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.

Development of an intergeneric conjugal transfer system for rimocidin-producing Streptomyces rimosus.

AIMS: To develop an intergeneric conjugation system for rimocidin-producing Streptomyces rimosus. METHODS AND RESULTS: High efficiencies of conjugation [10(-2)-10(-3) transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40 degrees C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24-h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l(-1) MgCl(2) was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. CONCLUSION: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.

Effects of multivitamin supplementation for improvement of thiamin, riboflavin, and retinol nutrition in pediatric patients.

The effect of multivitamin supplementation on thiamin, riboflavin and retinol nutrition was studied in ten male chronic pediatric patients at Ramathibodi Hospital. MTV forte (Government Pharmaceutical Organization) was given 1 tablet daily for ten days and the effect was compared with the preceding ten-day control period. Anthropometric biochemical, clinical and dietary assessments were performed throughout the study. Biochemical assessments of vitamin nutrition at the beginning and the end of a 10-day non-supplementation period revealed multiple vitamin deficiencies in the subjects, though clinical manifestation was not seen. Hospital diets improved vitamin nutrition of some patients but most of them still showed subclinical vitamin deficiencies. Improvement of vitamin nutrition was clearly shown in most patients after MTV supplementation. Multiple vitamin supplements should be prescribed in high-risk patients since there was no definite pattern of vitamin deficiencies in various diseases and early detection of vitamin deficiency was difficult to perform. The dosage between 1-2 times of RDA was considered adequate in most of our patients.

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.

The malaria parasite monitored by photoacoustic spectroscopy.

Noninvasive photoacoustic spectroscopy was used to study the malaria parasites Plasmodium chabaudi and Plasmodium berghei, their pigment, and ferriprotoporphyrin IX, which is a by-product of the hemoglobin that the parasite ingests. The results indicate that the pigment consists of ferriprotophorphyrin self-aggregates and a noncovalent complex of ferriprotoporphyrin and protein. Spectra of chloroquine-treated parasites reveal in situ interaction between the drug and ferriprotoporphyrin. Chloroquine-resistant parasites, readily distinguishable by this method, appear to degrade hemoglobin only partially.

Interaction of antimalarial drugs with hemin.

Hemin (ferriprotoporphyrin IX) is shown to form complexes with the chloroquine class of antimalarial drugs. The Soret band of hemin becomes optically active upon the addition of chiral drugs. Results on the hemin-induced quenching of the fluorescence of chloroquine are consistent with the formation of a 2:1 hemin:drug complex with a formation constant of 1.4 x 10(7) at 298 K. Also a direct comparison of the drug-treated and drug-free parasites themselves, by the noninvasive photoacoustic spectroscopic method, reveals an in vivo interaction between endogenous hemin and the added drug.

Effects of hypochlorite on thiamine and its derivatives.

Thiamine and its phosphorylated derivatives reacted with hypochlorite with reaction rates following the order: thiamine greater than thiamine monophosphate greater than thiamine diphosphate from pH 4.0 to 6.5. At least one unknown transient intermediate was formed and at least one non-thiochrome product was fluorescent. Chemiluminescence was also observed.

Kinetics of bisulphite cleavage of the three biological phosphorylated derivatives of thiamine.

Bisulphite/sulphite cleaves thiamine and its biological phosphorylated derivatives into the same pyrimidine moiety and their respective thiazole moiety with pseudo-first order kinetic constants following the order of thiamine greater than thiamine monophosphate greater than thiamine diphosphate greater than thiamine triphosphate over the pH range of 3.9 to 7.0. The activation energy was 14.8 kcal/mol for all four thiamines at pH 5.2 and over the temperature range of 25 to 65 degrees C. The rate differences may be rationalized in terms of differences in the pH-dependent electronic, charge and conformation distributions of the various thiamine species.

Reactions between phosphorylated thiamines and tannic acid as followed by difference spectroscopy.

Caffeic acid, catechin, chlorogenic acid, hydroquinone (also benzoquinone) and tannic acid were used to react with thiamine and the ultraviolet-visible difference spectra were recorded. The time-course of the thiamine-polyphenol interactions was studied by following the difference absorbance change with time. The rate of reaction as determined by difference spectroscopy was the same as that found by chemical assay. The spectroscopic method showed that thiamine, thiamine monophosphate and thiamine diphosphate reaction rate with tannic acid followed the order thiamine greater than thiamine monophosphate greater than thiamine diphosphate up to pH 7.4.

Molecular complexes of quinoline antimalarials with iron-porphyrin components of protease-digested methemoglobin.

Chloroquine, quinine, mefloquine and quinacrine have been found by difference spectroscopy to interact with hemozoin from Plasmodium berghei, trypsin and pronase-digested methemoglobin, hemin, heme, protoporphyrin IX and hematoporphyrin. These drugs also compete with one another in their binding to hemin. It is proposed that the iron-porphyrin moiety of digested hemoglobin is a common binding site for the accumulation of the schizontocidal drugs in the autophagosomes of the malarial parasite.

Binding of antimalarial drugs to hemozoin from Plasmodium berghei.

Chloroquine, quinacrine and mefloquine bind to Plasmodium berghei hemozoin, hemin, hemi, protoporphyrin IX and protease digested methemoglobin. This binding may be the basis for drug accumulation and action in the parasite.