. Despite geographical isolation for millions of years, and different fish species interactions within and between continents, the predictability of these parasite assemblages indicates they are shaped by a biology, especially feeding patterns, common to both perch species. This is evidence that parasite assemblages comprised of nonhost-specific parasites in freshwater fishes are not merely stochastic assemblages but have key components that are predictable at this broad continental scale.

Crepidostomum percopsisi n. sp. (Digenea: Allocreadiidae) from the trout perch (Percopsis omiscomaycus) of Dauphin Lake, Canada.

Crepidostomum percopsisi n. sp. is described from the small intestine of the trout perch (Percopsis omiscomaycus) in Dauphin Lake, Manitoba. It is morphologically similar to Crepidostomum isostomum, which has been reported from the trout perch and several other species of fish. It differs from C. isostomum based on the vitellaria confined to the hindbody of the worm, size and shape of the cirrus, size of the testes, and its greater body length. A comparison of our specimens with those illustrated and identified as C. isostomum from trout perch indicates that such specimens are identical to larger specimens of C. percopsisi recovered by us from trout perch in May. To date, C. percopsisi has only been reported from the trout perch of Dauphin Lake, Lake Winnipeg, and Oneida Lake, which suggests host specificity.

Diclybothrium atriatum n. sp. (Monogenea:Diclybothriidae) from North American acipenserid fishes with observations on Diclybothrium armatum and Diclybothrium hamulatum.

Diclybothrium atriatum n. sp. is described from North American acipenserid fishes, Acipenser fulvescens Rafinesque (lake sturgeon) and Acipenser brevirostrum Le Sueur (shortnose sturgeon). Diclybothrium atriatum is distinguished from other diclybothriids by the presence of a unique preovarial structure that we name the ventral atrial organ. This structure possesses a ventral orifice leading to a deep groove that widens internally as a crypt, bordered by densely staining granular bodies and more deeply situated glandular cells. Previous records of Diclybothrium armatum Leuckart, 1835 examined in this study from North American acipenserids are referable to D. atriatum. Studies on the anatomy of this and other species of Diclybothrium Leuckart, 1835, D. armatum and Diclybothrium hamulatum (Simer, 1929), reveal the presence of paired multichanelled sperm ducts that are shown to be characteristic of the Diclybothriidae Bykhovskii and Gusev, 1950. Paired sperm ducts, while common in Turbellaria with multiple testes, are unusual amongst Monogenea. The endemicity of Diclybothrium atriatum and specificity for A. fulvescens and A. brevirostrum suggests that speciation of this parasite may have occurred following the isolation of Acipenser L. spp. in central and eastern North America.

Observations on the morphology, systematics, and biogeography of the genus Truttaedacnitis (Nematoda:Cucullanidae).

All species of Truttaedacnitis Petter, 1974, were examined for a phylogenetic analysis. Morphological studies showed that Truttaedacnitis truttae (Fabricius, 1794) from North America possessed a reduced button-shaped or nipple-shaped caudal mucron that is in contrast to the sharp spike-shaped mucron commonly reported in Eurasian specimens. Histological studies showed that the excretory pore in Truttaedacnitis sphaerocephala (Rudolphi, 1809) was situated near the level of the pseudobuccal capsule between the 2 parts of the ventral cephalic ridge. Cucullanus heterodonti Johnston and Mawson, 1943 is transferred to Truttaedacnitis based on the presence of prominent cephalic plates separated by sutures. An hypothesis regarding interrelationships among Truttaedacnitis spp. was developed using phylogenetic systematics. The anterior excretory pore is a synapomorphy supporting the monophyly of Truttaedacnitis clitellarius (Ward and Magath, 1917) and T. sphaerocephala parasitizing sturgeons. The arrangement of cephalic plates indicates that Truttaedacnitis pybusae Anderson, 1992 and T. truttae may form a monophyletic group and further supports the monophyly of T. clitellarius and T. sphaerocephala. Using unordered character states of different cephalic plates produced a cladogram that suggested that species in holarctic/circumboreal families (Acipenseridae, Salmonidae, Petromyzontidae) may be monophyletic. The systematics and biogeography of the various species of Truttaedacnitis and their host specificity indicate that associations in this parasite genus are very ancient and of a relictual nature, determined largely by past continental and oceanic configurations, and a combination of vicariance and dispersal.

Dietary alpha-linolenic and linoleic acids competitively affect metabolism of polyunsaturated fatty acids in Arctic charr (Salvelinus alpinus).

We investigated the effects of various levels of dietary 18:2(n-6) (linoleic acid) and 18: 3(n-3) (alpha-linolenic acid) on the growth, feeding, body composition and metabolism of polyunsaturated fatty acids in liver and muscle of Arctic charr. Fish were fed 8 or 12 casein-based semipurified diets in two separate experiments for 12 or 14 wk, respectively. Low levels of dietary 18:2(n-6) and 18:3(n-3) supplemented with pure methyl esters or linseed (flax) oil had an additive effect on fish growth. For each 100 g of diet, the addition of 1.6-1.7 g of 18:3(n-3) and 2.6 g of 18:2(n-6) led to significantly lower fish growth compared with the treatment diets with lower amounts of 18:2(n-6). Whereas 18:2(n-6) was converted to 20:4(n-6) and 22: 5(n-6) in the absence or presence of dietary 18: 3(n-3), the dominant product of 18:3(n-3) conversion was consistently 22:6(n-3). High levels of dietary 18: 3(n-3) markedly inhibited the conversion of 18:2(n-6), whereas the inhibition of dietary 18:2(n-6) on 18: 3(n-3) conversion was noted only when the ratio of dietary 18:2(n-6) to 18:3(n-3) changed from 1.0 to 1.5. Feeding diets rich in 18:3(n-3) led to a marked accumulation of 18:3(n-3) and 18:4(n-3) in fish muscle but a negligible change in 20:5(n-3) and 22:6(n-3), regardless of the level of 18:3(n-3) in the diets.

Effects of dietary n-3 polyunsaturated fatty acids on lipid and fatty acid composition and haematology of juvenile Arctic charr Salvelinus alpinus (L.).

The effect of dietary n-3 and n-6 polyunsaturated fatty acids (PUFAs) on juvenile Arctic charr Salvelinus alpinus (L.) were investigated with respect to essential fatty acid (EFA) deficiency and lipid metabolism using one commercial and 12 casein-based test diets. Arctic charr with mean weight of 1.6g were fed test diets for 12 weeks at 10°C. At the end of the feeding, blood, liver, muscle and whole fish were sampled to determine haematocrit, haemoglobin, water content, lipid and fatty acid composition. Charr fed diets containing 0-1.0% n-3 PUFAs showed typical EFA deficiency signs: fatty liver or elevated water content in whole body or substantial accumulation of 20:3n-9 in liver polar lipids. These signs were less apparent or disappeared when charr were fed diets containing ≥ 2.0% 18:3n-3. No correlation was found between dietary PUFAs and haematocrit or haemoglobin values. Significant changes in fatty acid composition of liver polar lipids in charr fed dietary PUFAs indicate that charr can convert 18:3n-3, 18:2n-6 and 20:5n-3 into long-chain PUFAs. While charr had a direct incorporation of dietary 22:6n-3 into liver and muscle there appears to be preferential utilization of n-3 PUFAs for elongation and desaturation. The conversion of 18:4n-3 was less in muscle than in livers. These findings, combined with data on growth and feed efficiency reported previously by Yang and Dick (1993), indicate that charr require 1-2% dietary 18:3n-3 (dry weight). Small amounts of dietary 18:2n-6 (up to 0.7%) did not have detrimental effects on charr.

The mucosal and systemic response to phosphorylcholine in mice infected with Trichinella spiralis.

The presence of phosphorylcholine (PC) in Trichinella was confirmed by ELISA and Western blot experiments with the PC-specific myeloma TEPC-15. Anti-PC antibody production was detected in ELISA by cross-reaction with the PC-positive somatic polysaccharide of Aspergillus and the synthetic conjugate phosphorylcholine-bovine serum albumin conjugate and by inhibition with phosphorylcholine chloride (PCCl). The kinetics of the serum and mucosal anti-PC immunoglobulin response were determined following infection of CFW mice. Anti-PC IgA was a minor fraction of the serum response. In primary infections IgG binding to Trichinella antigen was partially inhibited by PCCl incubation, but by Day 6 following challenge infections, incubation with PCCl did not reduce IgG binding. PCCl incubation also reduced serum IgM binding to Trichinella antigen following primary infections, and in contrast to IgG, a reduction occurred following challenge infection as well. Following primary and challenge infections PCCl incubations also reduced bile IgA binding to Trichinella antigen. The kinetics and subclass distribution of the anti-Trichinella PC response were equivalent to the group I response reported for synthetic PC-protein conjugates. Anti-PC IgA production indicates that class switching occurred without maturation of the response. Immunization by feeding Trichinella antigen plus cholera toxin, in contrast to infection with larvae, did not affect anti-PC antibody production following infection. Since the response was not anamnestic and the serum IgG response was not downregulated, larval infection and antigen feeding differ in the anti-PC responses they induce. The anti-PC response does not appear to be protective in Trichinella infections in mice.

Trichinella spiralis: the effect of oral immunization and the adjuvancy of cholera toxin on the mucosal and systemic immune response of mice.

The effect of oral immunization and the adjuvancy of cholera toxin (CT) were examined in mice infected with Trichinella spiralis. The mean of total muscle larvae was reduced by 36% in mice infected with Trichinella larvae in combination with CT. In mice fed soluble, particulate, or soluble/particulate antigens in combination with CT on Days 0, 14, and 21, and challenged with Trichinella larvae on Day 28, there was a significant reduction in adult worm fecundity (50%), worm size (20-30%), and the mean of total muscle larvae (75%) but no apparent effect on the rate of expulsion on Day 6 postchallenge. Following antigen feeding, but prior to challenge with Trichinella (Day 28), the immunoglobulin response was positive in only a small proportion of mice. On Day 6 following challenge with Trichinella larvae, the biliary immunoglobulin response was enhanced approximately 10-fold (P < 0.05) in all antigen-feeding treatments which included CT, compared with nonimmunized controls or antigen-feeding treatments which did not include CT. Similarly, the serum IgG response was enhanced following challenge with Trichinella larvae in treatments fed soluble, particulate, or soluble/particulate antigens with CT. The response was also enhanced in the particulate, but not in soluble or soluble/particulate antigen treatments without CT.

Characterization of cholinesterases from the parasitic nematode Trichinella spiralis.

1. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors. 2. The 5.3 and 13S forms identified in Trichinella extracts are also found in C. elegans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident. 3. The level of cholinesterases in excretory/secretory products is low. 4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.

Trichinella spiralis: dose dependence and kinetics of the mucosal immune response in mice.

The role of the mucosal immune response in helminth infections is not clear. In this study, the dose dependence and kinetics of the mucosal immune response to Trichinella spiralis were determined in experimentally infected Swiss Webster and BALB/c mice. The primary mucosal isotype was sIgA, although IgG was also detected, and primary infections with 10 and 150 larvae produced an anamnestic response on challenge. The mucosal and systemic immunoglobulin responses were dose dependent in both primary and challenge infections. The fecundity and length of worms and the rate of expulsion from the gut were determined on Day 6 postchallenge in Swiss Webster mice. Adult worm recovery and fecundity were reduced by greater than 50% and worm length by 28% in mice infected and challenged with 10 larvae and by 90, 85, and 35%, respectively, in mice infected and challenged with 150 larvae. The rate of expulsion was correlated with the size of both primary and challenge doses and a reduction in fecundity was correlated with the size of the primary dose only. The reduction in worm length did not differ significantly between the infection doses, but the trend was similar to that for expulsion. In BALB/c mice the expulsion response was dissociated from a reduction in fecundity and worm length, the latter two being positively correlated with sIgA levels, supporting a role for sIgA and/or IgG in these effects. However, expulsion does not appear to be dependent on the mucosal immunoglobulin response.

The use of the polymerase chain reaction to identify porcine isolates of Trichinella.

A method was developed to identify domestic isolates of Trichinella using the polymerase chain reaction. Oligonucleotide primers, based on the repetitive DNA sequence (pPRA) from the P1 isolate of Trichinella, were used to amplify genomic DNA from 13 domestic isolates and tested against sylvatic isolates of Trichinella. Pattern differences were observed among domestic isolates, indicating divergence of this repetitive sequence. The primers were specific for domestic Trichinella as no amplification was detected for sylvatic isolates or Trichinella pseudospiralis. It was possible to identify an isolate from a single larva following digestion or in situ in muscle tissue.

Use of confidence ellipses to detect effects of parasites on the growth of yellow perch, Perca flavescens.

Determining the causes of mortality in populations of fish is inherently difficult. To simplify the determination of whether parasite-induced mortality occurs, parasitologists have relied on 3 types of subjective analyses of graphs. Peaked host age-parasite intensity curves concomitant with a decrease in the degree of dispersion (measured by variance-to-mean ratio) of parasites in older age-classes of fishes, a slope of less than 2.0 for a log-log graph of variance versus mean intensity of infection, and differences between truncated and nontruncated forms of a theoretical frequency distribution for the parasite are considered indicators of parasite-induced mortality in fishes. The nematode Raphidascaris acus causes significant parasite-induced mortality in natural populations of yellow perch (Perca flavescens) in Dauphin Lake, Manitoba, Canada. Using this fish-parasite system we present a comparison of some of the graphical techniques used by parasitologists to detect parasite-induced mortality and show how confidence ellipses based on the parameters beta 0 and beta 1 of a linear model for growth of yellow perch (weight = beta 0 + beta 1 x age) can be used to compare many growth curves simultaneously. When plotted in a bivariate fashion (beta 0 vs. beta 1), vertical displacement of confidence ellipses along the ordinate (beta 1) are due to sublethal effects on growth of fishes in response to parasites, whereas lateral shifts along the abscissa (beta 0) are suggestive of parasite-induced mortality.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of gill nets, fyke nets, and mark-recapture methods to estimate the number of Hirudinea and Crustacea on fish.

Twenty species of fishes (n = 20,759) were collected from Dauphin Lake, Manitoba, Canada, to determine the types and numbers of ectoparasites they harbored. Counts of ectoparasites on fishes collected with different gear were compared to evaluate different methods of collection and to estimate rates of recruitment of ectoparasites by fishes. Ectoparasites were found on 11 species of fishes and the majority of these were parasitic leeches (Myzobdella moorei, Cystobranchus verilli, and Placobdella montifera) and parasitic Crustacea (Argulus appendiculosus and Lernaea cyprinacea). Some fishes also were infested by neascus-type metacercariae (blackspot) or had tumors (lymphocystis). The prevalence of ectoparasites was correlated with the abundance, feeding habits, and spatial distribution of fish species. Argulus appendiculosus and blackspot were more prevalent on benthic fishes, whereas M. moorei and tumors were more prevalent on limnetic fishes. Mark-recapture records showed that fishes occupying shallow (less than or equal to 1.5 m) water had a higher prevalence of infestation and 28 of 29 infected fishes caught by gill nets were captured in shallow water. Placobdella montifera was the only ectoparasite found on fishes from deep (1.5-3.5 m) water and the only species that was acquired by fishes previously released with no ectoparasite (2 of 239 fishes). The littoral zone (less than or equal to 1.5 m) comprises only 14% of the surface area and 3% of the volume of Dauphin Lake, yet 72% of all gill-netted fishes harboring ectoparasites were collected there. Intensities of ectoparasites estimated from gill net and pound net samples were similar, but prevalence of ectoparasites estimated from samples obtained with gill nets was lower.

Diphyllobothrium, Anisakis and other fish-borne parasitic zoonoses.

Fish-borne parasitic zoonoses such as anisakiasis and diphyllobothriasis occur infrequently in Canada and more work needs to be done on the interactions and transmission dynamics of marine and freshwater anisakids in North America. The diphyllobothriid tapeworms are primarily restricted to the northern Canada. Problems with the specific identification of these parasites from their fish hosts prompted the development of a series of nucleic acid probes. Use of the polymerase chain reaction proved to be quick, accurate and requires little skill, once developed.

A rapid method to determine the isotype and specificity of coproantibodies in mice infected with Trichinella or fed cholera toxin.

A method was developed to isolate and test coproantibodies from mice infected with Trichinella or mice fed cholera toxin. It was rapid as results were obtained within 3 h, and it was applicable to a single faecal pellet. It reduced the number of experimental animals required 7-fold, and reduced variability, since samples were obtained from the same experimental animal over time. As it was noninvasive, it did not alter normal gut physiology, nor the course of the parasitic infection. The immune response was monitored over time in individual mice, and specific sIgA and IgG isotypes were detected for both Trichinella and cholera toxin by ELISA.