Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.     

Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 16S rDNA sequencing.

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 LD50 value of isolate MR1 was 6.6 x 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.     

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included 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. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.     

Effect of nitrite on interaction between the giant freshwater prawn Macrobrachium rosenbergii and its pathogen Lactococcus garvieae

Addition of nitrite-N at 1.5 mg l(-1) in tryptic soy broth (TSB) significantly (p < 0.05) decreased the growth rate of the bacterial pathogen Lactococcus garvieae and significantly (p < 0.05) reduced mortality compared to zero nitrite controls when injected into giant freshwater prawns Macrobrachium rosenbergii at 5 x 10(5) colony-forming units (CFU) per prawn. In other experiments, whereby prawns were injected with TSB-grown L. garvieae (5 x 10(5) CFU prawn(-1)) and then held in water containing nitrite-N, mortality at 72 h post-injection was significantly (p < 0.05) higher for prawns held in water containing 1.68 mg l(-1) nitrite than at lower concentrations. Prawns exposed to different concentrations of nitrite-N were examined for THC (total hemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and bacterial clearance efficiency. No significant differences in THC and phenoloxidase activity were observed among treatments. With prawns exposed to nitrite-N for 168 h (7 d) at 1.59 mg l(-1), phagocytic activity and clearance efficiency decreased, while at 1.15 mg l(-1) or more, respiratory burst increased, generating the superoxide anion at levels considered cytoxic to the host. We conclude that nitrite-N at 1.68 mg l(-1) causes depression in the immune response and increased mortality in M. rosenbergii infected with L. garvieae.     

Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.     

Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.     

Effects of intrinsic and extrinsic factors on the haemocyte profile of the prawn, Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii was investigated for its total haemocyte count (THC) based on season, sex, size and feeding rate. The THC, when the prawns were subjected to injections of foreign materials was also investigated. The prawns displayed the highest and lowest THC in autumn and winter respectively, with no significant difference between male and female, or among animals with a body weight range of 7-115 g. The prawns displayed the lowest THC at D3 stage, and the highest in C stage. The prawns displayed the lowest THC when they were fed at 0.1% feeding rate among feeding rates of 0.1%, 0.5%, 1.0% and 1.5% body weight x day(-1). Prawns injected with carbon powder and Enterococcus showed increased THC during the first 6 h. Prawns injected with saline and carbon powder had the lowest THC after 30 h, and recovered to the normal value after 54 h. Prawns injected with Enterococcus showed the lowest THC after 42 h, and showed delayed recovery.     

Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp

The virulence of two Aeromonas strains (A. veronii and A. caviae) isolated from the hepatopancreas of apparently healthy giant freshwater prawns (Macrobrachium rosenbergii) was compared using a challenge by injections. For the A. veronii strain, challenge with 3.7 x 10(5) cells/g of body weight led to 100% mortality; for the A. caviae strain, 3.8 x 10(6) cells/g produced 100% mortality. The 50% lethal doses (LD50) were 2.0 x 10(3) cells/g for A. veronii and 51.2 x 10(3) cells/g for A. caviae. Use of different culture media (trypticase soy broth vs prawn muscle extract) did not significantly affect the virulence of A. veronii. Injection of a sublethal dose (1 x 10(3) cells/g) of A. veronii led to a significant decrease in the total hemocyte count (THC) between 4 and 24 h after injection. Saline injections also caused a similar though less decrease in THC. In the first 24 h after injection of A. veronii (1 x 10(3) cells/g), the change in the percentages of granulocytes (both granular cells and semigranular cells) in the hemolymph was significantly different. After a significant initial increase, the percentage of hyaline cells fell by a factor of 4, from 9 to 2%. Phenoloxidase activity increased fourfold immediately after injection and returned to preinjection levels at 24 h.     

Effect of benzalkonium chloride stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

Addition of benzalkonium chloride (BKC) at 0, 0.3, 0.6 and 1.0 mg l(-1) to tryptic soy broth (TSB) had no effect on growth of Lactococcus garvieae, a bacterial pathogen of the giant freshwater prawn Macrobrachium rosenbergii. However, injection of the cultured cells into prawns at a dose of 4 x 10(6) colony-forming units (cfu) prawn(-1) resulted in significantly higher mortality at 120 h (p < 0.05) in prawns injected with cells grown in the absence of BKC than in prawns injected with cells grown in the presence of BKC. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 3 x 10(5) cfu prawn(-1)) and then held in water containing BKC at 0, 0.3, 0.6 and 1.0 mg l(-1). After 120 h, mortality was significantly higher in all the BKC treatments than in the control without BKC. Prawns showed no significant differences in total hemocyte count (THC) or differential hemocyte count (DHC) amongst treatment and control groups. However, 96 h exposure to 0.3 mg l(-1) BKC or more resulted in a decrease in phenoloxidase activity and an increase in respiratory burst to levels considered to be cytoxic. In summary, exposure of L. garvieae to BKC at 0.3 mg l(-1) or more decreased its virulence to M. rosenbergii, while exposure of M. rosenbergii to BKC at 0.3 mg l(-1) or more increased its susceptibility to L. garvieae infection.     

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.     

Continuous Darkness Stimulates Body Growth of the Juvenile Giant Freshwater Prawn, Macrobrachiumrosenbergiide Man

-The effect of photoperiod on growth of juvenile giant freshwater prawns, Macrobrachium rosenbergii de Man, was tested. The prawns were divided into four groups and each group was reared under one of the following light-dark conditions: continuous darkness (L0:D24), 12 hr light: 12 hr dark (L12:D12), 16 hr light: 8 hr dark (L16:D8), and 20 hr light: 4 hr dark (L20:D4). Body size was determined at the age of 45, 75, and 110 days by measuring total length, orbital length, and carapace length; body weight was determined at the age of 110 days. At 110 days of age, the prawns reared under L0:D24 photoperiod were significantly longer and heavier than those reared under other light-dark conditions. The survival rate of the prawns reared under L0:D24 photoperiod was also higher than that of other groups. This study indicates a positive effect of continuous darkness on growth and survival rate of juvenile giant freshwater prawns, M. rosenbergii.     

Point prevalence, microbiology and antifungal susceptibility patterns of oral Candida isolates colonizing or infecting Mexican HIV/AIDS patients and healthy persons

We have conducted a longitudinal study over a 3-year period to address the point prevalence, microbiological characteristics and antifungal susceptibility patterns of yeast isolates colonizing or infecting the oral cavities of 111 HIV-infected (51 adults, 60 children) and 201 non HIV-infected (109 adults, 92 children) Mexican persons. Regarding the epidemiology of oral candidiasis, Candida albicans was the most frequent species isolated. Seventy-one out of 85 isolates from colonized persons were C. albicans (83.5%), 27 isolates of them were from HIV-infected children and 44 from non HIV-infected patients. Sixty-two isolates belonged to serotype A which was the most prevalent serotype of C. albicans. Non-albicans species (Candida glabrata, Candida tropicalis and Candida parapsilosis, and Saccharomyces cerevisiae) were isolated from 16.5% of colonized patients and from 38.5% patients with candidiasis or Candida-related lesions. There were nine episodes of infection or colonization by at least 2 different yeast species. In the case of HIV/AIDS patients, it was determined that yeast carriage was not associated with the number of CD4+ cells or the viral load, but HAART reduced the prevalence of oral candidiasis. Overall, most patients harbored strains in vitro susceptible to fluconazole, however 10.8% of the yeasts were resistant to one or more azole antifungal agents and 29% were intermediate susceptible to them. On the contrary, 5-fluorocytosine was very active against all isolates tested, and amphotericin B was active against 97.9% of them.     

Prevention of lethal murine candidiasis using HP (2-20), an antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1

Peptide HP (2-20), A(2)KKVFKRLEKLFSKIQNDK(20), is a cationic antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein 1, HpRpL1. Native peptide HP (2-20) and its synthetic derivatives have been shown in vitro to exhibit potent killing activity against Gram-positive, Gram-negative and yeast cells, thus, making them promising candidates for treatment of polymicrobial infections. However, the therapeutic potential of peptide HP (2-20) or its synthetic derivatives in any animal model of either bacterial or fungal diseases has not yet been investigated. In this study, we demonstrate that synthetic peptide amide HP (2-20), administered in six doses (300microg each; one intraperitoneal dose at the time of the infection, followed by five intravenous doses at 12h intervals) to CBA/J male mice experimentally infected with a lethal inoculum ( [Formula: see text] CFU) of Candida albicans, delayed the onset of disease, suppressed disease progression, and greatly increased survival rate and time (16.6% by day 14), as compared with the untreated infected control mice (100% mortality by day 5). Further, using isotonic buffer systems differing in ionic strength, peptide HP (2-20) was shown in vitro to exhibit an ionic strength-dependent hemolytic activity, previously not detected. Repeated intravenous administration of uninfected control CBA/J male mice with peptide HP (2-20), however, caused neither morbidity nor mortality. These findings strongly evidence the therapeutic efficacy and safety values of peptide HP (2-20) as a lead drug for the treatment of acquired candidiasis.     

Occurrence and dominance of yeast species in naturally fermented milk from the Tibetan Plateau of China

To determine which yeasts are present in the naturally fermented milks of China, 69 samples made by the nomads of Tibet were collected from the Tibetan Plateau in China. From these samples, 225 strains of yeast were isolated and identified using conventional microbiological analysis and gene sequencing analysis of the D1/D2 domain of the large subunit (26S) ribosomal DNA. The results showed that the total concentration of yeasts in these samples ranged from 5.01 to 8.97 log10 colony-forming units (CFU)/mL (6.91?± 1.02 log10 CFU/mL; mean?± SD). The number of cultivable yeasts was higher in the samples from Qinghai (7.55?± 0.75 log10 CFU/mL) than those from Tibet (6.21?± 0.79 log10 CFU/mL, P?< 0.05). Moreover, there were 15 phylotypes in these 69 samples. Among these phylotypes, Kluyveromyces marxianus (49.3%, frequency percentage), Saccharomyces cerevisiae (62.3%), and Pichia fermentans (46.4%) appeared frequently and can be considered the most common culturable species in naturally fermented milk products. Traditional fermented Mongolian cow milk featured a wide diversity of yeast species, including Issatchenkia orientalis, Kazachstania unisporus, Rhodotorula mucilaginosa, Candida pararugosa, Torulaspora delbrueckii, Geotrichum sp., Kazachstania unisporus, Geotrichum fragrans, Debaryomyces hansenii, Yarrowia lipolytica, Trichosporon gracile, and Pichia membranifaciens. This study provides new data on yeast composition in naturally fermented milk and shows the yeast biodiversity of fermented milk products from the Tibetan Plateau of China.     

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.     

Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina

Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta-mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta-mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.     

Trichlorfon, an organophosphorus insecticide, depresses the immune responses and resistance to Lactococcus garvieae of the giant freshwater prawn Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.     

News & Notes: Virulence of Vibrio alginolyticus Isolated from Diseased Tiger Prawn,Penaeus monodon

Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan. A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar. This strain was characterized and identified to be Vibrio alginolyticus. The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide. The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P. monodon) and kuruma prawns (P. japonicus) with LD₅₀ values of 1.13 × 10⁵, 2.46 × 10⁵ CFU/g, and 0.23, 0.63 μg protein/g prawn body weight, respectively.     

Transfer of foreign gene to giant freshwater prawn (Macrobrachium rosenbergii) by spermatophore-microinjection

We developed a spermatophore-microinjection (SMI) technique that allows exogenous DNA fragments to be transferred easily into the giant freshwater prawn (Macrobrachium rosenbergii), an important aquacultural shellfish and aquatic invertebrate model. From 28 to 1, 000 ng of the circular plasmid pGL, in a total volume of 1 microl, were directly microinjected into spermatophores. Fertilization and hatching of prawns created with SMI were completed in vivo. Fertilization and hatching rates in the SMI treatments did not differ from those of the untreated control group. The genomes of free swimming, SMI-created larvae (21 days after fertilization) were analyzed using PCR and Southern blot analyses. A product with a molecular mass of 680 bp was amplified. It corresponded to amplifications of pGL, and Southern blot analysis revealed that the amplified band was positive. The gene transfer rate was primarily dependent on the concentration of DNA during SMI. The higher the concentration of pGL, the higher the rate of gene transfer. PCR and Southern blot analyses detected the existence of foreign DNA in 16 of 23 samples (70%) of genomic DNA isolated from hatched larvae in the 750 ng pGL SMI treatment. SMI, described here for the first time, is the simplest and most efficient method for mass producing transgenic giant freshwater prawns.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).