An epizootic bacterial infection in the giant freshwater prawn Macrobranchium rosenbergii occurred in Taiwan from May to June 1999. The cumulative mortality was approximately 30 to 75%. The diseased prawns showed opaque and whitish muscles and were approximately 2 mo old with total lengths from 5 to 6 cm. Histopathologically, they showed marked edema and necrotic lesions with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Three isolates from diseased prawns at different farms were tested using the API 20 Strepsystem and conventional tests and identified as Lactococcus garvieae. Experimental infections with these isolates gave gross signs and histopathological changes similar to those seen in the naturally infected prawns. The LD 50 value of isolate MR1 was 6.6 × 10 5 colony forming units/prawn. Identification of MR1 was confirmed by a PCR assay for L. garvieae that gave the expected amplicon of 1100 bp. In addition, its 16S rDNA sequence (GenBank accession number AF283499) gave 99% sequence identity to Enterococcus seriolicida (synonym L. garvieae; GenBank accession number AF061005). This is the first report of confirmed L. garvieae infection in prawn aquaculture. KEY WORDS: Lactococcus garvieae · Giant freshwater prawn · Macrobranchium rosenbergii · PCR · 16S rDNA sequencing Resale or republication not permitted without written consent of the publisherDis Aquat Org 45: [45][46][47][48][49][50][51][52] 2001 ola quinqueradiata in Japan was named Enterococcus seriolicida (Kusuda et al. 1991) after previously being classified as Streptococcus (Kusuda et al. 1976). However, taxonomic studies based on DNA-DNA hybridization (Eldar et al. 1996) and sequence analysis of 16S rRNA (Domenech et al. 1993) indicated E. seriolicida (Kusuda et al. 1991) was synonymous with L. garvieae (Eldar et al. 1996). In Spain, a serious septicemic disease of Scopthalmus maximus or large turbot (100 g to 3 kg) was accredited to an Enterococcus-like bacterium (Nieto et al. 1995) that was also found to match with L. garvieae (Eldar et al. 1996). Zlotkin et al. (1998) subsequently developed a polymerase chain reaction (PCR) assay based on unique regions of the L. garvieae 16S rRNA gene that allow for its specific identification.The giant freshwater prawn Macrobranchium rosenbergii is commercially cultured throughout the world and intensively so in Taiwan, where production, however, decreased by 47 to 52% from 1992 to 1995 (New 1995, Taiwan Fisheries Bureau 1996 due to disease problems. Two diseases have been linked to production decline. One is a yeast infection that occurs mostly in the cool season (October to March), with symptoms including a yellow exoskeleton, a swollen hepatopancreas (HP), milky hemolymph and opaque and whitish muscles (Shu 1993, Cheng & Chen 1998a. The other is an Enteroccoccus-like infection that occurs mostly in the hot season (June to September, especially during phytoplankton blooms) and causes muscle ...

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.

Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and M. kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, M. kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M. kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and M. kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.

Seventy-six Taiwanese bacterial isolates including 74 from diseased, cultured, aquatic animals (54 grey mullet Mugil cephalus, 3 basket mullet Chelon alatus, 2 tilapia Oreochromis niloticus, 1 grouper Epinephelus coioides, 2 yellowfin seabream Acanthopagrus latus, 1 Borneo mullet Chelon macrolepis, 1 bullfrog Rana catesbeiana, 1 Japanese eel Anguilla japonica, and 9 giant freshwater prawns Macrobrachium rosenbergii), 1 wild-caught seafood species (squid muscle collected from a restaurant) and 1 human isolate (from a patient with a history of consuming raw squid in the previously mentioned restaurant), all collected between 1999 and 2006, were confirmed by PCR assay to be Lactococcus garvieae. The phenotypic characterization was determined by rabbit anti-KG+ and KG− serums, and 74 of the 76 Taiwanese strains displayed a KG− phenotype. The genetic characterization was investigated by pulsed-field gel electrophoresis (PFGE). Genomic DNA was digested with restriction endonucleases Apa I and Sma I and separated by PFGE. Ten different L. garvieae pulsotypes were identified. Predominant pulsotypes A1a/S1a were obtained from > 96% of strains (52 of 54) from grey mullet, demonstrating a clonal dissemination of L. garvieae in grey mullet in Taiwan. In experimental challenges with grey mullet and tilapia, L. garvieae pulsotypes A1/S1 and A11/S11 showed higher virulence compared with other pulsotypes.KEY WORDS: Lactococcosis · Pulsed-field gel electrophoresis · Pulsotype · Grey mullet · Tilapia · Giant freshwater prawn Resale or republication not permitted without written consent of the publisher

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus. The monacolin K biosynthetic gene cluster, mokA-mokI, has been characterized in Monascus pilosus. The mokH gene encoding Zn(II)2Cys6 binuclear DNA binding protein is assumed to be an activator for monacolin K production. In this study, the mokH gene was cloned and driven by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter for overexpression in M. pilosus. The transformants containing an extra copy of the mokH gene were obtained and verified by PCR and Southern hybridization. The transcripts of mokH in the transformants were expressed significantly higher than those of the wild-type strain. The transformants were stably inherited through the next generation, as determined by observation of the enhanced green fluorescent protein (EGFP). The transformant T-mokH1 also showed a 1.7-fold higher production of monacolin K than the wild-type strain in a time course analysis. Analysis of the RT-PCR products demonstrated that the monacolin K biosynthetic genes in the transformant were expressed to a greater extent than those in the wild-type strain. These results indicated that mokH upregulated the transcription of monacolin K biosynthetic genes and increased monacolin K production.

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A new species of Kudoa lutjanus n. sp. (Myxosporea) is described from the brain and internal organs of cultured red snapper Lutjanus erythropterus from Taiwan. The fish, 260 to 390 g in weight, exhibited anorexia and poor appetite and swam in the surface water during outbreaks. Cumulative mortality was about 1% during a period of 3 wk. The red snapper exhibited numerous creamy-white pseudocysts, 0.003 to 0.65 cm (n = 100) in diameter, in the eye, swim bladder, muscle and other internal organs, but especially in the brain. The number of pseudocysts per infected fish was not correlated with fish size or condition. Mature spores were quadrate in apical view and suboval in side view, measuring 8.2 ± 0.59 µm in width and 7.3 ± 0.53 µm in length. The 4 valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 3.62 ± 0.49 µm in length and 2.2 ± 0.49 µm in width. Mild inflammatory responses or liquefaction of host tissue were associated with K. lutjanus n. sp. infection. The junction of shell valves appeared as overlapping, straight lines. The polar filament formed 2 to 3 coils. A general PCR (polymerase chain reaction) primer for Kudoa amplified the small subunit (SSU) rDNA sequences, and the amplified gene was sequenced. It was evident from the phylogenetic tree that the 3 strains tested, AOD93020M, AOD93028M and AOD93028B, were identical and belonged to the Kudoa SS rRNA subgroup. The evolutionary tree showed that these strains form a unique clade, at a distance from other Kudoa species and myxosporeans. The spore's morphological and ultrastructural characteristics, as well as the SS rDNA properties of the isolates, were also essentially identical and served to distinguish them from representative Kudoa. It is, therefore, proposed that the strains isolated from the diseased red snapper be assigned to a new species. KEY WORDS: Myxosporea · Kudoa lutjanus n. sp. · Brain · Red snapper · Lutjanus erythropterus Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [115][116][117][118][119][120][121][122][123][124] 2005 a polar capsule (Hervio et al. 1997, Moran et al. 1999a, Pampoulie et al. 1999, Swearer & Robertson 1999, Whipps et al. 2004. Most Kudoa species appear in the skeletal musculature and have been connected with post mortem myoliquefaction in commercially valuable marine fishes (Moran et al. 1999a). Kudoa thyrsites Gilchrist, 1924 has been recorded from 27 fish species dispersed worldwide (Whitaker et al. 1994). For example, this parasite has both a worldwide distribution and a wide host range (Kabata & Whitaker 1989, Kent et al. 1994, Moran & Kent 1999, Moran et al. 1999b. K. thyrsites does not normally cause mortality, although 1 case of high mortality in farmed Atlantic salmon Salmon salar L. has been observed (Harrell & Scott 1985). The few remaining Kudoa species have been identified from tissues including the gills, brain, gallbladder, pericardial cavity and ovary (Lom & Dyková 1992, Swearer & Robertson 1...

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