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Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

There is an intense interest in developing materials for safe and effective delivery of polynucleotides using non-viral vectors. Mineralization of organic templates has long been used to produce complex materials with outstanding biocompatibility. However, a lack of control over mineral growth has limited the applicability of mineralized materials to a few in vitro applications. With better control over mineral growth and surface functionalization, mineralized vectors have advanced significantly in recent years. Here, we review the recent progress in chemical synthesis, physicochemical properties, and applications of mineralized materials in gene therapy, focusing on structure-function relationships. We contrast the classical understanding of the mineralization mechanism with recent ideas of mineralization. A brief introduction to gene delivery is summarized, followed by a detailed survey of current mineralized vectors. The vectors derived from calcium phosphate are articulated and compared to other minerals with unique features. Advanced mineral vectors derived from templated mineralization and specialty coatings are critically analyzed. Mineral systems beyond the co-precipitation are explored as more complex multicomponent systems. Finally, we conclude with a perspective on the future of mineralized vectors by carefully demarcating the boundaries of our knowledge and highlighting ambiguous areas in mineralized vectors. STATEMENT OF SIGNIFICANCE: Therapy by gene-based medicines is increasingly utilized to cure diseases that are not alleviated by conventional drug therapy. Gene medicines, however, rely on macromolecular nucleic acids that are too large and too hydrophilic for cellular uptake. Without tailored materials, they are not functional for therapy. One emerging class of nucleic acid delivery system is mineral-based materials. The fact that they can undergo controlled dissolution with minimal footprint in biological systems are making them attractive for clinical use, where safety is utmost importance. In this submission, we will review the emerging synthesis technology and the range of new generation minerals for use in gene medicines., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© 2015 The Fisheries Society of the British Isles.)

The authors measured thyroid hormone (TH) levels in plasma, whole carcass, and tissues of cultured 2-year-old immature lake sturgeon held in fresh water and in serum of adults at spawning time from the Winnipeg River. Circulating thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels were low (T4 approximately 0.3 ng/ml, T3 approximately 0.2 ng/ml) in all cultured fish and most adults, but a few of the latter had exceptionally high T4 and T3 levels. The percentages of blood TH found in erythrocytes were 19.5% (T4), 6.1% (T3) and 6.9% (reverse T3 = rT3). Equilibrium dialysis showed much higher percentages of plasma free (F) FT4 (1.1%), FT3 (0.4%), and FrT3 (3,3',5'-triiodothyronine = rT3, 3.0%) for sturgeon than for rainbow trout, indicating more limited TH binding to sturgeon plasma sites. However, concentrations of FT4 and FT3 were close to those reported for salmonids. T3 levels exceeded T4 levels in most extrathyroidal tissues of cultured sturgeon but in most cases were less than 0.1 ng/g and 10 to 100 times lower than reported for salmonids; only the whole brain T3 concentration (5.6 ng/g) approached that of salmonids. The digested thyroid contained 21.3 ng T3/g and 2.4 ng T4/g. The authors conclude that lake sturgeon have a low circulating reserve of bound TH but have FT4 and FT3 concentrations close to those of salmonids. The high thyroidal T3:T4 ratio and low tissue T4 levels suggest that, in contrast to teleosts studied to date, the thyroid may be a significant direct source of T3, the primary TH in sturgeon tissues. High serum T4 and T3 levels in some sturgeon at spawning time may suggest a thyroid role in reproduction.

The authors studied the properties and tissue distribution of thyroid hormone (TH) deiodination activities measured in vitro at subnanomolar substrate levels for cultured 2-year-old lake sturgeon held at 12 to 15 degrees. We also studied the deiodination responses to an exogenous 3,5,3'-triiodothyronine (T3) challenge and to a diet-induced growth suppression. Thyroxine (T4) outer-ring deiodination (T4ORD), T4 inner-ring deiodination (T4IRD), T3IRD, and 3,3',5'-triiodothyronine (rT3)ORD activities were evident in liver and intestine. Their properties resembled those of teleosts. T3IRD and T4IRD activities predominated in brain. Low or negligible deiodination in any form occurred in gill, skeletal muscle, kidney, notochord, or immature gonad. Only T4ORD activity was evident in the thyroid, suggesting that it secretes some T3. T3ORD and rT3IRD activities were undetectable in any tissues. Hepatic T4ORD activity varied during the photophase and was highest during late morning. A dietary T3 challenge that doubled plasma T3 levels decreased hepatic T4ORD activity without altering any other deiodination pathways in liver, intestine, or brain. A diet change from trout pellets to ocean zooplankton reduced somatic growth and plasma T3 levels and increased hepatic and intestinal T3IRD activities and hepatic rT3ORD activity but did not alter hepatic or intestinal T4ORD activity. The authors conclude that plasma T3 in lake sturgeon can be derived both from the thyroid and from hepatic (and intestinal) T4ORD activity, which varies with sampling time and downregulates in response to a T3 challenge. However, a reduction in plasma T3 accompanying a change in diet and reduced growth was not due to a decrease in T4ORD activity; rather, it was due to an increase in hepatic and intestinal T3IRD activities. These results suggest a difference in emphasis in thyroidal regulation between sturgeon and certain teleosts.

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.

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).

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. 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. 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.

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.

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.

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 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.

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.

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.

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.

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)

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.

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.

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.

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.

Polypodium sp. is reported for the first time from Canadian waters. It occurred in lake sturgeon (Acipenser fulvescens) eggs, from the Nelson River, Manitoba, and from Cumberland House, Saskatchewan. The prevalence of this parasite was 100% in females with eggs of 3.0 mm diameter or more. One, 4, 25, and 179 infected eggs were collected in gonads with eggs of this size class, but infected eggs were not apparent in individuals with smaller eggs. This is the northernmost occurrence of this parasite reported in North America.

Nonresident (introduced) largemouth bass (Micropterus salmoides) from Boundary Reservoir, Saskatchewan were examined for helminths. Four species of parasites were found (Diplostomum sp., Proteocephalus ambloplitis, Pomphorhynchus bulbocolli, and Contracaecum sp.). Contracaecum sp. larvae were absent in age-0 and age-1 bass, but prevalence and mean intensity increased with age for bass age-2 or older. Similarly, the prevalence and mean intensity of P. ambloplitis plerocercoids in bass were low until age-2; older bass harbored significantly more plerocercoids. Analysis of stomach contents indicates that this pattern of recruitment for Contracaecum sp. and P. ambloplitis is probably due to increased feeding by largemouth bass on aquatic insects and cannibalism after age-2, respectively. Although Contracaecum sp. may have been established in the reservoir prior to the introduction of bass, we are certain that P. ambloplitis was introduced via stocking with infected fingerlings.

Two Trichinella isolates from humans in France were characterized using reproductive capacity indices and a combination of molecular methods. The isolate TRLL hybridized with the pig type-specific probe pPra and had pig type restriction profiles and rDNA patterns. It was therefore identified as a domestic or pig type isolate. The isolate CTRD-85 had similarities and differences in restriction profiles and rDNA patterns with both AF1 and Trichinella nelsoni and was identified as a sylvatic type. Pattern comparisons also show that T. nelsoni is similar to variants of the North American sylvatic type.

The positions of 3 cestode species, Triaenophorus crassus Forel, Triaenophorus nodulosus (Pallas), and Proteocephalus pinguis LaRue, and a nematode, Raphidascaris acus (Bloch), within the intestine of naturally infected pike (Esox lucius L.) were evaluated with respect to the presence or absence and state of digestion of host stomach contents. The positions of scolices and strobilae of the cestodes did not vary with respect to host stomach contents. By contrast, R. acus was anterior (near the stomach) when the stomach contained partially digested items, posterior when the stomach was empty, and in an intermediate position when the stomach contained only intact items. These results are interpreted as migration of R. acus, but not T. crassus, T. nodulosus, or P. pinguis, in response to feeding activity of the host. Adult and larval R. acus migrated, but the extent of migration was reduced in hosts harboring T. crassus, more so for larval than adult R. acus.

A system to designate and define isolates of Trichinella spiralis is proposed. The designation gives the host from which the isolate was recovered, geographic origin, and year of recovery. Isolates of T. spiralis recovered from frozen muscles from four species of wild carnivores had low and different infectivity to laboratory mice. Viable larvae of T. spiralis were obtained from muscle samples of marten, wolverine, polar bear and arctic fox which had been frozen for 5, 6, 12 and 14 mo, respectively.

The immune response that expels the tapeworm Hymenolepis citelli from the small intestine of its host the white-footed deer mouse is genetically controlled. Patent infections with this tapeworm occur only in individuals that are homozygous for a recessive allele expressed at a single gene locus. By studying this natural host-parasite system in the laboratory it was shown that host genetics contributes to parasite overdispersion in a host population in the absence of all other ecological variables. Thus, the substantive influence of the proportions of resistant and susceptible genotypes in the host population must be considered when developing parasite population models of transmission or control measures.

Larvae of the nematode Raphidascaris acus were found free or encapsulated in the liver of yellow perch. Blood vessels were distorted or destroyed during larval migrations and larvae were eventually encapsulated in a thick-walled whitish nodule. Successful walling-off of the parasite resulted in the formation of a collagenous nodule and a complete loss of the worm. No mortality of perch was associated with larval R. acus but the introduction of susceptible fishes into a lake harboring this parasite may be important in some stocking programs.

Samples of muscle from 4,773 specimens of 18 species of wild mammals from Ontario were examined for Trichinella. One of 12 mink (Mustela vison), 83 of 1,821 fisher (Martes pennanti) and 68 of 1,980 marten (Martes americana) had T. spiralis. Prevalences of infections by Trichinella were determined for fisher and marten from the Algonquin region, over a 10-yr period. Prevalences ranged from 0.9-9.2% in fisher and 1.3-8.7% in marten indicating that the parasite is well-established in the region. Prevalences of Trichinella increased with age of both fisher and marten. Intensities determined for the 1981-1982 sample ranged from 0.4-15.8 larvae/g for fisher and 22.4-159.7 larvae/g for marten. Higher intensities were not correlated with older hosts. Fisher and marten appeared to be the key hosts maintaining Trichinella in the Algonquin region, but transmission dynamics were unclear. Transmission may include an unidentified small rodent or other host and natural carrion-feeding.

Rainbow trout (Salmo gairdneri) stocked into a small prairie dug-out were examined for helminths at harvest time. Five species of helminths were found (Diplostomum sp., Diplostomulum scheuringi, Clinostomum complanatum, Crepidostomum farionis and Pomphorhynchus bulbocolli); C. farionis and P. bulbocolli mature in fish while the remaining species utilize fish-eating birds as definitive hosts. Examination of stomach contents indicated that all invertebrate hosts required for the life-cycles of these helminths were present in the dug-out. The most conspicuous and prevalent helminth was C. complanatum as fish were "wormy" and unmarketable due to the presence of high numbers of metacercariae. Metacercariae of C. complanatum were recovered from most organs of rainbow trout. The low survival of stocked fish suggests that C. complanatum may cause some host mortality, but the condition of infected and uninfected fish was similar. It appears that a community of ichthyoparasites can become established in a population of stocked fish in a single growing season in north temperate regions.

The immune response of inbred mice was studied following infection with Trichinella spiralis var. pseudospiralis (TP) or with isolates of T. spiralis derived from a pig or from an arctic fox. Animals given a primary infection with 1 isolate of Trichinella and challenged 21 days later with the same or different isolates responded more quickly by expelling worms from the homologous challenge. In addition, although mesenteric lymph node cells from mice infected with each isolate of Trichinella would proliferate in vitro when cultured with antigen derived from each of the others, the strongest proliferation response always occurred when cells were cultured in the presence of antigen prepared from the specific isolate used to infect the mouse from which the cells were derived. In addition, it was possible to prepare monoclonal antibodies that recognized an antigen expressed by TP which was not shared by T. spiralis isolates and vice versa. Collectively, these data support the conclusion that the differences observed in the kinetics of immune responsiveness to different Trichinella isolates are referable, at least in part, to differences among the isolates in the expression of functionally relevant antigens.

Intestinal distribution of Trichinella spiralis was studied in mature and suckling Sprague-Dawley rats. Position of Trichinella along the small intestine was defined by a median value for the population. A range, 90% of total population, gave a relatively precise determination of microhabitat width. The normal position of Trichinella following an oral infection is in the duodenum, but position of Trichinella in mature rats is more anterior than position of Trichinella in suckling rats. Intestinal inoculation of Trichinella larvae into the jejunum and ileum revealed that worms remained at the site of inoculation and did not migrate to the duodenum. Trichinella is able to utilize the entire small intestine but its normal position is the duodenum. The position of Trichinella in the anterior region of the small intestine probably is related to physiological factors of the intestine and thereby host dominated.

Six species of parasites were recovered 4 mo after walleye fry were stocked in Heming Lake, Manitoba. The species of parasites acquired most rapidly were those that were non-host-specific and common to the indigenous populations of both walleye and yellow perch (Perca flavescens). Parasite species overlap (Jaccard's indices) was greatest within age groups of walleye and yellow perch, but was also high between older walleye and yellow perch. The higher numbers of parasites recruited by stocked walleye, particularly ones known to induce pathology, raises questions on the success of walleye introductions to aquatic systems with a diverse indigenous parasite fauna and a fish population with a large proportion of yellow perch.

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

The objectives of this study were to determine if the raccoon was a useful experimental animal for infections of Trichinella and to determine if the ability of Trichinella to survive freezing conditions, known to occur in wild animals, could be duplicated under laboratory conditions. The isolates of Trichinella used in this study were from pigs, polar bear, wolverine, arctic fox and T. spiralis var. pseudospiralis originally isolated from a raccoon in the USSR. The raccoon was found to be a useful experimental host for Trichinella as it was easily maintained under experimental conditions and was readily infected. Infectivity indices were lower in raccoons than in laboratory mice. Those isolates of Trichinella with the longest association with laboratory mice had the lowest infectivity indices. The isolate of Trichinella from an arctic fox retained its ability to survive freezing temperatures when introduced into raccoons held under experimental conditions. The type of host, method of passing the parasite and perhaps a special genetic characteristic of arctic isolates seem to be important factors influencing their ability to survive freezing temperatures.

We examined a host system (peromyscus maniculatus) in which a single autosomal gene controls susceptibility or resistance to infection by the cestode parasite Hymenolepis citelli. Parasite deaths of both primary and secondary (challenge) infections were examined, using a probabilistic model, to see if deaths were random and uncorrelated within each genotype. Within susceptible hosts, post-patent survival of primary worms was not random and heterogeneity was due to among-host effects rather than parasite-age effects, suggesting a second genetic or immunological process. Secondary infections in susceptible hosts appear to be lost randomly, independent of primary infection age or burden. The loss rate is similar to that for primary worms. The lack of correlation or alteration in (susceptible) host response to primary and secondary infections suggests that the latter are eliminated by a process that differs from that acting on primary worms. In resistant hosts, parasite survival rates suggest that the immunological process elicited by the primary infection also acts to eliminate the secondary infection more rapidly. Suggestions are made for improving experimental methods when dealing with mixed genotype populations. Experiments should permit direct estimation of genotype frequency and of parasite death rates within each genotype. This separation of host type is particularly important when studying correlation of successive worm burdens since any host or treatment mixture (genotype, age, sex, size or infection dose) may induce correlations that could be mistakenly interpreted.

In the United Kingdom, potable water put into supply is required to be 'wholesome'. The term 'wholesome' is interpreted as clear, palatable and safe to drink. About 99% of potable supplies are provided by Regional Water Authorities and Water Companies (for England and Wales), Regional Councils and Island Councils (for Scotland) and the Department of the Environment (NI) (for Northern Ireland). These water authorities draw their raw water from upland surface waters, lowland surface waters (including lakes and rivers) and underground waters. Although each source provides approximately one-third of supply, the proportion varies considerably in different parts of the UK. Consequently the control of potential health risks related to drinking water also varies according to the source of supply. The paper describes briefly the treatment practice for the various sources, including disinfection practice. More specifically the paper describes current UK practice or developments in the control or investigation of plumbosolvency, fluoridation, nitrate, trihalomethanes, other organic micropollutants, sodium, asbestos and tar linings in pipes. The possibilities for the surveillance of the 1% of private supplies are also discussed.

Biological characteristics of Trichinella isolate (wolverine: 55 degrees 00'N, 100 degrees 00'W, 1979) were established in Crl: COBS CFW (SW) mice. Comparison of the wolverine isolate's biological characteristics with another Trichinella isolate (polar bear), both from closely related geographic areas, revealed there were stable and reproducible genetic differences between isolates. Differences were most pronounced for degree of infections causing a 50% mortality of mice, larval production by females in vitro, reproductive capacity indices, and survival of muscle larvae. Species and strain of host altered characteristics of the wolverine isolate such as worm position, sex ratios, % recovery, larval production by females in vitro, and reproductive capacity indices. Genetic differences between isolates of Trichinella and interaction with host genetics raises interesting questions on Trichinella speciation. Differences reported here, are best interpreted for the time being at least, as part of the normal biological variability of the species Trichinella spiralis.