Biomedical subjects

P T Woo

Publications and source records attributed to P T Woo.

At least 19 recordsLinked to original sources

PubMed2001

Cryptobiosis and its control in North American fishes.

Cryptobiosis is caused by the haemoflagellates Cryptobia bullocki and Cryptobia salmositica. These parasites infect food fishes (e.g. flounders, salmon) on both the Atlantic and Pacific coasts of North America and clinical signs of the disease include anaemia, and abdominal distention with ascites. The virulent factor in salmonid cryptobiosis, caused by C. salmositica, is a secretory metalloprotease (200 kDa). Fish mortality may be up to 100% in the absence of treatment, consequently strategies have been developed to protect them from disease/mortality. A single dose of a live vaccine protects fish for at least 2 years, and it is via the production of complement-fixing antibodies, enhanced phagocytosis and cell-mediated cytotoxicity. Inhibition of the parasite's cysteine protease by a monoclonal antibody reduces multiplication, infectivity and survival of the parasite. Consequently, the recombinant cysteine protease (49 kDa) of the parasite will be tested as a potential vaccine. The trypanocidal drug, isometamidium chloride (1.0 mg/kg), is effective (therapeutic and prophylactic) against C. salmositica in chinook salmon. Its efficacy is significantly enhanced if it is conjugated either to a monoclonal antibody or to polyclonal antibodies from immune fish. Selective breeding of Cryptobia-resistant brook charr (innate resistance to infection) is possible, and the resistant factor(s) is controlled by a dominant Mendelian locus. In these resistant charr the parasite is lysed via the alternate pathway of complement activation (innate immunity to infection). There are also Cryptobia-tolerant charr, fish that are susceptible to infection but have no clinical disease (innate resistance to disease). In these fish, one of the natural anti-proteases, alpha2-macroglobulin, neutralises the metalloprotease secreted by C. salmositica. Production of transgenic Cryptobia-tolerant salmon is an option to vaccination and or chemotherapy. Also, transgenic pathogen-tolerant animals may be an alternate strategy against other pathogens where the disease mechanism is similar to cryptobiosis.

Animals↗

PubMed2001

Therapeutic and prophylactic effects of isometamidium chloride (Samorin) against the hemoflagellate Cryptobia salmositica in chinook salmon (Oncorhynchus tshawytscha) and the effects of the drug on uninfected rainbow trout (O. mykiss).

A series of compounds (triphenylmethanes, thiazines, xanthenes, benzidines, phenanthridiniums, napthalamines, and diamidines) were screened for in vitro toxicity against Cryptobia salmositica. Isometamidium chloride (Samorin) was cryptobiacidal at low concentrations and was examined for therapeutic and prophylactic activities against C. salmositica in chinook salmon (Oncorhynchus tshawytscha). An intramuscular dose (1.0 mg/kg) of Samorin 3 weeks post-infection significantly reduced the parasitemia in adult chinook. A higher dose (2.5 mg/kg) eliminated the infection in 30% of adult fish and parasitemias were significantly reduced in the remaining infected fish. Juvenile chinook treated with 1.0 mg Samorin/kg at 2-3 weeks post-infection survived, while 100% of untreated control fish died from cryptobiosis. The high dose (2.5 mg/kg) was lethal to small fish (98.93 +/- 12.09 g) and 50% died within 24 h of treatment, while all large fish (168.38 +/- 13.87 g) survived. Samorin (1.0 mg/kg) did not affect growth, food consumption, complement, or hematocrit values in uninfected rainbow trout (O. mykiss).

Animals↗

PubMed2001

In vitro secretion of metabolic end-products by piscine haemoflagellates Cryptobia salmositica and C. bullocki (Kinetoplastida: Bodonidae) and the relationship of these products to the pH in the medium.

Pathogenic and nonpathogenic strains of Cryptobia salmositica Katz, 1951 and C. bullocki Strout, 1965 produced hydrogen peroxide, pyruvate and lactate under in vitro conditions in Minimum Essential Medium (MEM). As parasite number increased, the phenol red in the medium changed from red to yellow. This change was not associated with a decrease in pH, or an increase in pyruvate or lactate, but was correlated with an increased secretion of hydrogen peroxide. Parasites incubated at 10 degrees C in medium at pH 6.0, 6.5, 7.0 and 7.3 were active for about I week with decreasing activity in the absence of serum. Parasites in saline (pH 6.0, 6.5, 7.0 and 7.3) were nonmotile within 24 h and were dead in about 1 week. This suggests that these Cryptobia spp. are sensitive to changes in pH and require medium which is buffered, either with serum or Hepes.

Animals↗

PubMed2001

The in vitro effects of isometamidium chloride (Samorin) on the piscine hemoflagellate Cryptobia salmositica (Kinetoplastida, Bodonina).

Isometamidium chloride (Samorin) is therapeutic in rainbow trout (Oncorhynchus mykiss) during preclinical and chronic cryptobiosis. However, the toxic mechanism of isometamidium on Cryptobia salmositica has not been elucidated. The objective of the present study was to examine the in vitro effects of isometamidium on C. salmositica. Under in vitro conditions, isometamidium chloride reduced the infectivity of C. salmositica suspended in whole fish blood. It accumulated rapidly in the kinetoplast (within 1 min) and caused disruption and decantenation of kinetoplast DNA. The in vitro cryptobiacidal activity of isometamidium was reduced when parasites were incubated in medium containing serum supplement, suggesting that isometamidium also binds to plasma proteins. Isometamidium altered glycoprotein receptors (epitopes) for antibodies on the surface of C. salmositica and thus protected some of the parasites from lysis by complement-fixing antibodies. In vitro oxygen consumption and carbon dioxide production decreased in drug-exposed C. salmositica, with increased products of glycolysis, i.e., lactate and pyruvate, after exposure to isometamidium. This suggests that some C. salmositica switched from aerobic respiration to glycolysis when the mitochondrion was damaged by isometamidium.

Animals↗

PubMed2000

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae).

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.

Aerobiosis↗

PubMed2000

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

Animals↗

PubMed1999

The therapeutic use of isometamidium chloride against Cryptobia salmositica in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss injected intramuscularly with isometamidium chloride (0.01 or 0.1 mg kg-1) at 3 wk post-infection and given a booster 2 wk later had significantly lower parasitaemias than infected controls. Packed cell volume increased after treatment and remained higher than in infected controls. The concentration of isometamidium in plasma was highest at 2 wk after injection and then declined. An intramuscular dose of 1.0 mg kg-1 of isometamidium chloride at 1, 2 and 3 wk postinfection (preclinical) significantly reduced the parasitaemia in rainbow trout 2 wk after treatment. A booster at 9 wk postinfection (chronic disease phase) reduced the parasitaemia further in all fish. The packed cell volume in these fish was higher than in infected controls. Treatment at 5, 6, and 7 wk postinfection (acute disease) had no effects and parasitaemias in treated fish were higher than in infected controls; also, anti-Cryptobia salmositica antibodies and titres of complement-fixing antibody were higher in these than in infected controls. Incubation of immune plasma or complement with isometamidium for 3 h did not affect the lytic titres of complement-fixing antibodies nor rainbow trout complement.

Animals↗

PubMed1998

Identification of carbohydrates on the surface membrane of pathogenic and nonpathogenic piscine haemoflagellates, Cryptobia salmositica, C. bullocki and C. catostomi (Kinetoplastida).

Carbohydrates and protein glycoconjugates on the cell membranes of Cryptobia salmositica, C. bullocki and C. catostomi were analyzed using 13 highly purified lectins (unlabelled or digoxigenin/biotin labelled). No agglutinations were observed with C. salmositica, C. bullocki and C. catostomi using lectin TPA (Tetragonolobus purpureas agglutinin, for alpha-L-fucose). C. salmositica was agglutinated by 3 of 12 lectins [Con A, for alpha-man and alpha-D-glc; PSA, for alpha-man; PWM, for (glcNAc)3], while C. bullocki was agglutinated by 8 lectins and C. catostomi was agglutinated by 10 lectins. Glycoconjugate analysis with digoxigenin or biotin labelled lectins showed a species-specific staining pattern in pathogenic and nonpathogenic Cryptobia spp. The nonpathogenic C. catostomi had the strongest reaction. These results indicate that the surface carbohydrate residues and glycoconjugate compositions on Cryptobia spp. are different between species they may be related to the virulence of the parasite.

Agglutination↗

PubMed1998

Characterization of a 200 kDa glycoprotein (Cs-gp200) on the pathogenic piscine haemoflagellate Cryptobia salmositica.

The 200 kDa antigenic doublet of the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951, was detected using a monoclonal antibody (MAb-001) in 1-dimensional SDS-PAGE. This antigenic doublet is a glycoprotein since it was susceptible to both protease K and to sodium m-periodate oxidation and is designated Cs-gp200. It has a PI (isoelectric point) value of about 5.5 (using 2-dimensional gel electrophoresis). It migrated faster under reducing conditions than under nonreducing conditions and was partitioned into the aqueous phase in Triton X-114 phase separation. It is a secretory-excretory product since it was detected in a non-protein culture medium with C. salmositica. These results suggest that the Cs-gp200 is a glycoprotein consisting of carbohydrate determinants and conformational polypeptide with internal disulphide bonds. It is a hydrophilic antigen, is a secretory product of the parasite, and plays a important role in antibody elicitation in immunized fish.

Animals↗

PubMed1998

Biochemical characterisation of an epitope on the surface membrane antigen (Cs-gp200) of the pathogenic piscine haemoflagellate Cryptobia salmositica Katz 1951.

A protective surface antigen (200 kDa) on C. salmositica was detected using a monoclonal antibody (mAb-001). Enzymatic studies on the epitope indicated that it was sensitive to nonspecific protease K and to site-specific trypsin and protease V8 but not to alpha-chymotrypsin. The reactivity of the epitope with mAb-001 was not affected when the antigen was denatured with 8 M urea; however, reduction of the antigen with dithiothreitol destroyed the epitope. The epitope was susceptible to sodium m-periodate oxidation and N-glycosidase F, but not to O-glycosidase or neuraminidase. It was also sensitive to mild potassium hydrochloride hydrolysis and to phospholipase C, which is specific for phosphatidylinositol. These results suggest that the epitope consists of a polypeptide, a carbohydrate, and probably a phospholipid. The asparagine-bound N-glycosidically linked hybrid-type carbohydrate chain has the minimum length of a chitobiose core unit. There is probably a phosphatidylinositol residue which anchors the polypeptide to the surface membrane. The antigen is extensively posttranslationally modified.

Amidohydrolases↗

PubMed1998

Characterization of purified metallo- and cysteine proteases from the pathogenic haemoflagellate Cryptobia salmositica Katz 1951.

The purified metalloprotease and the partially purified cysteine protease from pathogenic Cryptobia salmositica were characterized. Using haemoglobin gel electrophoresis, we detected five enzymatic bands in crude parasite lysate; one protease (200 kDa) yielded a metalloprotease band and other four, cysteine protease bands (97, 70, 66 and 49 kDa). Both the metalloprotease and the cysteine protease had high levels of proteolytic activity against azocasein, haemoglobin and fibrinogen. The metalloprotease had high levels of activity against azocoll and gelatin but a low degree of activity against albumin. In contrast, the cysteine protease had extensive activity against albumin but low levels of activity against azocoll and gelatin. The metallo- and cysteine proteases had no activity against Pz-peptide, a specific substrate for bacterial collagenase. The optimal pH for the metalloprotease and the cysteine protease was 7.0 and 5.0, respectively. The metalloprotease was inhibited by metal-chelating agents and excess of zinc ions but was activated by calcium ions. The cysteine protease was inhibited by thiol-blocking agents. The natural antiprotease alpha2-macroglobulin, but not alpha1-protease inhibitor, inhibited the activity of both proteases from C. salmositica. The optimal in vitro temperature for the purified metalloprotease was 30 degrees C.

Animals↗

PubMed1998

Protection against Cryptobia (Trypanoplasma) salmositica and Salmonid Cryptobiosis.

Cryptobia (Trypanoplasma) salmositica is a haemoflagellate that causes morbidity and mortality in salmon, Oncorhynchus spp, on the Pacific coast of North America. In this review, Patrick Woo briefly describes the pathogen, its transmissions (either indirectly via its leech vector, Piscicola salmositica, or directly between fish) and the clinical signs of the disease. He then outlines strategies that have been developed to protect fish against the pathogen, and the mechanism of innate resistance to disease in Cryptobia-tolerant fish. Protective strategies include the breeding of fish that are resistant to infection, and the use of an attenuated C. salmositica strain to protect susceptible fish from disease for at least two years. He ends the review with suggestions for further research that include the use of the leech vector to deliver the vaccine and the development of more novel protective strategies (eg. immunochemotherapy, anti-idiotype vaccine) against cryptobiosis.

Journal Article↗

PubMed1998

The in vitro effects of crystal violet on the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida).

Crystal violet does not inhibit in vitro multiplication of a nonpathogenic strain of Cryptobia salmositica at low concentrations (0.01 microM and 0.001 microM) but multiplication is inhibited at higher concentrations (> or = 0.05 microM). In contrast, the pathogenic strain of C. salmositica does not multiply in vitro when incubated with crystal violet (0.001 microM, 0.01 microM and 0.05 microM). The infectivity of the pathogenic strain is significantly reduced after in vitro exposure to crystal violet. Crystal violet lyses C. salmositica (100.0 microM) and causes lesions on mitochondrial and nuclear membranes of the parasite. Pathogenic strains of Cryptobia salmositica and C. bullocki are more susceptible to lysis after in vitro exposure to crystal violet than are nonpathogenic strains of Cryptobia salmositica and C. catostomi.

Animals↗

PubMed1998

Improved culture media for piscine hemoflagellates, Cryptobia and Trypanosoma (Kinetoplastida).

Increasing the Hepes buffer in minimum essential medium from 25 mM to 100 mM yielded a significantly larger number of Cryptobia salmositica. Cryptobia salmositica (pathogenic and nonpathogenic strains), Cryptobia bullocki, and Trypanosoma danilewskyi did not multiply either in heat-inactivated trout plasma (< or =25%) or in less than 10% fresh trout plasma. Both strains of C. salmositica multiplied better in 10% fresh trout plasma than in 25% heat-inactivated fetal bovine serum. In contrast, C. bullocki and T. danilewskyi multiplied better in 25% fetal bovine serum; 10% fetal bovine serum did not significantly reduce multiplication of C. bullocki. The nonpathogenic vaccine strain of C. salmositica cultured in 10% fresh trout plasma still protected rainbow trout from high parasitemia when challenged with the pathogen.

Analysis of Variance↗

PubMed1997

Surface charge and surface carbohydrates of Cyptobia salmositica virulent and avirulent forms and of C. bullocki (Kinetoplastida: Cryptobiidae).

The surface charge and surface carbohydrate residues of the virulent (freshly isolated from the fish blood) and avirulent forms (from culture) of Cryptobia salmositica and one strain of C. bullocki were studied. Measurements of the zeta potential of parasites showed that C. bullocki and the virulent form of C. salmositica had a net negative surface charge of about -15 mV, whereas the attenuated form of C. salmositica showed a surface charge of -7.9 mV. Enzymatic treatments of parasites with neuraminidase, trypsin, or phospholipase C indicated the presence of sialic acid residues, phosphate groups, and protein glycoconjugates as components of the Cryptobia surface that accounted for their surface charge. Residues of alpha-D-man, alpha- and beta-D-gal, alpha-D-galNAc, alpha-L-fuc, and D-glcNAc could be detected on the surface of all parasites by specific fluorescein isothiocyanate (FITC)- and colloidal gold-labeled lectins. The cell surface of the avirulent form of C. salmositica showed the strongest reactivity to almost all lectins tested. A remarkable binding pattern of lectins in the anterior region of parasites was observed.

Animals↗

PubMed1997

Natural anti-proteases in rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis and the in vitro neutralization of fish alpha 2-macroglobulin by the metalloprotease from the pathogenic haemoflagellate, Cryptobia salmositica.

Natural anti-proteases (alpha 1-protease inhibitor (alpha 1-PI; alpha 1-antitrypsin) and alpha 2-macroglobulin (alpha 2-M)) were found in the blood of rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis. The alpha 2-M inhibited Cryptobia salmositica proteases and was significantly higher in brook charr than in rainbow trout. Under in vitro conditions it took longer for the same number of parasites to neutralize the alpha 2-M in charr than in trout blood. The haemolysis which occurred when C. salmositica was incubated in the blood of rainbow trout was due to neutralization of alpha 2-M. This in vitro study also showed that it was the metalloprotease of C. salmositica that lysed red blood cells and the plasma of the two species of fishes initially prevented haemolysis by inhibiting the proteolytic activity. We suggest that the natural plasma alpha 2-M plays an important role in defence against cryptobiosis in fishes.

Animals↗

PubMed1997

Immunization against parasitic diseases of fish.

Parasitologists have not, in the past, exploited the immune system to protect fish against parasitic diseases. In the past few years, however, there has been an increased interest in adopting this strategy, and we have made steady and promising progress against a few parasites which are of economic importance. Amyloodinium ocellatum is an ectoparasitic dinoflagellate on brackish and marine fishes, which may also cause problems to aquarium fishes. Antiserum from fish inoculated intraperitoneally (i.p.) with living dinospores of the parasite immobilizes and agglutinates living dinospores; it also reduces parasite infectivity in cell culture. Cryptobia salmositica is a pathogenic haemoflagellate of salmonids on the Pacific coast of North America, causing mortality in semi-natural and intensive salmon culture facilities. A live attenuated vaccine inoculated i.p. protects susceptible juvenile and adult fish for at least 24 months. The protection involves production of complement fixing antibodies, phagocytosis, and antibody-dependent and antibody-independent T-cell cytotoxicity. A monoclonal antibody against a surface membrane glycoprotein (199-200 kDa is therapeutic in that it significantly reduces parasitaemias when inoculated into fish with acute disease. Ichthyophthirius multifiliis is an ectoparasitic ciliate of freshwater fishes with world wide distribution, usually causing disease when fish are stressed and/or when environmental conditions are favourable for parasite multiplication. Live theronts injected into the body cavity protect fish, and monoclonal antibodies with immobilizing activity upon parasites have been developed. There is some evidence of passive transfer of protective immunity from immune to naive fish, and to eggs. Diplostomum spathaceum is an intestinal parasite of gulls; the metacercaria stage of the parasite encyst and causes disease and mortality in numerous species of freshwater fish in Europe and in North America. Fish injected i.p. with sonicated/killed cercariae or metacercariae have fewer metacercariae in the eyes and survives longer. Lepeophtheirus salmonis and Caligus elongatus are parasitic copepods (sea lice), and they are important parasites of Atlantic salmon in cage cultures. A vaccine against fish lice is plausible, and the efficacy of about 20 candidate antigens in protecting fish is being tested.

Animals↗

PubMed1997

Complement fixing antibody production in thymectomized Oncorhynchus mykiss, vaccinated against or infected with the pathogenic haemoflagellate Cryptobia salmositica.

Short-term thymectomized (two months after thymectomy) adult rainbow trout, Oncorhynchus mykiss (Walbaum) infected with the flagellate Cryptobia salmositica Katz, 1951 responded well during primary infection with C. salmositica and recovered fish also showed secondary response (rapid production of complement fixing antibody after homologous challenge) five months after recovery. Complement fixing antibody was detected during primary and secondary responses and the titres of complement fixing antibody in thymectomized fish were significantly lower than those in infected intact fish. The parasitaemia in thymectomized infected fish was significantly lower than in intact infected fish. Both recovered thymectomized fish and intact fish were protected from cryptobiosis when they were challenged. Similarly, long-term thymectomized fish (nine months after thymectomy) vaccinated with an attenuated strain of C. salmositica were protected from cryptobiosis. There were no significant difference (P > 0.05) in parasitaemia, packed cell volume and complement fixing antibody titres between vaccinated/challenged thymectomized and vaccinated/challenged intact fish. Hence, thymectomy in adult rainbow trout did not decrease the detectable complement fixing antibody against C. salmositica in long-term thymectomized fish but reduced the detectable protective antibody in short-term thymectomized fish.

Animals↗