Histopathology and culturable bacteria associated with "big belly" and "skin nodule" syndromes in ornamental Siamese fighting fish, Betta splendens.

The Siamese fighting fish (Betta splendens) is one of the popular aquarium ornamental fish in the global trade. Large numbers of ornamental fish farmed in central Thailand suffered from two common syndromes; preliminarily named skin nodule syndrome (SNS) and big belly syndrome (BBS): they showed noticeable clinical signs of abnormal appearances resulting in depressed saleability. Since very few specifics are known about causative agents of these syndromes, this study aimed at investigating histopathological features and culturable bacteria associated with these fish infected in the process of farming. Histopathologically, SNS fish consistently exhibited necrosis and severe melanization in the muscles and multiple internal organs. Whereas BBS fish exhibited either typical granulomas or tissue damage associated with acid-fast stained bacteria and Gram negative bacteria, respectively. Six different Gram negative bacterial species were recovered from BBS fish while 23 bacterial species belonging to 14 genera were recovered from fish suffering from SNS. Most of the culturable bacteria are new to betta fish and some of them are known to be marine bacteria, suggesting possible entry route via a contaminated live feed, commercial Artemia shrimp. The true causative agents of these syndromes remain unclear. However, histopathological changes and existence of a wide range of bacteria associated with the naturally diseased fish suggest involvement of multiple bacterial infections.

Phylogenetic analysis of parasitic trematodes of the genus Euclinostomum found in Trichopsis and Betta fish.

Many species of fish in the world are infected with digenean trematodes belonging to the genera Clinostomum and Euclinostomum. In this study, metacercariae, identified as Euclinostomum sp. on the basis of morphological characteristics and molecular data, were taken from 3 osphronemid fish- Trichopsis vittata , Trichopsis schalleri, and Betta imbellis, in Thailand. Phylogenetic analysis based on a mitochondrial gene (cytochrome c oxidase subunit I) and 2 nuclear genes (18S rDNA and ITS-internal transcribed spacer) of these Euclinostomum parasites indicated a clear distinction from those belonging to the Clinostomum genus. These are the first records of partial mitochondrial and nuclear DNA sequences of an Euclinostomum sp.

Molecular and morphological evidence supports the species status of the Mahachai fighter Betta sp. Mahachai and reveals new species of Betta from Thailand.

Two regions of mitochondrial (mt) DNA, cytochrome c oxidase subunit 1 (COI) and 16S rRNA, were sequenced in nine species of Betta from Thailand and Indonesia. Most species showed little intraspecific COI variation (adjusted mean = 0.48%) including the putative species Betta sp. Mahachai, but one species (Betta smaragdina) included three lineages showing much greater divergence (7.03-13.48%) that probably represent overlooked species. These findings were confirmed by maximum likelihood analysis and Bayesian inference, which revealed well-supported corresponding monophyletic clades. Based on these results and morphological differences, the putative species Betta sp. Mahachai from central Thailand is a species distinct from other members of the B. splendens group and represents a new and hitherto undescribed species. Furthermore, this study also demonstrated the probable existence of two overlooked Betta species found in the Khorat plateau basin, illustrating the utility of mitochondrial genetic markers in the revelation of overlooked diversity.

Serine hydroxymethyltransferase from pyrimethamine-sensitive and -resistant strains of Plasmodium chabaudi.

Serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified from a pyrimethamine sensitive strain of Plasmodium chabaudi. Km values of 2.91 and 1.08 mM were determined for tetrahydrofolate and serine, respectively. The effects of pH, of temperature and of some potential inhibitors were determined. The enzyme was also partially purified from a pyrimethamine-resistant strain of P. chabaudi and subjected to the same regime. No differences between the enzymes from the two sources could be detected. It would appear that the changes in properties in the enzymes dihydrofolate reductase and thymidylate synthetase associated with the development of drug resistance in P. chabaudi were not reflected in any obvious alterations in serine hydroxymethyltransferase.

Types of collagen in Opisthorchis viverrini infected hamster liver.

Levels and types of collagen from normal and Opisthorchis viverrini infected hamster livers were compared. Normal liver contained approximately twice as much type I collagen than type III collagen. Upon infection by O. viverrini, both type I and type III collagen were elevated, but the increase in type III was proportionately larger than type I collagen. Of the 3-fold increase in total collagen content of infected livers, type I and type III collagen increased 2- and 4-fold, respectively. As a result, the ratio of type I to type III collagen changed from 2 in normal liver to 1.1 in the livers of animals infected with O. viverrini. The extent of the increase in both type I and type III collagen was found to depend on the infection times and on the number of worms present. In livers infected with 50 metacercariae of O. viverrini, both collagen types increased gradually with duration of infection and reached plateau after 4 months of infection. In livers from 3 month infections, with 15 worms or less, both types of collagen increased directly with the number of worms recovered. Levels of type I and type III collagen did not increase in infections with more than 20 worms.

Biochemical aspects of drug action and resistance in malaria parasites.

Biochemical aspects of action of antifolates and 4-aminoquinolines and their resistance in the malaria parasites are reviewed, with emphasis on pyrimethamine and chloroquine respectively. Resistance to pyrimethamine has been shown to be associated with either an increase in the amount of parasite dihydrofolate reductase or a reduced affinity of the enzyme for drug binding, in line with the presence of a distinctive pathway for folate metabolism. The theories for drug synergism in the folate pathway are discussed with respect to resistance to pyrimethamine and its combination with sulpha drugs. The biochemical basis for chloroquine resistance is still unclear, reflecting incomplete understanding of its mechanism of action. Data implicating the role of haemozoin and other components as a putative chloroquine receptor of the parasites are reviewed, and possible explanations for resistance are discussed.

Comparative studies on thymidylate synthetase from P. berghei and mouse reticulocytes.

Partially purified thymidylate synthetase from Plasmodium berghei and mouse reticulocytes was characterized. The mol. wt of the enzyme from P. berghei was about twice that from mouse reticulocytes. The optimum pH of the enzyme from P. berghei was found to be 6.5-7.5 while that from the host was 7.0-8.0. The enzyme from P. berghei was more susceptible to pH denaturation than the enzyme from reticulocytes. The enzyme from both sources differed in their Km values for substrates. The enzyme from reticulocytes was less sensitive to inhibition by substrate analogs than that from P. berghei.

Impairment of acetylcholine synthesis in thiamine deficient rats developed by prolonged tea consumption.

The synthesis of whole brain acetylcholine is reduced in thiamine deficient rats produced by prolonged administration of tea. In those rats fed a normal diet and given tea (1:50, w/v) instead of drinking water for 20 weeks, the conversion of [14C] pyruvate to [14C]acetylcholine decreased by 35%. However, no neurological symptoms were observed. Administration of tea to rats fed a thiamine half-deficient diet for 7-8 weeks caused not only 60% decrease in acetylcholine synthesis but also neurological symptoms. This decreased synthesis of acetylcholine is related to a decline in pyruvate dehydrogenase activity. The results suggest that prolonged administration of tea to rats cause an impairment of acetyl CoA production resulting in a deficit in acetylcholine synthesizing capacity.

Increased efficacy of liposomes-encapsulated praziquantel in treatment of opisthorchiasis in hamsters.

An increased efficacy of liposomes-encapsulated praziquantel was observed in the treatment of hamster opisthorchiasis. A single intracardial dose of 1.5 mg/kg of encapsulated praziquantel is as effective as an intracardial dose of 30 mg/kg or an oral dose of 100 mg/kg of free praziquantel. The suppressing activity of both free and liposomes-encapsulated praziquantel significantly decrease as the infection times increase from 1 to 5 weeks, suggesting that the young liver flukes are more susceptible to praziquantel than the adult flukes.

Liver procollagen prolyl hydroxylase in Opisthorchis viverrini infected hamsters after praziquantel administration.

Infection of hamsters by the human liver fluke Opisthorchis viverrini elevated liver procollagen prolyl hydroxylase activity, reflecting increased collagen biosynthesis. The increase was proportional to the intensity of infection. However, the infected liver procollagen prolyl hydroxylase activity decreased after administration of praziquantel 300 mg kg-1 body weight, and approached normal levels two weeks after treatment. In the infected hamsters, praziquantel, at a curative dose, caused a transient increase in serum aminotransferase levels and a small but persistent rise in serum alkaline phosphatase. The drug, however, did not cause changes in these enzyme activities in the uninfected hamsters.

Liver collagen in Opisthorchis viverrini infected hamsters following praziquantel treatment.

The dose of praziquantel required to achieve a 100% worm reduction in O. viverrini infected hamsters was found to be 300 mg/kg body weight. The drug was administered orally for 1 day by dividing a total dose into 3 equal doses at 4 h interval. The effect of praziquantel treatment on liver collagen was followed by measuring liver collagen content in at various intervals after administration of the drug. A decrease in collagen content in the infected livers occurred within a few weeks following the treatment suggesting a recovery from liver fibrosis.

Increased liver prolyl hydroxylase activity in hamsters infected with the human liver fluke Opisthorchis viverrini.

A parallel increase in liver collagen content and prolyl hydroxylase activity was observed in hamsters infected with the human liver fluke Opisthorchis viverrini. They were elevated at 2 weeks after infection, gradually increased to approximately 2-fold at 7-11 weeks of infection, and then declined as with duration of infection time increasing from 11 to 22 weeks.

Liver collagen turnover in hamsters during infection by the human liver fluke, Opisthorchis viverrini.

Hamsters infected with Opisthorchis viverrini, a liver fluke of man, showed an increased deposition of collagen in their livers. However, increased collagen breakdown as well as its synthesis were observed in the infected livers. Thus, stimulated synthesis might be the main factor responsible for the net increase in collagen content. Synthesis and degradation increased to a greater extent in short-term infection than in long-term infection whereas the hepatic collagen content was equally elevated in both cases. The results, therefore, suggested a difference in collagen metabolism of short-term and long-term infected livers.

Stimulation of Ca2+ uptake in the human liver fluke Opisthorchis viverrini by praziquantel.

45Ca2+ uptake by the human liver fluke Opisthorchis viverrini is enhanced by praziquantel. The drug-induced 45Ca2+ uptake was dependent on the presence of Ca2+ and was attenuated in the presence of 10 mM Mg2+. La3+ and vanadate at concentration of 1 mM partially reduced the amount of 45Ca2+ uptake into the liver fluke in response to praziquantel treatment. The stimulating effect of praziquantel was eliminated in the presence of 10 microM verapamil. These findings suggest that praziquantel increases the permeability of the liver fluke tegument to Ca2+ probably by interfering with the mechanism that regulates Ca2+ binding or transport across the tegumental membrane.

Ultrastructural investigations on the effects of praziquantel on human trematodes from Asia: Clonorchis sinensis, Metagonimus yokogawai, Opisthorchis viverrini, Paragonimus westermani and Schistosoma japonicum.

The effect of praziquantel (Biltricide) on the ultrastructure of trematodes pathogenic to man in Asia was investigated in: Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum, Metagonimus yokogawai, and Paragonimus westermani. The different parasites were isolated from their respective experimental hosts and uniformly incubated for 5, 15, 30 or 60 min at 37 degrees C in medium TC 199 containing 0, 1, 10 or 100 micrograms praziquantel/ml. All parasites exposed to praziquantel were contracted and displayed tegumental alterations. 1. Clonorchis sinensis, Opisthorchis viverrini and Schistosoma japonicum reacted with severe tegumental vacuolisation within 5 min after exposure to 1 microgram praziquantel/ml. The extent of the damage induced increased with exposure time. 2. In vivo treated Opisthorchis viverrini worms had the same damages as in vitro treated ones. 3. Metagonimus yokogawai displayed the same degree of vacuolisation after exposure to all concentrations of praziquantel. Complete destruction of the tegumental surface was not achieved, however, possibly because of the presence of numerous tegumental hooks. 4. Paragonimus westermani was least sensitive to praziquantel. Only very few vacuoles were formed after incubation in 100 micrograms/ml for 60 min. It is assumed that the very dense texture of the thick tegument is responsible for the relative refractoriness to praziquantel in vitro. However, in vivo the parasite is successfully eliminated by praziquantel.

Protein-catalyzed exchange of phosphatidylinositol between rat brain microsomes and myelin.

Exchange of phosphatidylinositol and phosphatidylcholine between microsomal and myelin membranes has been demonstrated. This exchange is reversible and catalyzed by soluble proteins from the brain homogenate precipitated at pH 5.1. The extent of exchange of phosphatidylinositol from microsomal membrane to myelin is dependent upon pH and temperature, with an optimum around pH 7 and at 50 degrees C. Maximum exchange was observed at approximately equal amounts of microsomal, myelin, and supernatant proteins. The extent of the catalyzed exchange increases 4- to 8-fold upon using sonicated or heat-treated myelin as an acceptor membrane. Heating of microsomal membranes results in no change. The extent of catalyzed exchange of phosphatidylcholine is less than that of the phosphatidylinositol. The exchange of other phospholipids and glycolipids between microsomal and myelin membranes cannot be demonstrated. The catalytic activity of the pH 5.1 supernatant proteins in rat brain for the exchange of phosphatidylinositol increases with age after birth and reaches a maximum around 21 days of age analogous to the process of myelination. The pH 5.1 supernatant proteins from quaking and jimpy mutant mice has normal catalytic activity.

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.